WO2007117444A2 - Protein detection by aptamers - Google Patents

Abstract

The invention provides a system for performing large scale proteomic expression studies. The methods of the invention are based on detecting and/or measuring proteins using a plurality of aptamers that recognize oligopeptide epitopes on a target protein. Further, the present invention relates to methods for diagnosing and staging diseases by detecting and/or measuring proteins associated with certain clinical conditions. Methods of selecting aptamers, diagnostic kits are also encompassed.

Description

PROTEIN DETECTION BY APTAMERS

1. INTRODUCTION

[001] The field of the invention is diagnostics, particularly binding assays for detecting and/or measuring a protein. The present invention relates to methods for determining the presence and/or amount of a protein by the binding of a plurality of aptamers. Further, the present invention relates to methods for diagnosing and staging diseases by detecting and/or measuring proteins associated with certain clinical conditions.

2. BACKGROUND OF THE INVENTION

[002] Recent advances in technology, instrumentation, molecular biology, and bioinformatics have made it possible to begin to analyze entire units of cellular components, such as the genome, transcriptome, and more recently, the proteome. These advances provide the opportunity to begin to monitor changes in human tissue proteomes that are associated with aging, disease, and other important physiological processes. The ultimate goals of proteomics are to comprehensively identify all proteins, their associated biological activities, post-translational modifications, and protein- protein interactions occurring in a given cell, and use the information for disease diagnosis, health monitoring and drug development.

[003] Two of the factors that contribute to the enormity of the challenge of proteomics and the very modest progress to date are the 100-fold increased level of complexity of the proteome as compared to the genome and the estimated 10¹⁰ dynamic range of protein concentrations. Despite major technological improvements, advances in understanding of the human proteome so far have been modest. The annual rate of FDA-approved plasma protein— based clinical diagnostic assays has actually stagnated in recent years. This clearly is at odds with popular expectations that advances in genomics and proteomics are transforming the clinical landscape through diagnostic application of knowledge on large numbers of new proteins.

[004] Proteomics entails the simultaneous separation of proteins from a biological sample, and the quantitation of the relative abundance of the proteins resolved during the separation. Proteomics currently relies heavily on two-dimensional (2-D) gel electrophoresis. 2D-gel electrophoresis and mass spectrometry have been widely used for measuring and analyzing large numbers of proteins for research purposes (Mears et al., Proteomics 4 (2004) 4019-4031; Lee et al., Proteomics 3 (2003) 2330-2338; McDonough et al., Proteomics 2 (2002) 978-987. These technologies have limitations on their speed, sensitivity, high throughput and reproducibility (Zhou et al., Trends Biotechnol 19 (2001) S34-39.) The commonly used stains for evaluating protein expression in 2-D gels (such as Coomassie Blue, colloidal gold and silver stain) do not provide the requisite dynamic range to be effective in this capacity. These stains are linear over only a 10- to 40-fold range, whereas the abundance of individual proteins differs by many orders of magnitude. In addition, low abundance proteins, such as transcription factors and kinases that are present in 1-2000 copies per cell, often represent species that perform important regulatory functions. The accurate detection of such low-abundance proteins is an important challenge to proteomics. [005] Recently, RNA and DNA-based aptamers were found to be as specific as antibodies for interacting with proteins (Berezovski et al, Anal Chem 75 (2003) 1382- 1386; Lee et al., Biochem Biophys Res Commun 327 (2005) 294-299; Sekiya et al., Nucleic Acids Symp Ser (2000) 163-164; Katahira et al., Nucleic Acids Symp Ser (1999) 269-270; Convery et al., Nat Struct Biol 5 (1998) 133-139; and Jiang et al., Anal Chem 75 (2003) 2112-2116). Nucleic acid aptamers have been isolated to recognize and bind to proteins, neuropeptides, and even small molecules (Proske et al., Y, J Biol Chem 277 (2002) 11416-11422; Huizenga et al., Biochemistry 34 (1995) 656-665; Sazani et al., J Am Chem Soc 126 (2004) 8370-8371.19). These aptamers have been used in protein detection and protein arrays (Brody et al., MoI Diagn 4 (1999) 381-388; Liu et al., Angew Chem Int Ed Engl 44 (2005) 4333-4338; Xu et al., Anal Chem 77 (2005) 5107-5113; Stadtherr et al., Anal Chem 77 (2005) 3437-3443; Collett et al., Methods 37 (2005) 4-15; McCauley et al., Anal Biochem 319 (2003) 244-250; Ikebukuro et al., Biosens Bioelectron 20 (2005) 2168-2172; and Bock et al., Proteomics 4 (2004) 609- 618.). However, unlike sequence based DNA microarrays that cover most of the genes in the genome, these protein arrays only cover a very small fraction of proteins in the proteome. It has been estimated that post-transcriptional and post-translational modifications may generate millions of different proteinaceous entities in a mammalian proteome (Phelan et al., Proteomics 3 (2003) 2123-2134 and Godovac-Zimmermann et al., Mass Spectrom Rev 20 (2001) 1-57). Therefore, it is difficult and labor-intensive to identify and generate the specific molecular partners for every protein in the proteome. Indeed, there is an existing need for technology that can carry out protein expression analysis efficiently at the proteomic level.

[006] The present invention offers a system for developing aptamer reagents that is economical for large scale proteomic studies as well as methods of using the aptamers in highly sensitive and specific protein assays. Citation of any reference in Section 2 of this application is not to be construed as an admission that such reference is prior art to the present application

3. SUMMARY OF THE INVENTION

[007] The invention provides a system for determining the presence and/or amount of a protein by the specific binding of a plurality of aptamers. The system can be applied to large scale studies of protein expression and the methods of the invention are useful for diagnosing and staging diseases by detecting and/or measuring proteins associated with certain clinical conditions.

[008] In one embodiment, the system of the invention comprises a library of aptamers wherein each aptamer binds specifically to an oligopeptide, and accessory reagents that facilitate detection and measurement of the binding of the aptamers to a target. DNA aptamers are generally preferred.

[009] In another embodiment, the invention provides a method for detecting or measuring a target protein, comprising contacting at least two aptamers with a sample comprising the target protein, wherein said at least two aptamers each binds to a different oligopeptide epitope on the target protein under the appropriate conditions; detecting or measuring binding of the at least two aptamers to said target protein; wherein detection or measurement of the binding indicates presence or amount, respectively of the target protein. The specificity improves as the number of aptamers in the set increases. Thus, in a preferred embodiment, a set of four different aptamers are used in the method. In another preferred embodiment, a set of five different aptamers are used in the method. In various embodiments, the plurality of different aptamers can be contacted with the sample individually in any order, in groups sequentially, or simultaneously. The binding conditions can optionally be changed between contacting steps which use different aptamers. Unbound aptamers can optionally be removed prior to the following contacting step or detecting step. In certain embodiments, the method provide one or more pretreatment step(s) to remove contaminants from the sample, to change reaction conditions, to disassociate molecular complexes comprising the target and/or to denature the target in the sample.

[010] The method also provides the use of a variety of detection schemes for the binding of aptamers to the target, such as hybridization assays and nucleic acid amplification. To improve specificity of the method, it is preferred that the method detects the concurrent binding of all members of the aptamer set to the target. The sensitivity of the method is generally improved with nucleic acid amplification. A preferred method employs proximity-dependent ligation of the bound aptamers followed by nucleic acid amplification. Accordingly, the detecting or measuring step of the method further comprises ligating specifically the ends of neighboring aptamer pairs, directly or indirectly via a connector, to form a reporter template; amplifying the reporter template to generate a detectable or proportionate amount of reporter nucleic acids, wherein the presence or amount of the reporter nucleic acid indicates the presence or amount, respectively of the target protein.

[011] In another embodiment, the present invention provides for a method of diagnosing a disease or disorder in a subject comprising the steps of contacting at least two aptamers with a sample from the subject that might or might not contain a target, wherein said at least two aptamers each binds to a different oligopeptide epitope on the target protein under the appropriate conditions; detecting or measuring binding of the at least two aptamers to said target protein; and detecting or measuring binding of the at least two aptamers to the target, wherein detection or measurement of binding indicates presence or amount, respectively, of the target; and wherein the disease or disorder is determined to be present when the absence, presence or amount of the target differs from a control value representing the amount of target present in an analogous sample from a subject not having the disease or disorder. Preferably, a set of four or five different aptamers is used in the diagnostic methods of the invention.

[012] In yet another embodiment, the present invention provides for a method of staging a disease or disorder in a subject comprising the steps of contacting at least two aptamers with a sample from the subject that might or might not contain the target, wherein said at least two aptamers each binds to a different epitope on the target protein under the appropriate conditions; detecting or measuring binding of the at least two aptamers to said target protein; and detecting or measuring binding of the at least two aptamers to the target, wherein detection or measurement of binding indicates presence or amount, respectively, of the target; and wherein the stage of a disease or disorder is determined when the absence, presence or amount of the target is compared with the amount of target present in an analogous sample from a subject having no disease and/or disorder or having a particular stage of the disease or disorder. Preferably, a set of four or five different aptamers are used in the disease staging methods of the invention. [013] In yet another embodiment, the invention provides a method for enriching or isolating a target that has at least one protein component, comprising the steps of contacting at least two aptamers, preferably four aptamers, with a sample comprising the target, wherein said at least two aptamers each binds to a different epitope on the protein in the target under the appropriate conditions; separating the bound target from the bulk of the sample; and eluting the aptamers from the target; and recovering the target. Typically, at least one of the aptamers is immobilized on a solid phase which conveniently allows the separation of the bound and unbound materials. [014] In yet another embodiment, the invention provides a method for selecting aptamers that bind specifically to an oligopeptide consisting of 3, 4, 5, 6, 7, or 8 amino acid residues, comprising providing a mixture of oligonucleotides of unknown, non- predetermined or substantially non-predetermined nucleotide sequence, said mixture comprising a quantity of oligonucleotides sufficiently to statistically provide the presence of at least one oligonucleotide that is capable of binding said an oligonucleotide; incubating said mixture of oligonucleotides with said oligonucleotide under conditions wherein some oligonucleotides bind said target, said target-bound oligonucleotides defining an aptamer population; recovering said aptamers in substantially single stranded form; amplifying said aptamers to facilitate isolation; and repeating the incubation, recovery and amplification steps a plurality of times, typically three to four, or until a certain binding affinity of the aptamers is obtained. [015] The present invention further provides a kit which comprises in a container, a plurality of aptamers useful for the specific detection of a target according to the methods of the invention. The kits of the invention may optionally comprise accessory reagents for facilitating the detection or measurement of the binding of the aptamers, a solid phase for immobilizing the aptamers. The kits of the invention may be designed specifically for diagnosing or staging a particular disease or disorder, detection of a pathogen or a protein toxin in a subject, in food, or in the environment (air, water), routine physical check up, detection of one or more proteins for epidemiological or proteomic research. 3.1. DEFINITIONS

[016] As used herein, the term "aptamers" refers to nucleic acid molecules having one or more regions that are capable of binding to a molecule of interest in an environment wherein other substances in the same environment are not bound to the nucleic acid molecules. Since the molecules of interest in the present invention are proteins, the term "aptamers" generally refers to oligonucleotides that bind specifically to an epitope on a protein or a segment of an oligopeptide of the protein. Preferably, the aptamers are non-naturally occurring. Preferably, the aptamers are not present in nature in an isolated form. An aptamer of the invention is not an oligonucleotide that has the known physiological function of being bound by the target protein. [017] The term "aptamer" refers in general to either an oligonucleotide of a single defined sequence, or a mixture of oligonucleotides wherein the mixture exhibits the properties of binding specifically to the target. Thus, as used herein "aptamer" denotes both singular and plural sequences of oligonucleotides. The term "aptamer family" is also used herein to particularly identify and refer to groups of aptamers that bind specifically to a common epitope and share certain structural characteristics, such as but not limited to, nucleotide sequence homology and/or secondary structure. [018] The term "aptamer set" or "a set of aptamers" when used herein with reference to a protein refers to a plurality of different aptamers each exhibiting binding specificity for a distinct oligopeptide segment on the protein.

[019] The term "epitope" refers to a binding site on a protein for an aptamer or an antibody. The term "oligopeptide epitope" refers to a binding site on a protein that is constiuted by a contiguous segment of oligopeptide.

[020] "Oligonucleotide" refers to polydeoxyribonucleotides (containing T- deoxy-D-ribose or modified forms thereof), i.e., DNA, to polyribonucleotides (containing D-ribose or modified forms thereof), i.e., RNA, and to any other type of polynucleotide which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base or abasic nucleotides. Single-stranded oligonucleotide refers to those oligonucleotides which contain a single covalently linked series of nucleotide residues.

[021] As used herein, the term "oligopeptide" refers to a linear peptide consisting of three, four, five, six, seven, or eight amino acid residues. A segment of an oligopeptide in a protein to which an aptamer binds is also referred to as an eptiope. [022] As used herein, the term "target" refers to the entity of interest in an assay, which comprises at least one protein component. A "target protein" is a protein of interest in an assay. A target or a target protein can be detected, measured, investigated, or captured by the methods of the invention.

[023] The standard single-letter, triple-letter codes for amino acids are used herein interchangeably, i.e., A = Ala = alanine; C = Cys = cysteine; D = Asp = aspartic acid; E = GIu = glutamic acid; F = Phe = phenylalanine; G = GIy = glycine; H = His = histidine; I = He = isoleucine; K = Lys = lysine; L = Leu = leucine ; M = Met = methionine; N = Asn = asparagine; P = Pro = proline; Q = GIn = glutamine; R = Arg = arginine; S = Ser = serine; T = Thr = threonine; V = VaI = valine; W = Trp = tryptophan; Y = Tyr = tyrosine.

4. DESCRIPTION OF THE FIGURES

[024] Figure 1 The binding of the aptamers to different tripeptide-affinity columns. Aptamers targeted to different tripeptides were analyzed by binding assay using tripeptide-affinity columns. Radiolabeled DNA aptamers were incubated with tripeptides in the affinity columns, then washed and eluted and the percentage of bound DNA aptamers was determined.

[025] Figure 2. Interaction between aptamers and proteins by structure- switching methods, a) Structure-switching signaling aptamer method was used to detect the interaction between mouse Cat D protein segments. The DGI-Aptamer, KAI- Aptamer, GEL- Aptamer or LAS-Aptamer (□: LAS-Aptamer and Cat D, ■: DGI- Aptamer and Cat D, o: KAI- Aptamer and Cat D, •: GEL- Aptamer and Cat D, T : KAI- Aptamer and BSA, : GEL-Aptamer and BSA) were used for the assay. Different concentrations of Cat D ( O, 1.4, 2.7, 4.1 g/L for KAI and GEL, 0, 0.16, 0.48, 0.96, 1.44, 1.92, 2.4 and 2.72 g/L for DGI and LAS) and BSA ( 0, 2.0, 4.2, 6.3 g/L) proteins were added. The concentrations of MAP, FDNA and QDNA were 160 nM, 320 nM and 480 nM, respectively. The fluorescence intensity of each sample was normalized with F/FO, where F is the fluorescence intensity of each sample and FO is the initial signal in the absence of target proteins, b) KAI- Aptamer was used in the assay. Dahp protein concentrations were 0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/L. BSA concentrations were 0 g/L ( ), 0.4 g/L (▼), 2.0 g/L (o) and 4.0 g/L (•). The fluorescence intensity of each sample was normalized with F/FO, where F is the fluorescence intensity of each sample and FO is the initial signal in the absence of protein targets. [026] Figure 3. Schematic representation of the multiple aptamers based proximity-dependent ligation assay. In Figure 3 A and 3B, LA-GEL-Aptamer, LA-KAI- Aptamer, LA-LAS-Aptamer and LA-DGI-Aptamer were generated by adding arms at each end of the corresponding aptamers. In Figure 3B and 3C, the arms (linking regions) anneal to the aptamer itself which could reduce the background ligation and increase the specificity of interaction with targets. Figure 3D. When the Cat D protein was added to the system, the linking regions of the four aptamers are ligated together to form a reporter template for rolling circle amplification. Dotted lines represent the interactions between the tripeptides and their aptamer ligands. Short lines with different shadings are connectors for the ligation of the aptamers.

[027] Figure 4 Detection of Cat D protein by the proximity-dependent multiple aptamers ligation assay. Figure 4A. Four aptamers-based proximity-dependent ligation assay. Lane 1: Molecular weight marker, Lane 2: 10.0 nmol truncated Cat D, Lane 3: 1.0 nmol truncated Cat D, Lane 4: 10.0 fmol Cat D, Lane 5: 1.0 finol Cat D, Lane 6: 100.0 amol Cat D, Lane 7: 1.0 amol Cat D, Lane 8: 140 nmol BSA. Figure 4B. Five aptamers- based proximity-dependent ligation assay. Lane 1: 140 nmol BSA, Lane 2: 72.5 ng mock transformed E. coli protein extract, Lane 3: 0.7 ng of mock transformed E. coli protein extract, Lane 4: 7.25 pg mock transformed E. coli protein extract, Lane 5: 72.5 ng Cat D vector transformed E. coli protein extract, Lane 6: 0.7 ng Cat D vector transformed E. coli protein extract, Lane 7: 7.25 pg Cat D vector transformed E. coli protein extract, Lane 8: Molecular weight marker.

[028] Figure 5 Capture of partially purified recombinant mouse Cat D protein with combinations of aptamers. The combinations of aptamers used in the capturing reactions were: 4: 20 mM biotin labeled DGI, KAI, GEL and LAS aptamers; 3: 20 mM biotin labeled LAS, KAI and GEL aptamers with 20 mM biotin labeled control oligos; 2: 20 mM biotin labeled KAI and GEL aptamers with 40 mM biotin labeled control oligos; 1 : 20 mM biotin labeled LAS aptamer with 60 mM biotin labeled control oligos; 1 : 20 mM biotin labeled DGI ptamers with 60 mM biotin labeled control oligos; 0: 80 mM biotin labeled control oligos. The Integrated Optical Density (IOD) of captured protein was measured by Lab Works 4.0 software (UVP). Results are displayed as mean ± s.d.(n=3). 5. DETAILED DESCRIPTION OF THE INVENTION

[029] The present invention relates to a system for detecting any protein of interest using a plurality of aptamers that are selected to bind a protein according to the amino acid sequence of the protein. The invention is directed to methods for detecting a protein of interest using a plurality of aptamers that bind the protein at various specific sites. It is expected that the systematic approach of the invention to develop aptamers for specific protein detection will accelerate many aspects of proteomic studies, and the development of assays in the fields of public health, clinical diagnostics, environmental protection, and food science.

[030] The invention provides that an aptamer that specifically binds an oligopeptide, such as a tripeptide, can recognize and interact with the segment of oligopeptide in a protein, provided that the aptamer can gain access to the oligopeptide segment in the protein. The invention also provides that when several aptamers with different oligopeptide specificities bind to a target protein that comprises the different oligopeptide segments, the specific binding of each of the different aptamers can be exploited to detect, measure, and/or capture the target protein in a highly specific manner. The presence of each of the oligopeptide epitopes on an entity is ascertained by the binding of the respective aptamers to the entity, which collectively identifies the target and indicates its presence in a sample.

[031] Accordingly, by detecting the specific binding of each of the aptamers to an entity, the identity of the entity can then be ascertained. Many reporting means, such as but not limited to, hybridization assays, nucleic acid amplification and/or nucleic acid staining, are well known in the art and the skilled artisan can appreciate that they can be readily used in the invention. The binding data obtained for each of the aptamers can be used collectively to determine the identity of the entity and the amount of the entity present in a sample. It is also contemplated that the methods of the invention can use any reporting means that generate a signal when concurrent binding of the aptamers to a target protein occur. By concurrent binding, it is meant that all members of a chosen set of aptamers are bound to the target at the same time when detection of aptamer binding is performed. In a specific embodiment, the invention provides a reporting means that comprises proximity-dependent ligation of the aptamers and nucleic acid amplification. [032] According to the invention, an aptamer is selected from a pool of nucleic acids of random sequences on the basis of the affinity of the binding of the aptamer to an oligopeptide. The oligopeptide can consist of three, four, five, six, seven, or eight amino acid residues. It is thought that binding affinities of aptamers may be limited as the peptide gets shorter and hence more flexible. The shortest peptide to which aptamers were obtained is substance P which has 11 amino acids. Although tripeptides are used herein to illustrate the invention, a system using oligopeptides with four, five, six, seven, or eight amino acid residues can also be implemented and applied similarly. Moreover, there is no requirement that a method of the invention be practiced with aptamers all exhibiting specificities of the same oligopeptide length, i.e., the length of the oligopeptides to which members of an aptamer set bind can be different. [033] To illustrate the systematic approach of the invention, a universal library of aptamers that will allow specific detection of protein of any amino acid sequence, requires aptamers that exhibit 8000 different tripeptide specificities. The number of specificities is calculated by the n* power of the number of naturally occurring amino acids in proteins, wherein n is the number of amino acid residues in the oligopeptide segment to which an aptamer in the library binds specifically. Accordingly, with tripeptide specificities, the number of specificities in a universal library is 20x20x20 = 8000. The number for tetrapeptide specificities reaches 160,000 and 20 times more with pentapeptide specificities and so on.

[034] Specific detection of a target protein is achieved by using aptamers which bind a combination of oligopeptide epitopes that are present only in the target protein. The presence of a target protein is thus detected by the concurrent binding of a set of aptamers on the protein. The selection of aptamers for binding a target protein is based on identifying one or more unique combinations of oligopeptide segments in the amino acid sequence of the protein. The occurrence of a combination of oligopeptide segments in one or more protein other than the target can be estimated by bioinformatics techniques known in the art using available amino acid sequence data or translated genomic DNA data of an organism. In certain embodiments of the invention, the close proximity of the bound aptamers on the target protein is exploited to generate a highly specific detectable signal.

[035] The number of aptamers required in a set that can uniquely identify a target protein depends on the complexity of the amino acid sequence of the target protein. When the detection is carried out in a mixture of proteins, the number of aptamers used in the set depends also on the amino acid sequences of the proteins in the mixture. The number of aptamers required to uniquely identify a target protein is reduced if the number of amino acid residues recognized by the aptamers is increased. It is estimated that fewer aptamers of tetrapeptide specificities are required than aptamers of tripeptide specificities to uniquely detect a given protein. For example, it is determined that a set of four aptamers that exhibit tripeptide specificities can detect a protein specifically in many applications. To improve the signal when working with a specimen containing a complex mixture of proteins, a set of five aptamers that exhibit tripeptide specificities can be used to detect specifically a target protein. Generally, using more aptamers in a set can lessen cross reactivity, thereby resulting in lower background levels, higher specificity and/or fewer false positives. [036] The methods of the invention do not require the availability of an universal library. Even a partial library of aptamers can be used to detect many different proteins. The number of combinations of oligopeptide segments, which represent the number of proteins that can be identified uniquely, is n!/r!(n-r)! , where n is the size of the available library and r is the number of different aptamers used to detect a protein. For example, if a library of aptamers with 50 different oligopeptide specificites is available, and a set of four aptamers are employed to detect a protein, the number of proteins detectable by the library is 50!/((50-4)!)4! which are 230,000. Accordingly, using a set of 4 aptamers from a universal library with 8000 tripeptide specificites, the system can detect 170,538,695,998,000 different proteins (i.e., approximately 1.7xlO¹⁴ combinations of tripeptides). Using a set of 5 aptamers concurrently and selecting the aptamers from the same library of 8000 aptamer specificities, a total of 272,725,482,640,001,600 different proteins (i.e., approximately 2.7 xlO¹⁷) can be detected. Accordingly, a feature of the system is the efficiency in which reagents that bind a large number of different proteins can be generated. This presents a benefit over the current method which requires the availability of the target protein and the individual tailoring of aptamers for every target protein. Using current technology, as the scale of the project increases, the number of available proteins and aptamers increase linearly with the number of targets to be detected in the project. As illustrated above, far fewer aptamers are required using the system of the invention and it is not essential to isolate the target protein in advance as long as a partial amino acid sequence is available. When presented with a large number of detectable proteins, it will be much quicker to deploy assays for each protein using an aptamer library of the invention than isolating or expressing each protein individually and selecting aptamers that bind each protein ab initio. The efficiency in reagent generation and assay deployment will facilitate the rapid development of large scale proteomic studies. The efficiency can also improve the speed and economics in the development of diagnostic assays for infectious diseases, particualy diseases that involve rare infectious agents or infectious agents that are rapidly and constantly mutating (such as HIV and influenza). The availability of readily-testable aptamer reagents eliminates the time to isolate the protein and/or make the antibody and allows binding assays to be set up quickly and inexpensively. It also reduce the danger in handling a target which is a toxin.

[037] Another advantage of the system is scalability. As described above, the system is based on the amino acid sequences of the target proteins. A user of the system can predict in advance the number of aptamers needed and even reduce the number of aptamers required if an aptamer can be used in several different sets of aptamers. An aptamer useful for detecting a protein can also be used in combination with other aptamers to detect many other different proteins.

[038] Yet another advantage of the system is flexibility. As described above, a target protein can be detected by a set of aptamers that bind to a combination of oligopeptide epitopes on the protein. If the protein is made of a relatively large number of amino acid residues (generally greater than 150 residues), a user of the system can rely on various alternative combinations of oligopeptide epitopes, and thus different sets of aptamers, to detect the same protein. A further level of flexibility is afforded by the observation that different aptamer families with the same oligopeptide specificity emerged in the selection process. Therefore, even for a particular oligopeptide epitope, a number of distinct aptamers are available for optimizing performance. Thus, when faced with varying assay conditions or unknown sample complexity, a user usually has several readily-testable options when assembling a set of aptamers for detecting the protein. Accordingly, the invention provides a method for assembling a set of aptamers that are useful for detecting specifically a target protein, wherein the aptamers are selected on the basis of their binding affinities to oligopeptide segments present in the target protein.

[039] The efficiency and flexibility of the system of the invention make it suitable for use in large scale proteomic projects, such as population screening, epidemiological testing, system biology, whole cell proteome characterization, etc. The high specificity and sensitivity of the methods of the invention can also be exploited in various diagnostic assays. In the field of clinical chemistry, the methods can be applied to detect proteins in body fluids and tissue samples, and the expression of biomarkers associated with various health conditions. The methods can also be used to develop companion diganostics for a drug or a course of treatment involving one or more drugs, especially in the treatment of cancer. Accordingly, the present invention also relates to a method for determining a diagnosis or prognosis of a disease or disorder by assaying the presence or amount of a target that is correlated with a disease or health condition, and comparing the presence or amount of the target in an experimental sample with a control value, wherein a diagnosis or prognosis for a disease or health condition is determined when the presence or amount of target in the experimental sample differs from the control value. The subject of the diagnostic methods of the invention is not limited to humans, but also include companion animals, such as dogs and cats, domesticated animals, wild animals.The methods can also be used to detect pathogens by their proteinaceous antigens, including but not limited to bacteria, viruses, fungi, prions, and protozoa, in a subject or in the environment, such as air, water, soil, or food samples. [040] As the methods of the invention are very versatile and quick to deploy, it is expected that they will also find application in many areas including but not limited to detection of exotic proteins, environmental protection, pest control, forensic uses, and protein detection under non-physiological conditions, such as industrial conditions. For example, it is hazardous to produce antibodies to toxins, and thus the methods of the invention can be used to develop assays for detecting or measuring toxins, spores, and agents of highly infectious diseases, rare tropical diseases, or biological weapons that comprise a protein component, in the environment, such as air, water, soil, or food. Many assays that currently employ antibodies can be replaced by the more robust methods of the invention that use oligonculeotides.

[041] Also contemplated are kits that comprise one or more aptamers of the invention that bind specific oligopeptides and that can be used in the detection methods of the invention. The kits can comprises an universal library of aptamers or a partial library of aptamers, or subsets of aptamers which form a part of a system. The kits can comprises one or more sets of aptamers that are used in combination in a method for detecting a target. All such kits may also comprise other reagents for the detection method.

[042] The disclosures of copending Chinese patent application no.

200610025342.4, filed March 31, 2006; and Chinese patent application no. 200610025911.5 filed April 21, 2006, are both incorporated herein by reference in their entirety. For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections which follow.

5.1. APTAMER SELECTION

[043] In general, the method for selecting the aptamers of the invention involves incubating an oligopeptide, such as a tripeptide, with a mixture of oligonucleotides under conditions wherein some but not all of the members of the oligonucleotide mixture form complexes with the oligopeptide. The invention differs from the conventional approach which uses the entire target protein or a substantial portion thereof in the selection of aptamers. According to the invention, the specificity of an aptamer under selection are directed not to a single protein but to an oligopeptide segment that may occur in the amino acid sequences of many proteins. [044] In one embodiment, the resulting complexes of an oligopeptide and oligonucleotides are separated from the uncomplexed oligonucleotides and the complexed oligonucleotides which constitute a collection of aptamers (having a plurality of different nucleotide sequences) is recovered from the complex and amplified. The resulting aptamer mixtures are used as the starting mixture for incubation with the oligopeptide and subjected to repeated iterations of this series of steps. When a desired degree of specificity is obtained, the aptamer mixtures can be used as a mixture or may be sequenced and synthetic forms of one or more aptamers prepared. In many instances, upon sequencing the mixtures of aptamers that bind the oligopeptide specifically, multiple families of aptamers emerge wherein members of each family share a consensus nucleotide sequence and/or a secondary structure.

[045] hi this most generalized form of the method, the oligonucleotides used as members of the starting mixture may be single-stranded or double-stranded DNA or RNA, or modified forms thereof. However, single-stranded DNA is preferred. The use of DNA eliminates the need for conversion of RNA aptamers to DNA by reverse transcriptase prior to amplification by polymerase chain reaction (PCR). Furthermore, DNA is less susceptible to nuclease degradation than RNA and more convenient where a ligation reaction is performed during detection.

[046] The oligonucleotides that bind to the target are separated from the rest of the mixture and recovered and amplified. Amplification may be conducted before or after separation from the oligopeptide. The oligonucleotides are conveniently amplified by PCR to give a pool of DNA sequences. The PCR method is well known in the art and described in, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, and 4,800,159 and Saiki, R. K., et al., Science (1988) 239:487-491, as well as Methods in Enzymology (1987) 155:335-350. Other methods of nucleic acid amplification known in the art may be employed. IfRNA is initially used, the amplified DNA sequences are transcribed into RNA. The recovered DNA or RNA, in the original single-stranded or duplex form, is then used in another round of selection and amplification. After a number of rounds of selection/amplification (usually from three to six), oligonucleotides that bind with an affinity in the millimolar to molar range can be obtained for most targets and affinities below the molar range are possible for some targets.

[047] Amplified sequences can be applied to sequencing gels to determine the structure of the aptamers being selected by the oligopeptide after any number of rounds, especially when an aptamer family has been observed. Amplified sequences can also be cloned and individual oligonucleotides sequenced. The entire process can then be repeated using the recovered and amplified oligomers as needed. Once an aptamer that binds specifically to a target has been selected, it may be recovered as DNA or RNA in single-stranded or duplex form using conventional techniques.

[048] Similarly, a selected aptamer may be sequenced and synthesized using one or more modified bases, modified sugars and modified linkages using conventional techniques. In addition to the portion of nucleotides that are involved in binding the oligopeptide, flanking nucleotides on either or both ends can be present for various purposes as described in the next section.

[049] The starting mixture of oligonucleotides may be of completely or partially undetermined sequence. Preferably, the oligonucleotides in the mixture each have a portion that comprises a randomized nucleotide sequence, generally including from about 15 to about 100 nucleotides, more preferably 30 to 90 nucleotides, and most preferably 40 to 80. The binding regions in the aptamers used in the example comprise 60 nucleotides. It is expected that the sequence variations in this portion of the oligonucleotides produce binding affinities to the oligopeptide with various magnitude and that are subjected to selection. In certain embodiments, the sequences in the binding portion are completely randomized, i.e., all possible sequences may be present. In other embodiments, techniques known in the art can generate a population of oligonucleotides which comprises a preponderance of certain sequences in the population, or a preponderance of certain bases at particular positions. [050] In various embodiments, the binding portion of each oligonucleotide that comprises the randomized sequence is preferably flanked by primer sequences that permit the application of the nucleic acid amplification (such as PCR) directly to the recovered oligonucleotides from the complex. The flanking sequences may also contain features that enable detection and reporting, and other features, such as restriction sites which permit the cloning of the amplified sequence. The primer regions generally comprise 10 to 30, more preferably 15 to 25, and most preferably 18 to 20, bases of a known sequence. Aptamers can also be selected using a pool of oligonucleotides that vary in length as the starting material.

[0511 The oligonucleotides of the starting mixture may be conventional oligonucleotides, most preferably single-stranded DNA, or may be modified forms of these conventional oligomers as described hereinabove. For oligonucleotides containing conventional phosphodiester linkages or closely related forms thereof, standard oligonucleotide synthesis techniques may be employed. Such techniques are well known in the art, such methods being described, for example, in Froehler, B., et al., Nucleic Acids Research (1986) 14:5399-5467; Nucleic Acids Research (1988) 16:4831-4839; Nucleosides and Nucleotides (1987) 6:287-291; Froehler, B., Tet Lett (1986) 27:5575- 5578. Oligonucleotides may also be synthesized using solution phase methods such as triester synthesis, known in the art. The nature of the mixture is determined by the manner of the conduct of synthesis. Randomization can be achieved, if desired, by supplying mixtures of nucleotides for the positions at which randomization is desired. Any proportion of nucleotides and any desired number of such nucleotides can be supplied at any particular step. Thus, any degree of randomization may be employed. Some positions may be randomized by mixtures of only two or three bases rather than the conventional four. Randomized positions may alternate with those which have been specified.

[052] Generally, the starting mixture of oligonucleotides subjected to the invention method will have a binding affinity for the target characterized by a Kd of 1 micromolar or greater. Binding affinities of the original mixture for target may range from about lOμM to lμM, but, the smaller the value of the dissociation constant, the more initial affinity there is in the starting material for the target. This may or may not be advantageous as specificity may be sacrificed by starting the procedure with materials with high binding affinity. Improvements in the binding affinity over one or several iterations of the above steps of at least a factor of 10 to 50, preferably of a factor of 100, and more preferably of a factor of 200 may be achieved. A ratio of binding affinity reflects the ratio of Kds of the comparative complexes. Even more preferred in the conduct of the method of the invention is the achievement of an enhancement of an affinity of a factor of 500 or more. The desired affinity of the aptamers of the invention falls within the range of from about 10OnM, to 1OnM, and to InM or from about lOOpM to 1 OpM, and to IpM.

[053] In various embodiments, the aptamers of the invention exhibit high binding affinity to a tripeptide but relatively much lower affinity to a tetrapeptide (or longer oligopeptide) that comprises the tripeptide sequence under the same conditions, and are hence, deemed to be specific for the tripeptide only and not the tetrapeptide or longer oligopeptides that comprise the tripeptide sequence. A difference in affinities that is 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 200-fold, 500-fold or 1, 000-fold is desired. For certain aptamers of the invention which bind specifically a tetrapeptide, they do not bind specifically to a longer oligopeptide even the longer oligopeptide comprises the same tetrapeptide sequence. An aptamer of the invention that binds a tetrapeptide specifically can also exhibit much lower binding affinity for a tripeptide, even the sequence of the tripeptide is a subsequence of the tetrapeptide. The same principle applies to the description of other aptamers of the invention that exhibit specifϊcites for oligopeptides consisting of five, six, seven, and eight amino acid residues. These features of the aptamers of the invention distinguish them from existing aptamers that are selected to bind a much larger polypeptide or protein. [054] As used herein, physiological conditions means the salt concentration and ionic strength in an aqueous solution which characterize fluids found in human metabolism commonly referred to as physiological buffer or physiological saline. In general, these are represented by an intracellular pH of 7.1 and salt concentrations (in mM) OfNa⁺ : 3-15; K⁺ : 140; Mg⁺² : 6.3; Ca⁺² : 10^"4 ; Cl^" : 3-15, and an extracellular pH of 7.4 and salt concentrations (in mM) OfNa⁺ : 145; K⁺ : 3; Mg⁺² : 1-2; Ca⁺² : 1-2; and CI^' : 110.

[055] However, the use of physiological conditions in the aptamer selection method is optional especially for those aptamers that are intended for binding to denatured target proteins. As is understood in the art, the concentration of various ions, in particular, the ionic strength, and the pH value affects the dissociation constant of the target/aptamer complex. [056] In various embodiments, a column or other support matrix having covalently or noncovalently coupled a target oligopeptide is synthesized. Any standard coupling reagent or procedure may be utilized, depending on the nature of the support and the target. For example, covalent binding may include the formation of disulfide, ether, ester or amide linkages. The length of the linkers used may be varied by conventional means.

[057] In another embodiment, a solution phase selection method can be applied which allows oligopeptide-oligonucleotide complex formation to occur in solution phase and detects the change in mobility of the oligopeptide in gel after binding to an aptamer. Separation is based at least in part on an effective increase in the mass of the bound aptamer in aptamer-target complex compared to unbound nucleic acid. In cases where the effective charge of the aptamer is reduced through interactions with the target molecule, a decreased rate of migration compared to uncomplexed nucleic acids can also contribute to separation of unbound species during electrophoresis on the gel. This method can be used in combination with the solid phase-based selection method especially after intermediate rounds of selection on columns.

[058] Solution phase selection offers certain advantages over solid phase-based selection: more accurate determination of Kd values for binding of an aptamer to its target; lesser amounts of target is required; the amount of target used for a selection can be more precisely determined and controlled than immobilized target. [059] Complexes between the aptamer and target are separated from uncomplexed aptamers using any suitable technique, depending on the method used for complexation. For example, if columns are used, non-binding species are simply washed from the column using an appropriate buffer. Specifically bound material can then be eluted. If binding occurs in solution, the complexes can be separated from the uncomplexed oligonucleotides using, for example, the mobility shift in electrophoresis technique (EMSA), described in Davis, R. L., et al., Cell (1990) 60:733. In this method, aptamer-target molecule complexes are run on a gel and the aptamers are removed from the region of the gel where the target migrates. Unbound oligonucleotides migrate outside these regions and are separated from the oligonucleotides that bound. Finally, if complexes are formed on filters, unbound aptamers are eluted using standard techniques and the desired aptamer recovered from the filters.

[060] This invention is not dependent on the methodology or mechanism by which the aptamers are selected. Nevertheless, the invention encompasses a method of identifying an aptamer that binds to an oligopeptide, such as a tripeptide. In one embodiment, the method comprises:

(0611 (a) providing a mixture of oligonucleotides of unknown, non- predetermined or substantially non-predetermined nucleotide sequence, said mixture comprising a quantity of oligonucleotides sufficiently to statistically provide the presence of at least one oligonucleotide that is capable of binding said an oligonucleotide target such as a tripeptide; (b) incubating said mixture of oligonucleotides with said oligonucleotide under conditions wherein some oligonucleotides bind said target, said target-bound oligonucleotides defining an aptamer population; (c) recovering said aptamers in substantially single stranded form; and (d) amplifying said aptamers to facilitate isolation.

[062] In another embodiment, the invention provides methods which add the following steps to steps (a)-(d) listed above:

[063] (e) repeating steps (a)-(d) using said first aptamers of step (d), or a portion thereof, to form a second pool of oligonucleotides for use in step (a), thereby generating a second aptamer population which may be used to repeat steps (a)-(d), and optionally (f) repeating steps (a)-(d) using said second aptamers of step (e), or a portion thereof, a sufficient number of times so as to identify an optimal aptamer population from which at least one consensus region may be identified in at least two of the aptamers from said optimal aptamer population, wherein the presence of the consensus region may be correlated with target binding and/or a certain secondary structure. [064] This method includes the optional steps for selectively attaching and/or removing flanking regions to aptamers, thereby permitting efficient, convenient aptamer recovery in high yield. One such method comprises, after separating oligonucleotides in the method above in substantially single stranded form from the pool capable of binding target; attaching a 5' linker of known sequence to a first (the 5') end of the oligonucleotides, the 5' linker having a first type II restriction enzyme recognition site at its 3' end, attaching a 3' linker of known sequence to a second (the 3') end of the oligonucleotides, the 3¹ linker having a second type II restriction enzyme recognition site different from the site at the 5' end; amplifying the oligonucleotides, thereby generating a duplex comprising a first (upper) strand, having a 5' linker complement portion, an oligonucleotide complement portion and a 3¹ linker complement portion, and a second (lower) strand, comprising a 5¹ linker portion, an oligonucleotide portion and a 3' linker portion; removing the 5' and the 3' linker portions from the oligonucleotides; and recovering the oligonucleotides in substantially single stranded form. [065] In another embodiment, the aptamer selection methods of the invention may comprise a negative selection step that removes from the starting oligonucleotide mixture certain oligonucleotides which bind to one or more undesired interfering oligopeptides from which the target oligopeptide is to be distinguished. This method is particularly useful in obtaining aptamers which can distinguish oligopeptides that differ in only one amino acid residue or that assumes a similar though non-identical secondary structure. Due to the small size of oligopeptides, such as tripeptides, it is useful to remove as much as possible aptamers that cross-react with other oligopeptides. In a non-limiting example of incorporating a negative selection into the selection method of the invention, the target oligopeptide will be incubated with an initial mixture of oligonucleotides and, the complexes formed are separated from uncomplexed oligonucleotides. The complexed oligonucleotides, which are now aptamers for the target oligopeptide, are recovered and amplified from the complex. The recovered aptamers are then mixed with the one or more undesired oligopeptide(s) from which the target is to be distinguished under conditions wherein members of the aptamer population which bind to said undesired oligopeptide(s) can form complexes. These complexes are then separated from the remaining aptamers. The resulting unbound second aptamer population is then recovered and amplified. A certain number of cross- reacting aptamers are expected to be removed from this second aptamer population. [066] In an alternative approach, the negative selection step may be conducted first, comprising mixing the original oligonucleotide mixture with the undesired oligopeptide(s) to form complexes with members of the oligonucleotide mixture which bind to the undesired oligopeptide(s); the uncomplexed oligonucleotides are then recovered and amplified, and incubated with the target oligopeptide under conditions wherein those members of the oligonucleotide mixture which bind the target oligopeptides are complexed. The resulting complexes are then removed from the uncomplexed oligonucleotides and the bound aptamer population is recovered and amplified as usual.

[067] The incorporation of positive and negative selection steps in the selection methods of the invention are particularly useful when generating a library of aptamers that bind oligopeptides that are different only by one or two amino acid residues. The oligonucleotides that are removed from an initial mixture because of their affinities to an undesired oligopeptide can be used in another selection project in a positive selection step where the oligopeptide undesired in the earlier selection step becomes the desired oligopeptide. Using this experimental design, a library of aptamers that bind a large number of related but different oligopeptides can be generated rapidly and economically. [068] In a specific embodiment, the aptamers of the invention are of the opposite chirality from the oligonucleotides that occur in nature, i.e., D isomers. When using this approach of aptamer selection, the first step involves synthesizing an enantiomer of an oligopeptide sequence (e.g., a D-amino acid peptide enantiomer of an L-amino acid peptide). The peptide is contacted with a candidate mixture of nucleic acids of natural handedness (i.e., D-DNA or D-RNA) under conditions appropriate for binding. The nucleic acids with high binding affinity for the D-amino acid peptide are isolated and their sequences are identified. Nucleic acids of non-natural handedness (i.e., L-DNA or L-RNA), which are mirror images of the high affinity D-DNAs or D-RNAs, are synthesized using enantio-deoxyribose phosphoramidites or enantio-ribose phosphoramidites to yield ligands of non-natural handedness which bind to the natural conformation of the oligopeptide. Aptamer of non-natural handedness are less susceptible to nuclease degradation by naturally occurring proteases and nucleases. See, U.S. patent 5,780,221, which is incorporated herein by reference in its entirety. [069J Aptamer selection methods known in the art that are similar to the method described above can also be used, for example, U.S. patents 5,270,163; 5,475,096; 5,567,588; 5,580,737; 5,637,459; 5,683,867; 5,705,337; 5,707,796; 5,756,291 and 5,861,254, which are incorporated herein by reference in their entirety. [070] Other methods known in the art for isolating aptamers can also be used.

In a non-limiting example, aptamers can be selected using an approach which excludes amplification steps between rounds of affinity selection. Such an approach applies the technique of homogenous free-solution separation by capillary electrophoresis to mixtures of oligopeptides and oligonucleotides. One advantage of the technique is the ability to obtain aptamers with predefined binding parameters (Kd, K_0n, K_0R) and doing the selection under non-physiological conditions more efficiently. See Drabovich et al., Anal. Chem. 2006, 78:3171-8 and; Berezovski et al., J. Am. Chem. Soc. 2006, 128:1410-1411, which are incorporated herein by reference in their entirety. [071] In order to detect a desired target protein, a set of oligopeptide segments that occur within the target protein must be chosen for binding by aptamers of the invention. Although not essential to the methods of the invention, it would be desirable to take into consideration the structure of the target when choosing oligopeptide segments for aptamer binding.

[072] First, it should be confirmed if the three-dimensional structure of the target has not been determined. This can be achieved by searching known X-ray and NMR structures in the Protein Data Bank (Brookhaven National Laboratory, Upton, N. Y) for an amino acid sequence identical to that of interest. Next, identification of proteins with significantly similar sequences whose structures are already known could be used. Any software packages known in the art can be used to perform similarity searches. If proteins of similar sequence (>20% identity) are found, the Protein Data Bank should again be searched to determine whether the structures of any of those proteins are known. If so, then comparative homology modeling (Greer, 1990, Proteins Struct. Funct. Genet. 7:317-334; Bajorath et al., 1993, Protein Sci. 2:1798-1810) can be used to develop a three-dimensional model of the desired sequence. [073J Many methods are known in the art for determining the secondary and tertiary structure of proteins, see for example, Current Protocols in Protein Science, Unit 2.3 Protein Secondary Structure Prediction by Krystek et al., John Wiley & Sons, Inc. 2003, and Current Protocols in Bioinformatics, Chapter 5, Modeling Structure From Sequence, John Wiley & Sons, Inc. 2006) which is incorporated herein by reference in its entirety. These include but are not limited to the hydrophobicity analysis to identify the hydrophobic and hydrophilic segments of a target protein and therefore determine those segments of the target that are mostly likely to be accessible to oligonucleotides without disrupting the native conformation of the protein (Heijne, G., J. MoI. Biol. 225:487-494 (1992); (Kyte, J., and Doolittle, R. F. (1982) J. MoI. Biol., 157:105-132)). Other computer-assisted structural assessment tools that can be used include but are not limited to the methods of Gamier et al. 1978, J. MoI. Biol. 120:97-120; Chou, P. Y. and Fasman, G. D. (1976) 47:251-276; Rost and Sander 1993, J. MoI. Biol. 232:584-599 (the EMBL neural network preduction scheme (PHD)); and Stultz et al., Protein Sci. 2:305- 314. Preferably, a combination of the Chou and Fasman interactive sequence analysis and PHD neural network predictions are carried out. Correlation of flexibility plots with homology plots and surface exposure profiles are helpful in identifying preferred segments along the amino acid sequence of a target protein.

5.2 APTAMER REAGENTS [074] The aptamers of the invention have one or more regions that are capable of forming complexes with a target in an environment wherein other substances in the same environment are not complexed to the oligonucleotide. The aptamers of the invention exhibit binding specificity to a defined oligopeptide, such as a tripeptide. Aptamers that are able to bind to the oligopeptide even when it is joined at one or both ends to other amino acid residues in a protein or polypeptide, are particularly useful in the detection methods described herein.

[075] The specificity of the binding is defined in terms of the comparative dissociation constants (Kd) of the aptamer for an oligopeptide as compared to the dissociation constant with respect to the aptamer and other oligopeptides (or oligopeptide segments) in the environment, or other molecules in general. Typically, the Kd of an aptamer for its target oligopeptide will be 2-fold, 5-fold, and preferably 10-fold less than that with respect to other oligopeptides or other molecules in the environment. Even more preferably the Kd will be 50-fold less, more preferably 100-fold less, more preferably 200-fold less, and most preferably 500-fold less. It is expected that many of the aptamers of the invention exhibit a Kd in the nanomolar, micromolar and millimolar range.

[076] It is contemplated that aptamers of the invention are selected to exhibit detectable differences in binding affinities towards oligopeptides that differ only in a single amino acid residue. For example, the aptamers used herein are expected to bind differentially to tripeptide segments that differ by one amino acid residue, in a protein. [077] The value of this dissociation constant can be determined directly by well- known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci, M., et al., Byte (1984) 9:340-362. Alternatively, a competitive binding assay for a target oligopeptide may be conducted with respect to substances known to bind the oligopeptide. The value of the concentration at which 50% inhibition occurs (Ki) is, under ideal conditions, equivalent to Kd. However, in no event can Ki be less than Kd. Thus, determination of Ki, sets a maximal value for the value of Kd. Under those circumstances where technical difficulties preclude accurate measurement of Kd, measurement of Ki can conveniently be substituted to provide an upper limit for Kd.

[078] Iⁿ general, a minimum of approximately 6 nucleotides, preferably 10, to

20 nucleotides, are necessary to effect specific binding. Generally, the aptamer can comprise from about 15 to about 100 nucleotides, more preferably 30 to 90 nucleotides, and most preferably 40 to 80.

[079] Aptamer reagents of the invention comprise an oligonucleotide. In addition to the nucleotides that are involved in the binding to the oligopeptide epitope on the protein, other non-protein binding nucleotides can be included in the aptamer to improve the performance of the method. Generally, one or more regions of the aptamers comprise a nucleotide sequence that can interact with an accessory molecule that facilitates a detection means of the invention. In one embodiment, an aptamer of the invention comprises linking region(s) at one or both ends of the oligonucleotide that hybridize to a connector oligonucleotide or the linking region of another aptamer. hi a specific embodiment, the sequence of one or both of the linking regions are independently complementary to an internal nucleotide sequence such that a secondary structure is formed which prevents the linking regions of the aptamer from interacting with an accessory molecule or another aptamer. This feature is illustrated in Figure 3b and Figure 3c. Prior to binding to an epitope, intramolecular interactions between different regions of an aptamer are favored over intermolecular interactions. However, the secondary structure is temporary and is disrupted when the aptamer binds to its epitope. As a result of binding to the epitope, the linking region(s) are free to hybridize to an accessory molecule or to another aptamer (see Figure 3d). Aptamers of the invention having such a feature produces lower background. In embodiments of the invention that involve proximity-dependent ligation of the aptamers, this feature prevents premature ligation of the aptamers and their corresponding connector oligonucleotides.

[080] Any oligonucleotides useful in the invention can be synthesized using established oligonucleotide synthesis methods. Methods to produce or synthesize oligonucleotides are well known in the art. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989) Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligeπ or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, Mass. or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triester methods), and Narang et al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method). Many of the oligonucleotides described herein are designed to be complementary to certain portions of other oligonucleotides or nucleic acids such that stable hybrids can be formed between them. The stability of these hybrids can be calculated using known methods such as those described in Lesnick and Freier, Biochemistry 34:10807-10815 (1995), McGraw et al., Biotechniques 8:674-678 (1990), and Rychlik et al., Nucleic Acids Res. 18:6409-6412 (1990).

[081] Structurally, the aptamers of the invention are oligonucleotides including but not limited to those with conventional bases, sugar residues and internucleotide linkages, but also those which contain modifications of any or all of these three aspects. "Modified" oligonucleotides comprise at least one modification of any or all of these three aspects. Modification of the aptamer can be carried our before selection or after selection. Modifications can also include 5' and 3' ends modification, such as capping. [082] The term "nucleoside" or "nucleotide" refer to ribonucleosides or ribonucleotides, deoxyribonucleosides or deoxyribonucleotides, or to any other nucleoside which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base. Nucleoside and "nucleotide include those moieties which contain not only the natively found purine and pyrimidine bases A, T, C, G and U, but also modified or analogous forms thereof. Modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Such purines and pyrimidines are generally known in the art and include but are not limited to, pseudoisocytosine, N^N^ethanocytosine, 8-hydroxy-N⁶methyladenine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 7-deazaadenine, 7- deazaguanine, 5-bromouracil, S-carboxymethylaminomethyl^-thiouracil, 5- carboxyrnethylaminomethyl uracil, dihydrouracil, inosine, N⁶-isopentenyl-adenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N^methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5- methoxy aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5¹- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio- N⁶-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, pseudouracil, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil- 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5- ethyluracil, 5-ethylcytosine, 5-butyluracil, 5-butylcytosine, 5-pentyluracil, 5- pentylcytosine, and 2,6-diaminopurine.

[083] The sugar residues in the oligonucleotides of the invention may also be other than conventional ribose and deoxyribose residues. Modifications in the sugar moiety, for example, wherein one or more of the hydroxy, groups are replaced with halogen, aliphatic groups, or functionalized as ethers, amines, and the like, are also included. In particular, substitution at the 2'-position of the furanose residue is particularly important with regard to enhanced nuclease stability. An exemplary, but not exhaustive list includes 2' substituted sugars such as 2'-O-methyl-, 2'-O-alkyl, 2'-O-allyl, 2'-S-alkyl, 2'-S-allyl, 2'-fluoro-, 2'-halo, or 2'-azido-ribose, carbocyclic sugar analogs, - anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside, ethyl riboside or propyl riboside. The stereochemistry of the sugar carbons may be other than that of D-ribose in one or more residues. Also included are analogs where the ribose or deoxyribose moiety is replaced by an alternate structure such as the 6-membered morpholino ring described in U.S. patent no. 5,034,506. [084] The oligonucleotides of the invention may be manufactured using conventional phosphodiester-linked nucleotides and synthesized using standard solid phase (or solution phase) oligonucleotide synthesis techniques, which are commonly commercially available. The oligonucletoides may also contain one or more alternative linkages such as phosphorothioate or phosphoramidate. Alternative linking groups include, but are not limited to embodiments wherein a moiety of the formula P(O)S, ("thioate"), or P(S)S ("dithioate") is used to join adjacent nucleotides through ~O~ or ~ S-. Other linkages that may be used include nonphosphorous-based intemucleotide linkages. Aptamers that comprise modified nucleotides are known in the art and can be used in the present invention, see, for example, U.S. patent no. 5,580,737; 5,660,986; 5,756,703; and 5,861,254, which are incorporated herein by reference in their entirety. [085] When a number of individual, distinct aptamer sequences for a single target molecule have been obtained and sequenced as described above, the sequences may be examined for consensus sequences. As used herein, "consensus sequence" refers to a nucleotide sequence or region (which may or may not be made up of contiguous nucleotides), which is found in one or more regions of at least two aptamers, the presence of which may be correlated with aptamer-to-target-binding or with aptamer structure. A consensus sequence may be as short as three nucleotides long. It also may be made up of one or more noncontiguous sequences with nucleotide sequences or polymers of hundreds of bases long interspersed between the consensus sequences. Consensus sequences may be identified by sequence comparisons between individual aptamer species, which comparisons may be aided by computer programs and other tools for modeling secondary and tertiary structure from sequence information. Generally, the consensus sequence will contain at least about 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides.

[086] As used herein "consensus sequence" means that certain positions, not necessarily contiguous, of an oligonucleotide are specified. By specified is meant that the composition of the position is other than completely random. Not all oligonucleotides in a mixture may have the same nucleotide at such position; for example, the consensus sequence may contain a known ratio of particular nucleotides. [087] When detecting a target comprising a nucleic acid component, it is ^• contemplated that the aptamers useful in the methods do not bind to the nucleic acid component via Watson-Crick base pairing or triple helix formation. It is also contemplated that when detecting a target protein that binds nucleic acid in vivo as its natural physiological function, the aptamers useful in the methods do not comprise the nucleotide sequence of the native nucleic acid that binds to such a target protein. [088] As described above, there are 8000 different tripeptides that are present in naturally occurring proteins. The conformation of as many as 1220 such tripeptides from high resolution (less than or equal two angstroms) have been studied. According to this study, the structure of tripeptides can be classified into three groups: rigid, non-rigid and intermediate, with very few tripeptides in the non-rigid group. Rigidity due to proline is well understood because of the side chain interacting covalently with the backbone and generally, proline in position 3 in the tripeptide, makes the tripeptide rigid. Methionine and tryptophan are fairly bulky suggesting good space filling is the cause for rigidity. Rigid tripeptides with glutamine invariably also have another polar side chain residue; consequently they form a weak ionic bond within the tripeptide. Rigidity of a tripeptide varies as the position of the tripeptide changes within a protein and across proteins and this fluctuation has been expressed by the standard deviations of the distances between the C_α and Cp atoms of each of the amino acids in the tripeptides (RiR₂, Ri R₃ and R2R₃). A dataset of 7964 tripeptides along with all the 12 relative average distances, standard deviations and frequencies is available at the URL http://www.au-kbc.org/research areas/bio/projects/protein/tri. html. This analytical framework can be applied to guide the selection of tripeptides, see, Anishetty et al., BMC Structural Biology 2002, 2:9, which is incorporated herein by reference in its entirety.

[089] In various embodiments, the invention provides aptamers that bind specifically to one of the 8000 tripeptides having an amino acid sequence (in single letter code) selected from the following: AAA, AAC, AAD, AAE, AAF, AAG, AAH, AAI, AAK, AAL, AAM, AAN, AAP, AAQ, AAR, AAS, AAT, AAV, AAW, AAY, ACA, ACC, ACD, ACE, ACF, ACG, ACH, ACI, ACK, ACL, ACM, ACN, ACP, ACQ, ACR, ACS, ACT, ACV, ACW, ACY, ADA, ADC, ADD, ADE, ADF, ADG, ADH, ADI, ADK, ADL, ADM, ADN, ADP, ADQ, ADR, ADS, ADT, ADV, ADW, ADY, AEA, AEC, AED, AEE, AEF, AEG, AEH, AEI, AEK, AEL, AEM, AEN, AEP, AEQ, AER, AES, AET, AEV, AEW, AEY, AFA, AFC, AFD, AFE, AFF, AFG, AFH, AFI, AFK, AFL, AFM, AFN, AFP, AFQ, AFR, AFS, AFT, AFV, AFW, AFY, AGA, AGC, AGD, AGE, AGF, AGG, AGH, AGI, AGK, AGL, AGM, AGN, AGP, AGQ, AGR, AGS, AGT, AGV, AGW, AGY, AHA, AHC, AHD, AHE, AHF, AHG, AHH, AHI, AHK, AHL, AHM, AHN, AHP, AHQ, AHR, AHS, AHT, AHV, AHW, AHY, AIA, AIC, AID, AIE, AIF, AIG, AIH, All, AIK, AIL, AIM, AIN, AIP, AIQ, AIR, AIS, AIT, AIV, AIW, AIY, AKA, AKC, Ala), AXE, AKF, AKG, AKH, AKI, AKK, AKL, AKM, AKN, AKP, AKQ, AKR, AKS, AKT, AKV, AKW, AKY, ALA, ALC, ALD, ALE, ALF, ALG, ALH, ALI, ALK, ALL, ALM, ALN, ALP, ALQ, ALR, ALS, ALT, ALV, ALW, ALY, AMA, AMC, AMD, AME, AMF, AMG, AMH, AMI, AMK, AML, AMM, AMN, AMP, AMQ, AMR, AMS, AMT, AMV, AMW, AMY, ANA, ANC, AND, ANE, ANF, ANG, ANH, ANI, ANK, ANL, ANM, ANN, ANP, ANQ, ANR, ANS, ANT, ANV, ANW, ANY, APA, APC, APD, APE, APF, APG, APH, API, APK, APL, APM, APN, APP, APQ, APR, APS, APT, APV, APW, APY, AQA, AQC, AQD, AQE, AQF, AQG, AQH, AQI, AQK, AQL, AQM, AQN, AQP, AQQ, AQR, AQS, AQT, AQV, AQW, AQY, ARA, ARC, ARD, ARE, ARF, ARG, ARH, ARI, ARK, ARL, ARM, ARN, ARP, ARQ, ARR, ARS, ART, ARV, ARW, ARY, ASA, ASC, ASD, ASE, ASF, ASG, ASH, ASI, ASK, ASL, ASM, ASN, ASP, ASQ, ASR, ASS, AST, ASV, ASW, ASY, ATA, ATC, ATD, ATE, ATF, ATG, ATH, ATI, ATK, ATL, ATM, ATN, ATP, ATQ, ATR, ATS, ATT, ATV, ATW, ATY, AVA, AVC, AVD, AVE, AVF, AVG, AVH, AVI, AVK, AVL, AVM, AVN, AVP, AVQ, AVR, AVS, AVT, AVV, AVW, AVY, AWA, AWC, AWD, AWE, AWF, AWG, AWH, AWI, AWK, AWL, AWM, AWN, AWP, AWQ, AWR, AWS, AWT, AWV, AWW, AWY, AYA, AYC, AYD, AYE, AYF, AYG, AYH, AYI, AYK, AYL, AYM, AYN, AYP, AYQ, AYR, AYS, AYT, AYV, AYW, AYY, CAA, CAC, CAD, CAE, CAF, CAG, CAH, CAI, CAK, CAL, CAM, CAN, CAP, CAQ, CAR, CAS, CAT, CAV, CAW, CAY, CCA, CCC, CCD, CCE, CCF, CCG, CCH, CCI, CCK, CCL, CCM, CCN, CCP, CCQ, CCR, CCS, CCT, CCV, CCW, CCY, CDA, CDC, CDD, CDE, CDF, CDG, CDH, CDI, CDK, CDL, CDM, CDN, CDP, CDQ, CDR, CDS, CDT, CDV, CDW, CDY, CEA, CEC, CED, CEE, CEF, CEG, CEH, CEI, CEK, CEL, CEM, CEN, CEP, CEQ, CER, CES, CET, CEV, CEW, CEY, CFA, CFC, CFD, CFE, CFF, CFG, CFH, CFI, CFK, CFL, CFM, CFN, CFP, CFQ, CFR, CFS, CFT, CFV, CFW, CFY, CGA, CGC, CGD, CGE, CGF, CGG, CGH, CGI, CGK, CGL, CGM, CGN, CGP, CGQ, CGR, CGS, CGT, CGV, CGW, CGY, CHA, CHC, CHD, CHE, CHF, CHG, CHH, CHI, CHK, CHL, CHM, CHN, CHP, CHQ, CHR, CHS, CHT, CHV, CHW, CHY, CIA, CIC, CID, CIE, CIF, CIG, CIH, CII, CIK, CIL, CIM, CIN, CIP, CIQ, CIR, CIS, CIT, CTV, CIW, CIY, CKA, CKC, CKD, CKE, CKF, CKG, CKH, CKI, CKK, CKL, CKM, CKN, CKP, CKQ, CKR, CKS, CKT, CKV, CKW, CKY, CLA, CLC, CLD, CLE, CLF, CLG, CLH, CLI, CLK, CLL, CLM, CLN, CLP, CLQ, CLR, CLS, CLT, CLV, CLW, CLY, CMA, CMC, CMD, CME, CMF, CMG, CMH, CMI, CMK, CML, CMM, CMN, CMP, CMQ, CMR, CMS, CMT, CMV, CMW, CMY, CNA, CNC, CND, CNE, CNF, CNG, CNH, CNI, CNK, CNL, CNM, CNN, CNP, CNQ, CNR, CNS, CNT, CNV, CNW, CNY, CPA, CPC, CPD, CPE, CPF, CPG, CPH, CPI, CPK, CPL, CPM, CPN, CPP, CPQ, CPR, CPS, CPT, CPV, CPW, CPY, CQA, CQC, CQD, CQE, CQF, CQG, CQH, CQI, CQK, CQL, CQM, CQN, CQP, CQQ, CQR, CQS, CQT, CQV, CQW, CQY, CRA, CRC, CRD, CRE, CRF, CRG, CRH, CRI, CRK, CRL, CRM, CRN, CRP, CRQ, CRR, CRS, CRT, CRV, CRW, CRY, CSA, CSC, CSD, CSE, CSF, CSG, CSH, CSI, CSK, CSL, CSM, CSN, CSP, CSQ, CSR, CSS, CST, CSV, CSW, CSY, CTA, CTC, CTD, CTE, CTF, CTG, CTH, CTI, CTK, CTL, CTM, CTN, CTP, CTQ, CTR, CTS, CTT, CTV, CTW, CTY, CVA, CVC, CVD, CVE, CVF, CVG, CVH, CVI, CVK, CVL, CVM, CVN, CVP, CVQ, CVR, CVS, CVT, CVV, CVW, CVY, CWA, CWC, CWD, CWE, CWF, CWG, CWH, CWI, CWK, CWL, CWM, CWN, CWP, CWQ, CWR, CWS, CWT, CWV, CWW, CWY, CYA, CYC, CYD, CYE, CYF, CYG, CYH, CYI, CYK, CYL, CYM, CYN, CYP, CYQ, CYR, CYS, CYT, CYV, CYW, CYY, DAA, DAC, DAD, DAE, DAF, DAG, DAH, DAI, DAK, DAL, DAM, DAN, DAP, DAQ, DAR, DAS, DAT, DAV, DAW, DAY, DCA, DCC, DCD, DCE, DCF, DCG, DCH, DCI, DCK, DCL, DCM, DCN, DCP, DCQ, DCR, DCS, DCT, DCV, DCW, DCY, DDA, DDC, DDD, DDE, DDF, DDG, DDH, DDI, DDK, DDL, DDM, DDN, DDP, DDQ, DDR, DDS, DDT, DDV, DDW, DDY, DEA, DEC, DED, DEE, DEF, DEG, DEH, DEI, DEK, DEL, DEM, DEN, DEP, DEQ, DER, DES, DET, DEV, DEW, DEY, DFA, DFC, DFD, DFE, DFF, DFG, DFH, DFI, DFK, DFL, DFM, DFN, DFP, DFQ, DFR, DFS, DFT, DFV, DFW, DFY, DGA, DGC, DGD, DGE, DGF, DGG, DGH, DGI, DGK, DGL, DGM, DGN, DGP, DGQ, DGR, DGS, DGT, DGV, DGW, DGY, DHA, DHC, DHD, DHE, DHF, DHG, DHH, DHI, DHK, DHL, DHM, DHN, DHP, DHQ, DHR, DHS, DHT, DHV, DHW, DHY, DIA, DIC, DID, DIE, DIF, DIG, DIH, DII, DIK, DIL, DIM, DIN, DIP, DIQ, DIR, DIS, DIT, DIV, DIW, DIY, DKA, DKC, DKD, DKE, DKF, DKG, DKH, DKI, DKK, DKL, DKM, DKN, DKP, DKQ, DKR, DKS, DKT, DKV, DKW, DKY, DLA, DLC, DLD, DLE, DLF, DLG, DLH, DLI, DLK, DLL, DLM, DLN, DLP, DLQ, DLR, DLS, DLT, DLV, DLW, DLY, DMA, DMC, DMD, DME, DMF, DMG, DMH, DMI, DMK, DML, DMM, DMN, DMP, DMQ, DMR, DMS, DMT, DMV, DMW, DMY, DNA, DNC, DND, DNE, DNF, DNG, DNH, DNI, DNK, DNL, DNM, DNN, DNP, DNQ, DNR, DNS, DNT, DNV, DNW, DNY, DPA, DPC, DPD, DPE, DPF, DPG, DPH, DPI, DPK, DPL, DPM, DPN, DPP, DPQ, DPR, DPS, DPT, DPV, DPW, DPY, DQA, DQC, DQD, DQE, DQF, DQG, DQH, DQI, DQK, DQL, DQM, DQN, DQP, DQQ, DQR, DQS, DQT, DQV, DQW, DQY, DRA, DRC, DRD, DRE, DRF, DRG, DRH, DRI, DRK, DRL, DRM, DRN, DRP, DRQ, DRR, DRS, DRT, DRV, DRW, DRY, DSA, DSC, DSD, DSE, DSF, DSG, DSH, DSI, DSK, DSL, DSM, DSN, DSP, DSQ, DSR, DSS, DST, DSV, DSW, DSY, DTA, DTC, DTD, DTE, DTF, DTG, DTH, DTI, DTK, DTL, DTM, DTN, DTP, DTQ, DTR, DTS, DTT, DTV, DTW, DTY, DVA, DVC, DVD, DVE, DVF, DVG, DVH, DVI, DVK, DVL, DVM, DVN, DVP, DVQ, DVR, DVS, DVT, DVV, DVW, DVY, DWA, DWC, DWD, DWE, DWF, DWG, DWH, DWI, DWK, DWL, DWM, DWN, DWP, DWQ, DWR, DWS, DWT, DWV, DWW, DWY, DYA, DYC, DYD, DYE, DYF, DYG, DYH, DYI, DYK, DYL, DYM, DYN, DYP, DYQ, DYR, DYS, DYT, DYV, DYW, DYY, EAA, EAC, EAD, EAE, EAF, EAG, EAH, EAI, EAK, EAL, EAM, EAN, EAP, EAQ, EAR, EAS, EAT, EAV, EAW, EAY, ECA, ECC, ECD, ECE, ECF, ECG, ECH, ECI, ECK, ECL, ECM, ECN, ECP, ECQ, ECR, ECS, ECT, ECV, ECW, ECY, EDA, EDC, EDD, EDE, EDF, EDG, EDH, EDI, EDK, EDL, EDM, EDN, EDP, EDQ, EDR, EDS, EDT, EDV, EDW, EDY, EEA, EEC, EED, EEE, EEF, EEG, EEH, EEI, EEK, EEL, EEM, EEN, EEP, EEQ, EER, EES, EET, EEV, EEW, EEY, EFA, EFC, EFD, EFE, EFF, EFG, EFH, EFI, EFK, EFL, EFM, EFN, EFP, EFQ, EFR, EFS, EFT, EFV, EFW, EFY, EGA, EGC, EGD, EGE, EGF, EGG, EGH, EGI, EGK, EGL, EGM, EGN, EGP, EGQ, EGR, EGS, EGT, EGV, EGW, EGY, EHA, EHC, EHD, EHE, EHF, EHG, EHH, EHI, EHK, EHL, EHM, EHN, EHP, EHQ, EHR, EHS, EHT, EHV, EHW, EHY, EIA, EIC, EID, EIE, EIF, EIG, EIH, EII, EIK, EIL, EIM, EIN, EIP, EIQ, EIR, EIS, EIT, EIV, EIW, EIY, EKA, EKC, EKD, EKE, EKF, EKG, EKH, EKI, EKK, EKL, EKM, EKN, EKP, EKQ, EKR, EKS, EKT, EKV, EKW, EKY, ELA, ELC, ELD, ELE, ELF, ELG, ELH, ELI, ELK, ELL, ELM, ELN, ELP, ELQ, ELR, ELS, ELT, ELV, ELW, ELY, EMA, EMC, EMD, EME, EMF, EMG, EMH, EMI, EMK, EML, EMM, EMN, EMP, EMQ, EMR, EMS, EMT, EMV, EMW, EMY, ENA, ENC, END, ENE, ENF, ENG, ENH, ENI, ENK, ENL, ENM, ENN, ENP, ENQ, ENR, ENS, ENT, ENV, ENW, ENY, EPA, EPC, EPD, EPE, EPF, EPG, EPH, EPI, EPK, EPL, EPM, EPN, EPP, EPQ, EPR, EPS, EPT, EPV, EPW, EPY, EQA, EQC, EQD, EQE, EQF, EQG, EQH, EQI, EQK, EQL, EQM, EQN, EQP, EQQ, EQR, EQS, EQT, EQV, EQW, EQY, ERA, ERC, ERD, ERE, ERF, ERG, ERH, ERI, ERK, ERL, ERM, ERN, ERP, ERQ, ERR, ERS, ERT, ERV, ERW, ERY, ESA, ESC, ESD, ESE, ESF, ESG, ESH, ESI, ESK, ESL, ESM, ESN, ESP, ESQ, ESR, ESS, EST, ESV, ESW, ESY, ETA, ETC, ETD, ETE, ETF, ETG, ETH, ETI, ETK, ETL, ETM, ETN, ETP, ETQ, ETR, ETS, ETT, ETV, ETW, ETY, EVA, EVC, EVD, EVE, EVF, EVG, EVH, EVI, EVK, EVL, EVM, EVN, EVP, EVQ, EVR, EVS, EVT, EVV, EVW, EVY, EWA, EWC, EWD, EWE, EWF, EWG, EWH, EWI, EWK, EWL, EWM, EWN, EWP, EWQ, EWR, EWS, EWT, EWV, EWW, EWY, EYA, EYC, EYD, EYE, EYF, EYG, EYH, EYI, EYK, EYL, EYM, EYN, EYP, EYQ, EYR, EYS, EYT, EYV, EYW, EYY, FAA, FAC, FAD, FAE, FAF, FAG, FAH, FAI, FAK, FAL, FAM, FAN, FAP, FAQ, FAR, FAS, FAT, FAV, FAW, FAY, FCA, FCC, FCD, FCE, FCF, FCG, FCH, FCI, FCK, FCL, FCM, FCN, FCP, FCQ, FCR, FCS, FCT, FCV, FCW, FCY, FDA, FDC, FDD, FDE, FDF, FDG, FDH, FDI, FDK, FDL, FDM, FDN, FDP, FDQ, FDR, FDS, FDT, FDV, FDW, FDY, FEA, FEC, FED, FEE, FEF, FEG, FEH, FEI, FEK, FEL, FEM, FEN, FEP, FEQ, FER, FES, FET, FEV, FEW, FEY, FFA, FFC, FFD, FFE, FFF, FFG, FFH, FFI, FFK, FFL, FFM, FFN, FFP, FFQ, FFR, FFS, FFT, FFV, FFW, FFY, FGA, FGC, FGD, FGE, FGF, FGG, FGH, FGI, FGK, FGL, FGM, FGN, FGP, FGQ, FGR, FGS, FGT, FGV, FGW, FGY, FHA, FHC, FHD, FHE, FHF, FHG, FHH, FHI, FHK, FHL, FHM, FHN, FHP, FHQ, FHR, FHS, FHT, FHV, FHW, FHY, FIA, FIC, FID, FIE, FIF, FIG, FIH, FII, FIK, FIL, FIM, FIN, FIP, FIQ, FIR, FIS, FIT, FIV, FIW, FIY, FKA, FKC, FKD, FKE, FKF, FKG, FKH, FKI, FKK, FKL, FKM, FKN, FKP, FKQ, FKR, FKS, FKT, FKV, FKW, FKY, FLA, FLC, FLD, FLE, FLF, FLG, FLH, FLI, FLK, FLL, FLM, FLN, FLP, FLQ, FLR, FLS, FLT, FLV, FLW, FLY, FMA, FMC, FMD, FME, FMF, FMG, FMH, FMI, FMK, FML, FMM, FMN, FMP, FMQ, FMR, FMS, FMT, FMV, FMW, FMY, FNA, FNC, FND, FNE, FNF, FNG, FNH, FNI, FNK, FNL, FNM, FNN, FNP, FNQ, FNR, FNS, FNT, FNV, FNW, FNY, FPA, FPC, FPD, FPE, FPF, FPG, FPH, FPI, FPK, FPL, FPM, FPN, FPP, FPQ, FPR, FPS, FPT, FPV, FPW, FPY, FQA, FQC, FQD, FQE, FQF, FQG, FQH, FQI, FQK, FQL, FQM, FQN, FQP, FQQ, FQR, FQS, FQT, FQV, FQW, FQY, FRA, FRC, FRD, FRE, FRF, FRG, FRH, FRI, FRK, FRL, FRM, FRN, FRP, FRQ, FRR, FRS, FRT, FRV, FRW, FRY, FSA, FSC, FSD, FSE, FSF, FSG, FSH, FSI, FSK, FSL, FSM, FSN, FSP, FSQ, FSR, FSS, FST, FSV, FSW, FSY, FTA, FTC, FTD, FTE, FTF, FTG, FTH, FTI, FTK, FTL, FTM, FTN, FTP, FTQ, FTR, FTS, FTT, FTV, FTW, FTY, FVA, FVC, FVD, FVE, FVF, FVG, FVH, FVI, FVK, FVL, FVM, FVN, FVP, FVQ, FVR, FVS, FVT, FVV, FVW, FVY, FWA, FWC, FWD, FWE, FWF, FWG, FWH, FWI, FWK, FWL, FWM, FWN, FWP, FWQ, FWR, FWS, FWT, FWV, FWW, FWY, FYA, FYC, FYD, FYE, FYF, FYG, FYH, FYI, FYK, FYL, FYM, FYN, FYP, FYQ, FYR, FYS, FYT, FYV, FYW, FYY, GAA, GAC, GAD, GAE, GAF, GAG, GAH, GAI, GAK, GAL, GAM, GAN, GAP, GAQ, GAR, GAS, GAT, GAV, GAW, GAY, GCA, GCC, GCD, GCE, GCF, GCG, GCH, GCI, GCK, GCL, GCM, GCN, GCP, GCQ, GCR, GCS, GCT, GCV, GCW, GCY, GDA, GDC, GDD, GDE, GDF, GDG, GDH, GDI, GDK, GDL, GDM, GDN, GDP, GDQ, GDR, GDS, GDT, GDV, GDW, GDY, GEA, GEC, GED, GEE, GEF, GEG, GEH, GEI, GEK, GEL, GEM, GEN, GEP, GEQ, GER, GES, GET, GEV, GEW, GEY, GFA, GFC, GFD, GFE, GFF, GFG, GFH, GFI, GFK, GFL, GFM, GFN, GFP, GFQ, GFR, GFS, GFT, GFV, GFW, GFY, GGA, GGC, GGD, GGE, GGF, GGG, GGH, GGI, GGK, GGL, GGM, GGN, GGP, GGQ, GGR, GGS, GGT, GGV, GGW, GGY, GHA, GHC, GHD, GHE, GHF, GHG, GHH, GHI, GHK, GHL, GHM, GHN, GHP, GHQ, GHR, GHS, GHT, GHV, GHW, GHY, GIA, GIC, GID, GIE, GIF, GIG, GIH, GII, GIK, GIL, GIM, GIN, GIP, GIQ, GIR, GIS, GIT, GIV, GIW, GIY, GKA, GKC, GKD, GKE, GKF, GKG, GKH, GKI, GKK, GKL, GKM, GKN, GKP, GKQ, GKR, GKS, GKT, GKV, GKW, GKY, GLA, GLC, GLD, GLE, GLF, GLG, GLH, GLI, GLK, GLL, GLM, GLN, GLP, GLQ, GLR, GLS, GLT, GLV, GLW, GLY, GMA, GMC, GMD, GME, GMF, GMG, GMH, GMI, GMK, GML, GMM, GMN, GMP, GMQ, GMR, GMS, GMT, GMV, GMW, GMY, GNA, GNC, GND, GNE, GNF, GNG, GNH, GNI, GNK, GNL, GNM, GNN, GNP, GNQ, GNR, GNS, GNT, GNV, GNW, GNY, GPA, GPC, GPD, GPE, GPF, GPG, GPH, GPI, GPK, GPL, GPM, GPN, GPP, GPQ, GPR, GPS, GPT, GPV, GPW, GPY, GQA, GQC, GQD, GQE, GQF, GQG, GQH, GQI, GQK, GQL, GQM, GQN, GQP, GQQ, GQR, GQS, GQT, GQV, GQW, GQY, GRA, GRC, GRD, GRE, GRF, GRG, GRH, GRI, GRK, GRL, GRM, GRN, GRP, GRQ, GRR, GRS, GRT, GRV, GRW, GRY, GSA, GSC, GSD, GSE, GSF, GSG, GSH, GSI, GSK, GSL, GSM, GSN, GSP, GSQ, GSR, GSS, GST, GSV, GSW, GSY, GTA, GTC, GTD, GTE, GTF, GTG, GTH, GTI, GTK, GTL, GTM, GTN, GTP, GTQ, GTR, GTS, GTT, GTV, GTW, GTY, GVA, GVC, GVD, GVE, GVF, GVG, GVH, GVI, GVK, GVL, GVM, GVN, GVP, GVQ, GVR, GVS, GVT, GVV, GVW, GVY, GWA, GWC, GWD, GWE, GWF, GWG, GWH, GWI, GWK, GWL, GWM, GWN, GWP, GWQ, GWR, GWS, GWT, GWV, GWW, GWY, GYA, GYC, GYD, GYE, GYF, GYG, GYH, GYI, GYK, GYL, GYM, GYN, GYP, GYQ, GYR, GYS, GYT, GYV, GYW, GYY, HAA, HAC, HAD, HAE, HAF, HAG, HAH, HAI, HAK, HAL, HAM, HAN, HAP, HAQ, HAR, HAS, HAT, HAV, HAW, HAY, HCA, HCC, HCD, HCE, HCF, HCG, HCH, HCI, HCK, HCL, HCM, HCN, HCP, HCQ, HCR, HCS, HCT, HCV, HCW, HCY, HDA, HDC, HDD, HDE, HDF, HDG, HDH, HDI, HDK, HDL, HDM, HDN, HDP, HDQ, HDR, HDS, HDT, HDV, HDW, HDY, HEA, HEC, HED, HEE, HEF, HEG, HEH, HEI, HEK, HEL, HEM, HEN, HEP, HEQ, HER, HES, HET, HEV, HEW, HEY, HFA, HFC, HFD, HFE, HFF, HFG, HFH, HFI, HFK, HFL, HFM, HFN, HFP, HFQ, HFR, HFS, HFT, HFV, HFW, HFY, HGA, HGC, HGD, HGE, HGF, HGG, HGH, HGI, HGK, HGL, HGM, HGN, HGP, HGQ, HGR, HGS, HGT, HGV, HGW, HGY, HHA, HHC, HHD, HHE, HHF, HHG, HHH, HHI, HHK, HHL, HHM, HHN, HHP, HHQ, HHR, HHS, HHT, HHV, HHW, HHY, HIA, HIC, HID, HIE, HIF, HIG, HIH, HII, HIK, HIL, HIM, HIN, HIP, HIQ, HIR, HIS, HIT, HIV, HIW, HIY, HKA, HKC, HKD, HKE, HKF, HKG, HKH, HKI, HKK, HKL, HKM, HKN, HKP, HKQ, HKR, HKS, HKT, HKV, HKW, HKY, HLA, HLC, HLD, HLE, HLF, HLG, HLH, HLI, HLK, HLL, HLM, HLN, HLP, HLQ, HLR, HLS, HLT, HLV, HLW, HLY, HMA, HMC, HMD, HME, HMF, HMG, HMH, HMI, HMK, HML, HMM, HMN, HMP, HMQ, HMR, HMS, HMT, HMV, HMW, HMY, HNA, HNC, HND, HNE, HNF, HNG, HNH, HNI, HNK, HNL, HNM, HNN, HNP, HNQ, HNR, HNS, HNT, HNV, HNW, HNY, HPA, HPC, HPD, HPE, HPF, HPG, HPH, HPI, HPK, HPL, HPM, HPN, HPP, ^* HPQ, HPR, HPS, HPT, HPV, HPW, HPY, HQA, HQC, HQD, HQE, HQF, HQG, HQH, HQI, HQK, HQL, HQM, HQN, HQP, HQQ, HQR, HQS, HQT, HQV, HQW, HQY, HRA, HRC, HRD, HRE, HRF, HRG, HRH, HRI, HRK, HRL, HRM, HRN, HRP, HRQ, HRR, HRS, HRT, HRV, HRW, HRY, HSA, HSC, HSD, HSE, HSF, HSG, HSH, HSI, HSK, HSL, HSM, HSN, HSP, HSQ, HSR, HSS, HST, HSV, HSW, HSY, HTA, HTC, HTD, HTE, HTF, HTG, HTH, HTI, HTK, HTL, HTM, HTN, HTP, HTQ, HTR, HTS, HTT, HTV, HTW, HTY, HVA, HVC, HVD, HVE, HVF, HVG, HVH, HVI, HVK, HVL, HVM, HVN, HVP, HVQ, HVR, HVS, HVT, HW, HVW, HVY, HWA, HWC, HWD, HWE, HWF, HWG, HWH, HWI, HWK, HWL, HWM, HWN, HWP, HWQ, HWR, HWS, HWT, HWV, HWW, HWY, HYA, HYC, HYD, HYE, HYF, HYG, HYH, HYI, HYK, HYL, HYM, HYN, HYP, HYQ, HYR, HYS, HYT, HYV, HYW, HYY, IAA, IAC, IAD, IAE, IAF, LAG, IAH, IAI, IAK, IAL, IAM, IAN, IAP, IAQ, IAR, IAS, IAT, IAV, IAW, IAY, ICA, ICC, ICD, ICE, ICF, ICG, ICH, ICI, ICK, ICL, ICM, ICN, ICP, ICQ, ICR, ICS, ICT, ICV, ICW, ICY, IDA, IDC, IDD, IDE, IDF, IDG, IDH, IDI, IDK, IDL, IDM, IDN, IDP, IDQ, IDR, IDS, IDT, IDV, IDW, IDY, IEA, IEC, IED, IEE, IEF, IEG, IEH, IEI, IEK, IEL, IEM, IEN, IEP, IEQ, IER, IES, IET, IEV, IEW, IEY, IFA, IFC, IFD, IFE, IFF, IFG, IFH, IFI, IFK, IFL, IFM, IFN, IFP, IFQ, IFR, IFS, IFT, IFV, IFW, IFY, IGA, IGC, IGD, IGE, IGF, IGG, IGH, IGI, IGK, IGL, IGM, IGN, IGP, IGQ, IGR, IGS, IGT, IGV, IGW, IGY, IHA, IHC, IHD, IHE, IHF, IHG, IHH, IHI, IHK, IHL, IHM, IHN, IHP, IHQ, IHR, IHS, IHT, IHV, IHW, IHY, IIA, IIC, HD, HE, HF, HG,

HH, in, HK, HL, HM, iiN, iip, HQ, HR, iis, HT, πv, iiw, IIY, IKA, IKC, IKD, KE,

IKF, IKG, IKH, IKI, IKK, IKL, IKM, IKN, IKP, IKQ, IKR, IKS, IKT, IKV, IKW, IKY, ILA, ILC, ILD, ILE, ILF, ILG, ILH, ILI, ILK, ILL, ILM, ILN, ILP, ILQ, ILR, ILS, ILT, ILV, ILW, ILY, IMA, IMC, IMD, IME, IMF, IMG, IMH, IMI, IMK, IML, IMM, IMN, IMP, IMQ, IMR, IMS, IMT, IMV, IMW, IMY, INA, INC, IND, INE, INF, ING, INH, INI, INK, INL, INM, INN, INP, INQ, INR, INS, INT, INV, INW, INY, IPA, IPC, IPD, IPE, IPF, IPG, IPH, IPI, IPK, IPL, IPM, IPN, IPP, IPQ, IPR, IPS, IPT, IPV, IPW, IPY, IQA, IQC, IQD, IQE, IQF, IQG, IQH, IQI, IQK, IQL, IQM, IQN, IQP, IQQ, IQR, IQS, IQT, IQV, IQW, IQY, IRA, IRC, ERD, IRE, IRF, IRG, IRH, IRI, IRK, IRL, IRM, IRN, IRP, IRQ, IRR, IRS, IRT, IRV, IRW, IRY, ISA, ISC, ISD, ISE, ISF, ISG, ISH, ISI, ISK, ISL, ISM, ISN, ISP, ISQ, ISR, ISS, 1ST, ISV, ISW, ISY, ITA, ITC, ITD, ITE, ITF, ITG, ITH, ITI, ITK, ITL, ITM, ITN, ITP, ITQ, ITR, ITS, ITT, ITV, ITW, ITY, IVA, IVC, IVD, IVE, IVF, IVG, IVH, IVI, IVK, IVL, IVM, IVN, IVP, IVQ, IVR, IVS, IVT, IVV, IVW, IVY, IWA, IWC, IWD, IWE, IWF, IWG, IWH, IWI, IWK, IWL, IWM, IWN, IWP, IWQ, IWR, IWS, IWT, IWV, IWW, IWY, IYA, IYC, IYD, IYE, IYF, IYG, IYH, IYI, IYK, IYL, IYM, IYN, IYP, IYQ, IYR, IYS, IYT, IYV, IYW, IYY, KAA, KAC, KAD, KAE, KAF, KAG, KAH, KAI, KAK, KAL, KAM, KAN, KAP, KAQ, KAR, KAS, KAT, KAV, KAW, KAY, KCA, KCC, KCD, KCE, KCF, KCG, KCH, KCI, KCK, KCL, KCM, KCN, KCP, KCQ, KCR, KCS, KCT, KCV, KCW, KCY, KDA, KDC, KDD, KDE, KDF, KDG, KDH, KDI, KDK, KDL, KDM, KDN, KDP, KDQ, KDR, KDS, KDT, KDV, KDW, KDY, KEA, KEC, KED, KEE, KEF, KEG, KEH, KEI, KEK, KEL, KEM, KEN, KEP, KEQ, KER, KES, KET, KEV, KEW, KEY, KFA, KFC, KFD, KFE, KFF, KFG, KFH, KFI, KFK, KFL, KFM, KFN, KFP, KFQ, KFR, KFS, KFT, KFV, KFW, KFY, KGA, KGC, KGD, KGE, KGF, KGG, KGH, KGI, KGK, KGL, KGM, KGN, KGP, KGQ, KGR, KGS, KGT, KGV, KGW, KGY, KHA, KHC, KHD, KHE, KHF, KHG, KHH, KHI, KHK, KHL, KHM, KHN, KHP, KHQ, KHR, KHS, KHT, KHV, KHW, KHY, KIA, KIC, KID, KIE, KIF, KIG, KIH, KII, KIK, KIL, KIM, KIN, KIP, KIQ, KIR, KIS, KIT, KTV, KIW, KIY, KKA, KKC, KKD, KKE, KKF, KKG, KKH, KKI, KKK, KKL, KKM, KKN, KKP, KKQ, KKR, KKS, KKT, KKV, KKW, KKY, KLA, KLC, KLD, KLE, KLF, KLG, KLH, KLI, KLK, KLL, KLM, KLN, KLP, KLQ, KLR, KLS, KLT, KLV, KLW, KLY, KMA, KMC, KMD, KME, KMF, KMG, KMH, KMI, KMK, KML, KMM, KMN, KMP, KMQ, KMR, KMS, KMT, KMV, KMW, KMY, KNA, KNC, KND, KNE, KNF, KNG, KNH, KNI, KNK, KNL, KNM, KNN, KNP, KNQ, KNR, KNS, KNT, KNV, KNW, KNY, KPA, KPC, KPD, KPE, KPF, KPG, KPH, KPI, KPK, KPL, KPM, KPN, KPP, KPQ, KPR, KPS, KPT, KPV, KPW, KPY, KQA, KQC, KQD, KQE, KQF, KQG, KQH, KQI, KQK, KQL, KQM, KQN, KQP, KQQ, KQR, KQS, KQT, KQV, KQW, KQY, KRA, KRC, KRD, KRE, KRF, KRG, KRH, KRI, KRK, KRL, KRM, KRN, KRP, KRQ, KRR, KRS, KRT, KRV, KRW, KRY, KSA, KSC, KSD, KSE, KSF, KSG, KSH, KSI, KSK, KSL, KSM, KSN, KSP, KSQ, KSR, KSS, KST, KSV, KSW, KSY, KTA, KTC, KTD, KTE, KTF, KTG, KTH, KTI, KTK, KTL, KTM, KTN, KTP, KTQ, KTR, KTS, KTT, KTV, KTW, KTY, KVA, KVC, KVD, KVE, KVF, KVG, KVH, KVI, KVK, KVL, KVM, KVN, KVP, KVQ, KVR, KVS, KVT, KW, KVW, KVY, KWA, KWC, KWD, KWE, KWF, KWG, KWH, KWI, KWK, KWL, KWM, KWN, KWP, KWQ₅ KWR, KWS, KWT, KWV, KWW, KWY, KYA, KYC, KYD, KYE, KYF, KYG, KYH, KYI, KYK, KYL, KYM, KYN, KYP, KYQ, KYR, KYS, KYT, KYV, KYW, KYY, LAA, LAC, LAD, LAE, LAF, LAG, LAH, LAI, LAK, LAL, LAM, LAN, LAP, LAQ, LAR, LAS, LAT, LAV, LAW, LAY, LCA, LCC, LCD, LCE, LCF, LCG, LCH, LCI, LCK, LCL, LCM, LCN, LCP, LCQ, LCR, LCS, LCT, LCV, LCW, LCY, LDA, LDC, LDD, LDE, LDF, LDG, LDH, LDI, LDK, LDL, LDM, LDN, LDP, LDQ, LDR, LDS, LDT, LDV, LDW, LDY, LEA, LEC, LED, LEE, LEF, LEG, LEH, LEI, LEK, LEL, LEM, LEN, LEP, LEQ, LER, LES, LET, LEV, LEW, LEY, LFA, LFC, LFD, LFE, LFF, LFG, LFH, LFI, LFK, LFL, LFM, LFN, LFP, LFQ, LFR, LFS, LFT, LFV, LFW, LFY, LGA, LGC, LGD, LGE, LGF, LGG, LGH, LGI, LGK, LGL, LGM, LGN, LGP, LGQ, LGR, LGS, LGT, LGV, LGW, LGY, LHA, LHC, LHD, LHE, LHF, LHG, LHH, LHI, LHK, LHL, LHM, LHN, LHP, LHQ, LHR, LHS, LHT, LHV, LHW, LHY, LIA, LIC, LID, LIE, LIF, LIG, LIH, LII, LIK, LIL, LIM, LIN, LIP, LIQ, LIR, LIS, LIT, LIV, LIW, LIY, LKA, LKC, LKD, LKE, LKF, LKG, LKH, LKI, LKK, LKL, LKM, LKN, LKP, LKQ, LKR, LKS, LKT, LKV, LKW, LKY, LLA, LLC, LLD, LLE, LLF, LLG, LLH, LLI, LLK, LLL, LLM, LLN, LLP, LLQ, LLR, LLS, LLT, LLV, LLW, LLY, LMA, LMC, LMD, LME, LMF, LMG, LMH, LMI, LMK, LML, LMM, LMN, LMP, LMQ, LMR, LMS, LMT, LMV, LMW, LMY, LNA, LNC, LND, LNE, LNF, LNG, LNH, LNI, LNK, LNL, LNM, LNN, LNP, LNQ, LNR, LNS, LNT, LNV, LNW, LNY, LPA, LPC, LPD, LPE, LPF, LPG, LPH, LPI, LPK, LPL, LPM, LPN, LPP, LPQ, LPR, LPS, LPT, LPV, LPW, LPY, LQA, LQC, LQD, LQE, LQF, LQG, LQH, LQI, LQK, LQL, LQM, LQN, LQP, LQQ, LQR, LQS, LQT, LQV, LQW, LQY, LRA, LRC, LRD, LRE, LRF, LRG, LRH, LRI, LRK, LRL, LRM, LRN, LRP, LRQ, LRR, LRS, LRT, LRV, LRW, LRY, LSA, LSC, LSD, LSE, LSF, LSG, LSH, LSI, LSK, LSL, LSM, LSN, LSP, LSQ, LSR, LSS, LST, LSV, LSW, LSY, LTA, LTC, LTD, LTE, LTF, LTG, LTH, LTI, LTK, LTL, LTM, LTN, LTP, LTQ, LTR, LTS, LTT, LTV, LTW, LTY, LVA, LVC, LVD, LVE, LVF, LVG, LVH, LVI, LVK, LVL, LVM, LVN, LVP, LVQ, LVR, LVS, LVT, LVV, LVW, LVY, LWA, LWC, LWD, LWE, LWF, LWG, LWH, LWI, LWK, LWL, LWM, LWN, LWP, LWQ, LWR, LWS, LWT, LWV, LWW, LWY, LYA, LYC, LYD, LYE, LYF, LYG, LYH, LYI, LYK, LYL, LYM, LYN, LYP, LYQ, LYR, LYS, LYT, LYV, LYW, LYY, MAA, MAC, MAD, MAE, MAF, MAG, MAH, MAI, MAK, MAL, MAM, MAN, MAP, MAQ, MAR, MAS, MAT, MAV, MAW, MAY, MCA, MCC, MCD, MCE, MCF, MCG, MCH, MCI, MCK, MCL, MCM, MCN, MCP, MCQ, MCR, MCS, MCT, MCV, MCW, MCY, MDA, MDC, MDD, MDE, MDF, MDG, MDH, MDI, MDK, MDL, MDM, MDN, MDP, MDQ, MDR, MDS, MDT, MDV, MDW, MDY, MEA, MEC, MED, MEE, MEF, MEG, MEH, MEI, MEK, MEL, MEM, MEN, MEP, MEQ, MER, MES, MET, MEV, MEW, MEY, MFA, MFC, MFD, MFE, MFF, MFG, MFH, MFI, MFK, MFL, MFM, MFN, MFP, MFQ, MFR, MFS, MFT, MFV, MFW, MFY, MGA, MGC, MGD, MGE, MGF, MGG, MGH, MGI, MGK, MGL, MGM, MGN, MGP, MGQ, MGR, MGS, MGT, MGV, MGW, MGY, MHA, MHC, MHD, MHE, MHF, MHG, MHH, MHI, MHK, MHL, MHM, MHN, MHP, MHQ, MHR, MHS, MHT, MHV, MHW, MHY, MIA, MIC, MID, MIE, MIF, MIG, MIH, Mil, MIK, MIL, MIM, MIN, MIP, MIQ, MIR, MIS, MIT, MIV, MIW, MIY, MKA, MKC, MKD, MKE, MKF, MKG, MKH, MKI, MKK, MKL, MKM, MKN, MKP, MKQ, MKR, MKS, MKT, MKV, MKW, MKY, MLA, MLC, MLD, MLE, MLF, MLG, MLH, MLI, MLK, MLL, MLM, MLN, MLP, MLQ, MLR, MLS, MLT, MLV, MLW, MLY, MMA, MMC, MMD, MME, MMF, MMG, MMH, MMI, MMK, MML, MMM, MMN, MMP, MMQ, MMR, MMS, MMT, MMV, MMW, MMY, MNA, MNC, MND, MNE, MNF, MNG, MNH, MNI, MNK, MNL, MNM, MNN, MNP, MNQ, MNR, MNS, MNT, MNV, MNW, MNY, MPA, MPC, MPD, MPE, MPF, MPG, MPH, MPI, MPK, MPL, MPM, MPN, MPP, MPQ, MPR, MPS, MPT, MPV, MPW, MPY, MQA, MQC, MQD, MQE, MQF, MQG, MQH, MQI, MQK, MQL, MQM, MQN, MQP, MQQ, MQR, MQS, MQT, MQV, MQW, MQY, MRA, MRC, MRD, MRE, MRF, MRG, MRH, MRI, MRK, MRL, MRM, MRN, MRP, MRQ, MRR, MRS, MRT, MRV, MRW, MRY, MSA, MSC, MSD, MSE, MSF, MSG, MSH, MSI, MSK, MSL, MSM, MSN, MSP, MSQ, MSR, MSS, MST, MSV, MSW, MSY, MTA, MTC, MTD, MTE, MTF, MTG, MTH, MTI, MTK, MTL, MTM, MTN, MTP, MTQ, MTR, MTS, MTT, MTV, MTW, MTY, MVA, MVC, MVD, MVE, MVF, MVG, MVH, MVI, MVK, MVL, MVM, MVN, MVP, MVQ, MVR, MVS, MVT, MVV, MVW, MVY, MWA, MWC, MWD, MWE, MWF, MWG, MWH, MWI, MWK, MWL, MWM, MWN, MWP, MWQ, MWR, MWS, MWT, MWV, MWW, MWY, MYA, MYC, MYD, MYE, MYF, MYG, MYH, MYI, MYK, MYL, MYM, MYN, MYP, MYQ, MYR, MYS, MYT, MYV, MYW, MYY, NAA, NAC, NAD, NAE, NAF, NAG, NAH, NAI, NAK, NAL, NAM, NAN, NAP, NAQ, NAR, NAS, NAT, NAV, NAW, NAY, NCA, NCC, NCD, NCE, NCF, NCG, NCH, NCI, NCK, NCL, NCM, NCN, NCP, NCQ, NCR, NCS, NCT, NCV, NCW, NCY, NDA, NDC, NDD, NDE, NDF, NDG, NDH, NDI, NDK, NDL, NDM, NDN, NDP, NDQ, NDR, NDS, NDT, NDV, NDW, NDY, NEA, NEC, NED, NEE, NEF, NEG, NEH, NEI, NEK, NEL, NEM, NEN, NEP, NEQ, NER, NES, NET, NEV, NEW, NEY, NFA, NFC, NFD, NFE, NFF, NFG, NFH, NFI, NFK, NFL, NFM, NFN, NFP, NFQ, NFR, NFS, NFT, NFV, NFW, NFY, NGA, NGC, NGD, NGE, NGF, NGG, NGH, NGI, NGK, NGL, NGM, NGN, NGP, NGQ, NGR, NGS, NGT, NGV, NGW, NGY, NHA, NHC, NHD, NHE, NHF, NHG, NHH, NHI, NHK, NHL, NHM, NHN, NHP, NHQ, NHR, NHS, NHT, NHV, NHW, NHY, NIA, NIC, NID, NIE, NIF, NIG, NIH, Nil, NIK, NIL, NIM, NIN, NIP, NIQ, NIR, NIS, NIT, NIV, NIW, NIY, NKA, NKC, NKD, NKE, NKF, NKG, NKH, NKI, NKK, NKL, NKM, NKN, NKP, NKQ, NKR, NKS, NKT, NKV, NKW, NKY, NLA, NLC, NLD, NLE, NLF, NLG, NLH, NLI, NLK, NLL, NLM, NLN, NLP, NLQ, NLR, NLS, NLT, NLV, NLW, NLY, NMA, NMC, NMD, NME, NMF, NMG, NMH, NMI, NMK, NML, NMM, NMN, NMP, NMQ, NMR, NMS, NMT, NMV, NMW, NMY, NNA, NNC, NND, NNE, NNF, NNG, NNH, NNI, NNK, NNL, NNM, NNN, NNP, NNQ, NNR, NNS, NNT, NNV, NNW, NNY, NPA, NPC, NPD, NPE, NPF, NPG, NPH, NPI, NPK, NPL, NPM, NPN, NPP, NPQ, NPR, NPS, NPT, NPV, NPW, NPY, NQA, NQC, NQD, NQE, NQF, NQG, NQH, NQI, NQK, NQL, NQM, NQN, NQP, NQQ, NQR, NQS, NQT, NQV, NQW, NQY, NRA, NRC, NRD, NRE, NRF, NRG, NRH, NRI, NRK, NRL, NRM, NRN, NRP, NRQ, NRR, NRS, NRT, NRV, NRW, NRY, NSA, NSC, NSD, NSE, NSF, NSG, NSH, NSI, NSK, NSL, NSM, NSN, NSP, NSQ, NSR, NSS, NST, NSV, NSW, NSY, NTA, NTC, NTD, NTE, NTF, NTG, NTH, NTI, NTK, NTL, NTM, NTN, NTP, NTQ, NTR, NTS, NTT, NTV, NTW, NTY, NVA, NVC, NVD, NVE, NVF, NVG, NVH, NVI, NVK, NVL, NVM, NVN, NVP, NVQ, NVR, NVS, NVT, NVV, NVW, NVY, NWA, NWC, NWD, NWE, NWF, NWG, NWH, NWI, NWK, NWL, NWM, NWN, NWP, NWQ, NWR, NWS, NWT, NWV, NWW, NWY, NYA, NYC, NYD, NYE, NYF, NYG, NYH, NYI, NYK, NYL, NYM, NYN, NYP, NYQ, NYR, NYS, NYT, NYV, NYW, NYY, PAA, PAC, PAD, PAE, PAF, PAG, PAH, PAI, PAK, PAL, PAM, PAN, PAP, PAQ, PAR, PAS, PAT, PAV, PAW, PAY, PCA, PCC, PCD, PCE, PCF, PCG, PCH, PCI, PCK, PCL, PCM, PCN, PCP, PCQ, PCR, PCS, PCT, PCV, PCW, PCY, PDA, PDC, PDD, PDE, PDF, PDG, PDH, PDI, PDK, PDL, PDM, PDN, PDP, PDQ, PDR, PDS, PDT, PDV, PDW, PDY, PEA, PEC, PED, PEE, PEF, PEG, PEH, PEI, PEK, PEL, PEM, PEN, PEP, PEQ, PER, PES, PET, PEV, PEW, PEY, PFA, PFC, PFD, PFE, PFF, PFG, PFH, PFI, PFK, PFL, PFM, PFN, PFP, PFQ, PFR, PFS, PFT, PFV, PFW, PFY, PGA, PGC, PGD, PGE, PGF, PGG, PGH, PGI, PGK, PGL, PGM, PGN, PGP, PGQ, PGR, PGS, PGT, PGV, PGW, PGY, PHA, PHC, PHD, PHE, PHF, PHG, PHH, PHI, PHK, PHL, PHM, PHN, PHP, PHQ, PHR, PHS, PHT, PHV, PHW, PHY, PIA, PIC, PID, PIE, PIF, PIG, PIH, PII, PIK, PIL, PIM, PIN, PIP, PIQ, PIR, PIS, PIT, PIV, PIW, PIY, PKA, PKC, PKD, PKE, PKF, PKG, PKH, PKI, PKK, PKL, PKM, PKN, PKP, PKQ, PKR, PKS, PKT, PKV, PKW, PKY, PLA, PLC, PLD, PLE, PLF, PLG, PLH, PLI, PLK, PLL, PLM, PLN, PLP, PLQ, PLR, PLS, PLT, PLV, PLW, PLY, PMA, PMC, PMD, PME, PMF, PMG, PMH, PMI, PMK, PML, PMM, PMN, PMP, PMQ, PMR, PMS, PMT, PMV, PMW, PMY, PNA, PNC, PND, PNE, PNF, PNG, PNH, PNI, PNK, PNL, PNM, PNN, PNP, PNQ, PNR, PNS, PNT, PNV, PNW, PNY, PPA, PPC, PPD, PPE, PPF, PPG, PPH, PPI, PPK, PPL, PPM, PPN, PPP, PPQ, PPR, PPS, PPT, PPV, PPW, PPY, PQA, PQC, PQD, PQE, PQF, PQG, PQH, PQI, PQK, PQL, PQM, PQN, PQP, PQQ, PQR, PQS, PQT, PQV, PQW, PQY, PRA, PRC, PRD, PRE, PRF, PRG, PRH, PRI, PRK, PRL, PRM, PRN, PRP, PRQ, PRR, PRS, PRT, PRV, PRW, PRY, PSA, PSC, PSD, PSE, PSF, PSG, PSH, PSI, PSK, PSL, PSM, PSN, PSP, PSQ, PSR, PSS, PST, PSV, PSW, PSY, PTA, PTC, PTD, PTE, PTF, PTG, PTH, PTI, PTK, PTL, PTM, PTN, PTP, PTQ, PTR, PTS, PTT, PTV, PTW, PTY, PVA, PVC, PVD, PVE, PVF, PVG, PVH, PVI, PVK, PVL, PVM, PVN, PVP, PVQ, PVR, PVS, PVT, PVV, PVW, PVY, PWA, PWC, PWD, PWE, PWF, PWG, PWH, PWI, PWK, PWL, PWM, PWN, PWP, PWQ, PWR, PWS, PWT, PWV, PWW, PWY, PYA, PYC, PYD, PYE, PYF, PYG, PYH, PYI, PYK, PYL, PYM, PYN, PYP, PYQ, PYR, PYS, PYT, PYV, PYW, PYY, QAA, QAC, QAD, QAE, QAF, QAG, QAH, QAI, QAK, QAL, QAM, QAN, QAP, QAQ, QAR, QAS, QAT, QAV, QAW, QAY, QCA, QCC, QCD, QCE, QCF, QCG, QCH, QCI, QCK, QCL, QCM, QCN, QCP, QCQ, QCR, QCS, QCT, QCV, QCW, QCY, QDA, QDC, QDD, QDE, QDF, QDG, QDH, QDI, QDK, QDL, QDM, QDN, QDP, QDQ, QDR, QDS, QDT, QDV, QDW, QDY, QEA, QEC, QED, QEE, QEF, QEG, QEH, QEI, QEK, QEL, QEM, QEN, QEP, QEQ, QER, QES, QET, QEV, QEW, QEY, QFA, QFC, QFD, QFE, QFF, QFG, QFH, QFI, QFK, QFL, QFM, QFN, QFP, QFQ, QFR, QFS, QFT, QFV, QFW, QFY, QGA, QGC, QGD, QGE, QGF, QGG, QGH, QGI, QGK, QGL, QGM, QGN, QGP, QGQ, QGR, QGS, QGT, QGV, QGW, QGY, QHA, QHC, QHD, QHE, QHF, QHG, QHH, QHI, QHK, QHL, QHM, QHN, QHP, QHQ, QHR, QHS, QHT, QHV, QHW, QHY, QIA, QIC, QID, QIE, QIF, QIG, QIH, QII, QIK, QIL, QIM, QIN, QIP, QIQ, QIR, QIS, QIT, QIV, QIW, QIY, QKA, QKC, QKD, QKE, QKF, QKG, QKH, QKI, QKK, QKL, QKM, QKN, QKP, QKQ, QKR, QKS, QKT, QKV, QKW, QKY, QLA, QLC, QLD, QLE, QLF, QLG, QLH, QLI, QLK, QLL, QLM, QLN, QLP, QLQ, QLR, QLS, QLT, QLV, QLW, QLY, QMA, QMC, QMD, QME, QMF, QMG, QMH, QMI, QMK, QML, QMM, QMN, QMP, QMQ, QMR, QMS, QMT, QMV, QMW, QMY, QNA, QNC, QND, QNE, QNF, QNG, QNH, QNI, QNK, QNL, QNM, QNN, QNP, QNQ, QNR, QNS, QNT, QNV, QNW, QNY, QPA, QPC, QPD, QPE, QPF, QPG, QPH, QPI, QPK, QPL, QPM, QPN, QPP, QPQ, QPR, QPS, QPT, QPV, QPW, QPY, QQA, QQC, QQD, QQE, QQF, QQG, QQH, QQI, QQK, QQL, QQM, QQN, QQP, QQQ, QQR, QQS, QQT, QQV, QQW, QQY, QRA, QRC, QRD, QRE, QRF, QRG, QRH, QRI, QRK, QRL, QRM, QRN, QRP, QRQ, QRR, QRS, QRT, QRV, QRW, QRY, QSA, QSC, QSD, QSE, QSF, QSG, QSH, QSI, QSK, QSL, QSM, QSN, QSP, QSQ, QSR, QSS, QST, QSV, QSW, QSY, QTA, QTC, QTD, QTE, QTF, QTG, QTH, QTI, QTK, QTL, QTM, QTN, QTP, QTQ, QTR, QTS, QTT, QTV, QTW, QTY, QVA, QVC, QVD, QVE, QVF, QVG, QVH, QVI, QVK, QVL, QVM, QVN, QVP, QVQ, QVR, QVS, QVT, QVV, QVW, QVY, QWA, QWC, QWD, QWE, QWF, QWG, QWH, QWI, QWK, QWL, QWM, QWN, QWP, QWQ, QWR, QWS, QWT, QWV, QWW, QWY, QYA, QYC, QYD, QYE, QYF, QYG, QYH, QYI, QYK, QYL, QYM, QYN, QYP, QYQ, QYR, QYS, QYT, QYV, QYW, QYY, RAA, RAC, RAD, RAE, RAF, RAG, RAH, RAI, RAK, RAL, RAM, RAN, RAP, RAQ, RAR, RAS, RAT, RAV, RAW, RAY, RCA, RCC, RCD, RCE, RCF, RCG, RCH, RCI, RCK, RCL, RCM, RCN, RCP, RCQ, RCR, RCS, RCT, RCV, RCW, RCY, RDA, RDC, RDD, RDE, RDF, RDG, RDH, RDI, RDK, RDL, RDM, RDN, RDP, RDQ, RDR, RDS, RDT, RDV, RDW, RDY, REA, REC, RED, REE, REF, REG, REH, REI, REK, REL, REM, REN, REP, REQ, RER, RES, RET, REV, REW, REY, RFA, RFC, RFD, RFE, RFF, RFG, RFH, RFI, RFK, RFL, RFM, RFN, RFP, RFQ, RFR, RFS, RFT, RFV, RFW, RFY, RGA, RGC, RGD, RGE, RGF, RGG, RGH, RGI, RGK, RGL, RGM, RGN, RGP, RGQ, RGR, RGS, RGT, RGV, RGW, RGY, RHA, RHC, RHD, RHE, RHF, RHG, RHH, RHI, RHK, RHL, RHM, RHN, RHP, RHQ, RHR, RHS, RHT, RHV, RHW, RHY, RIA, RIC, RID, RIE, RIF, RIG, RIH, RII, RIK, RIL, RIM, RIN, RIP, RIQ, RIR, RIS, RIT, RIV, RIW, RIY, RKA, RKC, RKD, RKE, RKF, RKG, RKH, RKI, RKK, RKL, RKM, RKN, RKP, RKQ, RKR, RKS, RKT, RKV, RKW, RKY, RLA, RLC, RLD, RLE, RLF, RLG, RLH, RLI, RLK, RLL, RLM, RLN, RLP, RLQ, RLR, RLS, RLT, RLV, RLW, RLY, RMA, RMC, RMD, RME, RMF, RMG, RMH, RMI, RMK, RML, RMM, RMN, RMP, RMQ, RMR, RMS, RMT, RMV, RMW, RMY, RNA, RNC, RND, RNE, RNF, RNG, RNH, RNI, RNK, RNL, RNM, RNN, RNP, RNQ, RNR, RNS, RNT, RNV, RNW, RNY, RPA, RPC, RPD, RPE, RPF, RPG, RPH, RPI, RPK, RPL, RPM, RPN, RPP, RPQ, RPR, RPS, RPT, RPV, RPW, RPY, RQA, RQC, RQD, RQE, RQF, RQG, RQH, RQI, RQK, RQL, RQM, RQN, RQP, RQQ, RQR, RQS, RQT, RQV, RQW, RQY, RRA, RRC, RRD, RRE, RRF, RRG, RRH, RRI, RRK, RRL, RRM, RRN, RRP, RRQ, RRR, RRS, RRT, RRV, RRW, RRY, RSA, RSC, RSD, RSE, RSF, RSG, RSH, RSI, RSK, RSL, RSM, RSN, RSP, RSQ, RSR, RSS, RST, RSV, RSW, RSY, RTA, RTC, RTD, RTE, RTF, RTG, RTH, RTI, RTK, RTL, RTM, RTN, RTP, RTQ, RTR, RTS, RTT, RTV, RTW, RTY, RVA, RVC, RVD, RVE, RVF, RVG, RVH, RVI, RVK, RVL, RVM, RVN, RVP, RVQ, RVR, RVS, RVT, RVV, RVW, RVY, RWA, RWC, RWD, RWE, RWF, RWG, RWH, RWI, RWK, RWL, RWM, RWN, RWP, RWQ, RWR, RWS, RWT, RWV, RWW, RWY, RYA, RYC, RYD, RYE, RYF, RYG, RYH, RYI, RYK, RYL, RYM, RYN, RYP, RYQ, RYR, RYS, RYT, RYV, RYW, RYY, SAA, SAC, SAD, SAE, SAF, SAG, SAH, SAI, SAK, SAL, SAM, SAN, SAP, SAQ, SAR, SAS, SAT, SAV, SAW, SAY, SCA, SCC, SCD, SCE, SCF, SCG, SCH, SCI, SCK, SCL, SCM, SCN, SCP, SCQ, SCR, SCS, SCT, SCV₅ SCW, SCY, SDA, SDC, SDD, SDE, SDF, SDG, SDH, SDI, SDK, SDL, SDM, SDN, SDP, SDQ, SDR, SDS, SDT, SDV, SDW, SDY, SEA, SEC, SED, SEE, SEF, SEG, SEH, SEI, SEK, SEL, SEM, SEN, SEP, SEQ, SER, SES, SET, SEV, SEW, SEY, SFA, SFC, SFD, SFE, SFF, SFG, SFH, SFI, SFK, SFL, SFM, SFN, SFP, SFQ, SFR, SFS, SFT, SFV, SFW, SFY, SGA, SGC, SGD, SGE, SGF, SGG, SGH, SGI, SGK, SGL, SGM, SGN, SGP, SGQ, SGR, SGS, SGT, SGV, SGW, SGY, SHA, SHC, SHD, SHE, SHF, SHG, SHH, SHI, SHK, SHL, SHM, SHN, SHP, SHQ, SHR, SHS, SHT, SHV, SHW, SHY, SIA, SIC, SID, SIE, SIF, SIG, SIH, SII, SIK, SIL, SIM, SIN, SIP, SIQ, SIR, SIS, SIT, SIV, SIW, SIY, SKA, SKC, SKD, SKE, SKF, SKG, SKH, SKI, SKK, SKL, SKM, SKN, SKP, SKQ, SKR, SKS, SKT, SKV, SKW, SKY, SLA, SLC, SLD, SLE, SLF, SLG, SLH, SLI, SLK, SLL, SLM, SLN, SLP, SLQ, SLR, SLS, SLT, SLV, SLW, SLY, SMA, SMC, SMD, SME, SMF, SMG, SMH, SMI, SMK, SML, SMM, SMN, SMP, SMQ, SMR, SMS, SMT, SMV, SMW, SMY, SNA, SNC, SND, SNE, SNF, SNG, SNH, SNI, SNK, SNL, SNM, SNN, SNP, SNQ, SNR, SNS, SNT, SNV, SNW, SNY, SPA, SPC, SPD, SPE, SPF, SPG, SPH, SPI, SPK, SPL, SPM, SPN, SPP, SPQ, SPR, SPS, SPT, SPV, SPW, SPY, SQA, SQC, SQD, SQE, SQF, SQG, SQH, SQI, SQK, SQL, SQM, SQN, SQP, SQQ, SQR, SQS, SQT, SQV, SQW, SQY, SRA, SRC, SRD, SRE, SRF, SRG, SRH, SRI, SRK, SRL, SRM, SRN, SRP, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, SSA, SSC, SSD, SSE, SSF, SSG, SSH, SSI, SSK, SSL, SSM, SSN, SSP, SSQ, SSR, SSS, SST, SSV, SSW, SSY, STA, STC, STD, STE, STF, STG, STH, STI, STK, STL, STM, STN, STP, STQ, STR, STS, STT, STV, STW, STY, SVA, SVC, SVD, SVE, SVF, SVG, SVH, SVI, SVK, SVL, SVM, SVN, SVP, SVQ, SVR, SVS, SVT, SVV, SVW, SVY, SWA, SWC, SWD, SWE, SWF, SWG, SWH, SWI, SWK, SWL, SWM, SWN, SWP, SWQ, SWR, SWS, SWT, SWV, SWW, SWY, SYA, SYC, SYD, SYE, SYF, SYG, SYH, SYI, SYK, SYL, SYM, SYN, SYP, SYQ, SYR, SYS, SYT, SYV, SYW, SYY, TAA, TAC, TAD, TAE, TAF, TAG, TAH, TAI, TAK, TAL, TAM, TAN, TAP, TAQ, TAR, TAS, TAT, TAV, TAW, TAY, TCA, TCC, TCD, TCE, TCF, TCG, TCH, TCI, TCK, TCL, TCM, TCN, TCP, TCQ, TCR, TCS, TCT, TCV, TCW, TCY, TDA, TDC, TDD, TDE, TDF, TDG, TDH, TDI, TDK, TDL, TDM, TDN, TDP, TDQ, TDR, TDS, TDT, TDV, TDW, TDY, TEA, TEC, TED, TEE, TEF, TEG, TEH, TEI, TEK, TEL, TEM, TEN, TEP, TEQ, TER, TES, TET, TEV, TEW, TEY, TFA, TFC, TFD, TFE, TFF, TFG, TFH, TFI, TFK, TFL, TFM, TFN, TFP, TFQ, TFR, TFS, TFT, TFV, TFW, TFY, TGA, TGC, TGD, TGE, TGF, TGG, TGH, TGI, TGK, TGL, TGM, TGN, TGP, TGQ, TGR, TGS, TGT, TGV, TGW, TGY, THA, THC, THD, THE, THF, THG, THH, THI, THK, THL, THM, THN, THP, THQ, THR, THS, THT, THV, THW, THY, TIA, TIC, TID, TIE, TIF, TIG, TIH, TII, TIK, TIL, TIM, TIN, TIP, TIQ, TIR, TIS, TIT, TIV, TIW, TIY, TKA, TKC, TKD, TKE, TKF, TKG, TKH, TKI, TKK, TKL, TKM, TKN, TKP, TKQ, TKR, TKS, TKT, TKV, TKW, TKY, TLA, TLC, TLD, TLE, TLF, TLG, TLH, TLI, TLK, TLL, TLM, TLN, TLP, TLQ, TLR, TLS, TLT, TLV, TLW, TLY, TMA, TMC, TMD, TME, TMF, TMG, TMH, TMI, TMK, TML, TMM, TMN, TMP, TMQ, TMR, TMS, TMT, TMV, TMW, TMY, TNA, TNC, TND, TNE, TNF, TNG, TNH, TNI, TNK, TNL, TNM, TNN, TNP, TNQ, TNR, TNS, TNT, TNV, TNW, TNY, TPA, TPC, TPD, TPE, TPF, TPG, TPH, TPI, TPK, TPL, TPM, TPN, TPP, TPQ, TPR, TPS, TPT, TPV, TPW, TPY, TQA, TQC, TQD, TQE, TQF, TQG, TQH, TQI, TQK, TQL, TQM, TQN, TQP, TQQ, TQR, TQS, TQT, TQV, TQW, TQY, TRA, TRC, TRD, TRE, TRF, TRG, TRH, TRI, TRK, TRL, TRM, TRN, TRP, TRQ, TRR, TRS, TRT, TRV, TRW, TRY, TSA, TSC, TSD, TSE, TSF, TSG, TSH, TSI, TSK, TSL, TSM, TSN, TSP, TSQ, TSR, TSS, TST, TSV, TSW, TSY, TTA, TTC, TTD, TTE, TTF, TTG, TTH, TTI, TTK, TTL, TTM, TTN, TTP, TTQ, TTR, TTS, TTT, TTV, TTW, TTY, TVA, TVC, TVD, TVE, TVF, TVG, TVH, TVI, TVK, TVL, TVM, TVN, TVP, TVQ, TVR, TVS, TVT, TVV, TVW, TVY, TWA, TWC, TWD, TWE, TWF, TWG, TWH, TWI, TWK, TWL, TWM, TWN, TWP, TWQ, TWR, TWS, TWT, TWV, TWW, TWY, TYA, TYC, TYD, TYE, TYF, TYG, TYH, TYI, TYK, TYL, TYM, TYN, TYP, TYQ, TYR, TYS, TYT, TYV, TYW, TYY, VAA, VAC, VAD, VAE, VAF, VAG, VAH, VAI, VAK, VAL, VAM, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VAW, VAY, VCA, VCC, VCD, VCE, VCF, VCG, VCH, VCI, VCK, VCL, VCM, VCN, VCP, VCQ, VCR, VCS, VCT, VCV, VCW, VCY, VDA, VDC, VDD, VDE, VDF, VDG, VDH, VDI, VDK, VDL, VDM, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VDW, VDY, VEA, VEC, VED, VEE, VEF, VEG, VEH, VEI, VEK, VEL, VEM, VEN, VEP, VEQ, VER, VES, VET, VEV, VEW, VEY, VFA, VFC, VFD, VFE, VFF, VFG, VFH, VFI, VFK, VFL, VFM, VFN, VFP, VFQ, VFR, VFS, VFT, VFV, VFW, VFY, VGA, VGC, VGD, VGE, VGF, VGG, VGH, VGI, VGK, VGL, VGM, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VGW, VGY, VHA, VHC, VHD, VHE, VHF, VHG, VHH, VHI, VHK, VHL, VHM, VHN, VHP, VHQ, VHR, VHS, VHT, VHV, VHW, VHY, VIA, VIC, VID, VIE, VIF, VIG, VIH, VII, VIK, VIL, VIM, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VIW, VIY, VKA, VKC, VKD, VKE, VKF, VKG, VKH, VKI, VKK, VKL, VKM, VKN, VKP, VKQ, VKR, VKS, VKT, VKV, VKW, VKY, VLA, VLC, VLD, VLE, VLF, VLG, VLH, VLI, VLK, VLL, VLM, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VLW, VLY, VMA, VMC, VMD, VME, VMF, VMG, VMH, VMI, VMK, VML, VMM, VMN, VMP, VMQ, VMR, VMS, VMT, VMV, VMW, VMY, VNA, VNC, VND, VNE, VNF, VNG, VNH, VNI, VNK, VNL, VNM, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VNW, VNY, VPA, VPC, VPD, VPE, VPF, VPG, VPH, VPI, VPK, VPL, VPM, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VPW, VPY, VQA, VQC, VQD, VQE, VQF, VQG, VQH, VQI, VQK, VQL, VQM, VQN, VQP, VQQ, VQR, VQS, VQT, VQV, VQW, VQY, VRA, VRC, VRD, VRE, VRF, VRG, VRH, VRI, VRK, VRL, VRM, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VRW, VRY, VSA, VSC, VSD, VSE, VSF, VSG, VSH, VSI, VSK, VSL, VSM, VSN, VSP, VSQ, VSR, VSS, VST, VSV, VSW, VSY, VTA, VTC, VTD, VTE, VTF, VTG, VTH, VTI, VTK, VTL, VTM, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, VTW, VTY, VVA, VVC, WD, WE, WF, WG, VVH, WI, WK, VVL, VVM, VVN, VVP, VVQ, VVR, VVS, WT, VW, VVW, VVY, VWA, VWC, VWD, VWE, VWF, VWG, VWH, VWI, VWK, VWL, VWM, VWN, VWP, VWQ, VWR, VWS, VWT, VWV, VWW, VWY, VYA, VYC, VYD, VYE, VYF, VYG, VYH, VYI, VYK, VYL, VYM, VYN, VYP, VYQ, VYR, VYS, VYT, VYV, VYW, VYY, WAA, WAC, WAD, WAE, WAF, WAG, WAH, WAI, WAK, WAL, WAM, WAN, WAP, WAQ, WAR, WAS, WAT, WAV, WAW, WAY, WCA, WCC, WCD, WCE, WCF, WCG, WCH, WCI, WCK, WCL, WCM, WCN, WCP, WCQ, WCR, WCS, WCT, WCV, WCW, WCY, WDA, WDC, WDD, WDE, WDF, WDG, WDH, WDI, WDK, WDL, WDM, WDN, WDP, WDQ, WDR, WDS, WDT, WDV, WDW, WDY, WEA, WEC, WED, WEE, WEF, WEG, WEH, WEI, WEK, WEL, WEM, WEN, WEP, WEQ, WER, WES, WET, WEV, WEW, WEY, WFA, WFC, WFD, WFE, WFF, WFG, WFH, WFI, WFK, WFL, WFM, WFN, WFP, WFQ, WFR, WFS, WFT, WFV, WFW, WFY, WGA, WGC, WGD, WGE, WGF, WGG, WGH, WGI, WGK, WGL, WGM, WGN, WGP, WGQ, WGR, WGS, WGT, WGV, WGW, WGY, WHA, WHC, WHD, WHE, WHF, WHG, WHH, WHI, WHK, WHL, WHM, WHN, WHP, WHQ, WHR, WHS, WHT, WHV, WHW, WHY, WIA, WIC, WID, WIE, WIF, WIG, WIH, WII, WIK, WIL, WIM, WIN, WIP, WIQ, WIR, WIS, WIT, WIV, WIW, WIY, WKA, WKC, WKD, WKE, WKF, WKG, WKH, WKI, WKK, WKL, WKM, WKN, WKP, WKQ, WKR, WKS, WKT, WKV, WKW, WKY, WLA, WLC, WLD, WLE, WLF, WLG, WLH, WLI, WLK, WLL, WLM, WLN, WLP, WLQ, WLR, WLS, WLT, WLV, WLW, WLY, WMA, WMC, WMD, WME, WMF, WMG, WMH, WMI, WMK, WML, WMM, WMN, WMP, WMQ, WMR, WMS, WMT, WMV, WMW, WMY, WNA, WNC, WND, WNE, WNF, WNG, WNH, WNI, WNK, WNL, WNM, WNN, WNP, WNQ, WNR, WNS, WNT, WNV, WNW, WNY, WPA, WPC, WPD, WPE, WPF, WPG, WPH, WPI, WPK, WPL, WPM, WPN, WPP, WPQ, WPR, WPS, WPT, WPV, WPW, WPY, WQA, WQC, WQD, WQE, WQF, WQG, WQH, WQI, WQK, WQL, WQM, WQN, WQP, WQQ, WQR, WQS, WQT, WQV, WQW, WQY, WRA, WRC, WRD, WRE, WRF, WRG, WRH, WRI, WRK, WRL, WRM, WRN, WRP, WRQ, WRR, WRS, WRT, WRV, WRW, WRY, WSA, WSC, WSD, WSE, WSF, WSG, WSH, WSI, WSK, WSL, WSM, WSN, WSP, WSQ, WSR, WSS, WST, WSV, WSW, WSY, WTA, WTC, WTD, WTE, WTF, WTG, WTH, WTI, WTK, WTL, WTM, WTN, WTP, WTQ, WTR, WTS, WTT, WTV, WTW, WTY, WVA, WVC, WVD, WVE, WVF, WVG, WVH, WVI, WVK, WVL, WVM, WVN, WVP, WVQ, WVR, WVS, WVT, WW, WVW, WVY, WWA, WWC, WWD, WWE, WWF, WWG, WWH, WWI, WWK, WWL, WWM, WWN, WWP, WWQ, WWR, WWS, WWT, WWV, WWW, WWY, WYA, WYC, WYD, WYE, WYF, WYG, WYH, WYI, WYK, WYL, WYM, WYN, WYP, WYQ, WYR, WYS, WYT, WYV, WYW, WYY, YAA, YAC, YAD, YAE, YAF, YAG, YAH, YAI, YAK, YAL, YAM, YAN, YAP, YAQ, YAR, YAS, YAT, YAV, YAW, YAY, YCA, YCC, YCD, YCE, YCF, YCG, YCH, YCI, YCK, YCL, YCM, YCN, YCP, YCQ, YCR, YCS, YCT, YCV, YCW, YCY, YDA, YDC, YDD, YDE, YDF, YDG, YDH, YDI, YDK, YDL, YDM, YDN, YDP, YDQ, YDR, YDS, YDT, YDV, YDW, YDY, YEA, YEC, YED, YEE, YEF, YEG, YEH, YEI, YEK, YEL, YEM, YEN, YEP, YEQ, YER, YES, YET, YEV, YEW, YEY, YFA, YFC, YFD, YFE, YFF, YFG, YFH, YFI, YFK, YFL, YFM, YFN, YFP, YFQ, YFR, YFS, YFT; YFV, YFW, YFY, YGA, YGC, YGD, YGE, YGF, YGG, YGH, YGI, YGK, YGL, YGM, YGN, YGP, YGQ, YGR, YGS, YGT, YGV, YGW, YGY, YHA, YHC, YHD, YHE, YHF, YHG, YHH, YHI, YHK, YHL, YHM, YHN, YHP, YHQ, YHR, YHS, YHT, YHV, YHW, YHY, YIA, YIC, YID, YIE, YIF, YIG, YIH, YII, YIK, YIL, YIM, YIN, YIP, YIQ, YIR, YIS, YIT, YIV, YIW, YIY, YKA, YKC, YKD, YKE, YKF, YKG, YKH, YKI, YKK, YKL, YKM, YKN, YKP, YKQ, YKR, YKS, YKT, YKV, YKW, YKY, YLA, YLC, YLD, YLE, YLF, YLG, YLH, YLI, YLK, YLL, YLM, YLN, YLP, YLQ, YLR, YLS, YLT, YLV, YLW, YLY, YMA, YMC, YMD, YME, YMF, YMG, YMH, YMI, YMK, YML, YMM, YMN, YMP, YMQ, YMR, YMS, YMT, YMV, YMW, YMY, YNA, YNC, YND, YNE, YNF, YNG, YNH, YNI, YNK, YNL, YNM, YNN, YNP, YNQ, YNR, YNS, YNT, YNV, YNW, YNY, YPA, YPC, YPD, YPE, YPF, YPG, YPH, YPI, YPK, YPL, YPM, YPN, YPP, YPQ, YPR, YPS, YPT, YPV, YPW, YPY, YQA, YQC, YQD, YQE, YQF, YQG, YQH, YQI, YQK, YQL, YQM, YQN, YQP, YQQ, YQR, YQS, YQT, YQV, YQW, YQY, YRA, YRC, YRD, YRE, YRF, YRG, YRH, YRI, YRK, YRL, YRM, YRN, YRP, YRQ, YRR, YRS, YRT, YRV, YRW, YRY, YSA, YSC, YSD, YSE, YSF, YSG, YSH, YSI, YSK, YSL, YSM, YSN, YSP, YSQ, YSR, YSS, YST, YSV, YSW, YSY, YTA, YTC, YTD, YTE, YTF, YTG, YTH, YTI, YTK, YTL, YTM, YTN, YTP, YTQ, YTR, YTS, YTT, YTV, YTW, YTY, YVA, YVC, YVD, YVE, YVF, YVG, YVH, YVI, YVK, YVL, YVM, YVN, YVP, YVQ, YVR, YVS, YVT, YVV, YVW, YVY, YWA, YWC, YWD, YWE, YWF, YWG, YWH, YWI, YWK, YWL, YWM, YWN, YWP, YWQ, YWR, YWS, YWT, YWV, YWW, YWY, YYA, YYC, YYD, YYE, YYF, YYG, YYH, YYI, YYK, YYL, YYM, YYN, YYP, YYQ, YYR, YYS, YYT, YYV, YYW, YYY.

[090] Described below in Section 6 is an experiment wherein five oligopeptides that exist within the amino acid sequence of a target, i.e., cathepsin D, were chosen to develop aptamers. As described above, because the oligopeptides may also exist in other proteins, the aptamers developed in Section 6 that bind Leu-Ala-Ser (LAS), Asp- Gly-Ile (DGI), Gly-Glu-Leu (GEL) and Lys-Ala-Ile (KAI) specifically can also be used to detect other proteins which have one or more of the oligopeptides. A search revealed that a number of human proteins that are of potential diagnostic or proteomic interest (see Table 1) which carry the same set of four oligopeptides in their amino acid sequences. These human proteins can be detected using the existing aptamers of the invention.

[091] Table 1 : Human proteins that can be detected using aptamers that bind the tripeptides LAS, DGI, GEL and KAI.

5.3 METHODS OF DETECTION

[092] The present invention relates to a method for specifically detecting and/or measuring a target (i.e., molecule of interest being detected or measured in an analytical procedure) using a plurality of aptamers, wherein the aptamers are selected according to their binding affinities to oligopeptides and wherein the oligopeptides are present as segments of a polypeptide in the target. The target can be a non-covalent or covalent association of multiple molecules wherein at least one molecule is a protein which comprises the oligopeptide segments that are recognized by the aptamers. Many types of targets are contemplated and are discussed in details in the following section. [093] In one embodiment, the present invention is a method for detecting or measuring a target comprising contacting, in one or more steps, a plurality of aptamers with a sample containing the target under conditions that allow the aptamers to bind the target; and detecting or measuring the aptamers that are bound to the target; wherein the target comprises a plurality of oligopeptide epitopes to which the plurality of aptamers bind specifically, respectively. Each oligopeptide epitope of the protein present in the target consists essentially of an oligopeptide segment, wherein the sequence of the oligopeptide is used to select the aptamer that specifically recognizes and binds the oligopeptide epitope.

[0941 The methods of the invention employ a set of aptamers, preferably at least three different aptamers, more preferably four or five different aptamers. In a specific embodiment, four aptamers are used, each exhibiting specificity to a tripeptide epitope. In another specific embodiment, five different aptamers are used, each exhibiting specificity to a tripeptide epitope. In yet another embodiment, four or five aptamers are used, wherein the aptamers each independently exhibits specificity to a tripeptide, a tetrapeptide, a pentapetide, a hexapeptide, a heptapeptide or an octapeptide epitope. In yet another embodiment, four or five aptamers are used, wherein at least three of the aptamers each independently exhibits specificity to a tripeptide, a tetrapeptide, a pentapetide, a hexapeptide, a heptapeptide or an octapeptide, and at least one aptamer exhibits specificity to a polypeptide or a non-polypeptide molecule. [095] According to the invention, in a sample comprising a plurality of molecular entities, only the desired target would consists of all the oligopeptide segments (or epitopes) that are bound specifically by the respective aptamers. The collective binding of the aptamers to their respective epitopes on a target is exploited to allow detection of the target with high specificity. The binding of the aptamers can be detected by nucleic acid amplification, nucleic acid staining, hybridization assays, or a combination of the foregoing. In nucleic acid amplification, a reporter template is generated and then amplified to produce reporter nucleic acids which are detected and measured. In a hybridization assay, a labeled nucleic acid probe hybridizes specifically to an aptamer, a reporter template or a reporter nucleic acid. In a preferred embodiment, the proximity of the aptamers when bound to their respective epitopes on a target is exploited to generate an ampHfiable signal when all the aptamers are bound to their respective sites concurrently.

[096] Many targets exist in different conformations and some of these targets assume certain conformations only when bound covalently or non-covalently to a certain second molecule. Such allosteric changes are commonly found among many proteins of interest. The present invention can take advantage of such allosteric changes to indicate the status or activity of the target protein. By choosing a set of aptamers that includes at least one aptamer that binds specifically to an epitope on the target which epitope is revealed or masked by the allosteric change, and detecting/or measuring the concurrent binding of all the aptamers within the set, the allosteric changes of the target can be detected or measured. For example, if the allosteric change results in the masking of an epitope on a target to which one of the aptamers in the chosen aptamer set is expected to bind, then the lack of concurrent binding of all the aptamers indicate the allosteric change occurred in the target. In this example, the skilled person will recognize the benefit of using a control reaction to determine the presence of the target, or the total amount of the target present regardless of allosteric status. This can be accomplished by using an alternative aptamer with a specificity towards an oligopeptide segment present in the target wherein the binding of the aptamer is not sensitive to allosteric change. [097] In another embodiment where the target comprises more than one protein component (i.e., a first protein component, a second protein component and so on), and one or more aptamers that bind a second protein component is obtainable or available, the one or more aptamers that bind a second protein component can be used in combination with the set of aptamers that bind the oligopeptide segments of the first protein component. The detection means of the invention can be adapted to report the concurrent binding of the aptamer(s) that bind the first protein component and the aptamers of the second protein component, so as to detect the target. By replacing the aptamer(s) that bind(s) the second protein component with an aptamer that bind to an epitope that is revealed on a first protein component when not complexed with the second component, the uncomplexed first component can be detected. The use of such combinations of aptamers allows the detection of formation or dissociation of multiprotein complexes and permits the measurement of non-complexed and/or complexed form of a target.

[098] In a related embodiment, where the target comprises a non-protein component and an aptamer that binds this non-protein component is obtainable or available, this non-protein binding aptamer can be used in combination with the oligopeptide-binding aptamers of the invention. The detection means of the invention can be adapted to report the concurrent binding of this non-protein binding aptamer and the oligopeptide-binding aptamers to the components of the target, so as to detect the target. Aptamers have been reported to bind a large variety of molecules, including but not limited to, carbohydrates, lipids, natural products, small organic molecules, many of which forms a molecular complex with a protein. Such non-peptide binding aptamers can include additional nucleic acids or other accessory molecules to participate in the detection means of the invention. Accordingly, the methods of the invention can be used to detect such protein-containing hybrid targets.

[099] In another related embodiment, the methods of the invention can be exploited to detect a modified or variant form of a target which comprises (i) the protein component of the target without the non-protein component(s); or (ii) the first protein component of the target without other protein component(s). Accordingly, the invention provides methods to detect protein modification, to distinguish between unmodified forms and one or more different modified forms of the protein, and to measure the absolute and/or relative quantities of the unmodified and modified forms of the target. In such an embodiment, an aptamer that bind specifically to a modified site or a site of variation on the target is used. This aptamer is used with other aptamers in a first set of aptamers to detect or measure the modified form of the target. In a second set, all the aptamers used in the binding reaction are not sensitive to modification or variation of the target , and can thus measure the total amount of the target regardless of modification. The difference between the total amount of the target and the unmodified amount yields the modified amount of the target. The first set, second set and other alternative sets of aptamers can share a majority of the aptamers except those that differentially bind the modified and non-modified forms of the protein.

[0100] Samples, potentially containing a target that are useful for the assays of the present invention include, but are not limited to, an aqueous solution, soil, food, food ingredients, food residue, fecal matter, plant or animal cells, tissue or tissue extract, tissue culture, tissue culture extract or tissue culture medium. In one embodiment, the sample to be assayed for the presence and/or amount of a target is a patient sample. In a further embodiment, the patient sample is a biological fluid such as, but not limited to, blood, serum, lymph, plasma, milk, urine, saliva, pleural effusions, synovial fluid, spinal fluid, tissue infiltrations or tumor infiltrates. In another embodiment, the patient sample is a tissue or tissue extract, hi yet another embodiment, the patient sample is fecal matter. In a specific embodiment, the sample tissue is obtained from a biopsy. In various embodiments, the method of the invention further comprise collecting a sample from the subject, and/or processing the sample such that the target in the sample is more amenable to detection by the methods of the invention.

[0101] Aptamers of different oligopeptide specificities can be brought into contact with a sample simultaneously. Alternatively, individual or subsets of aptamers can be contacted with the sample sequentially. The aptamers can be contacted with the sample in any sequence prior to detection of binding. The term "contacting" or "bringing into contact" is used herein interchangeably with the following: introducing into, combined with, added to, mixed with, passed over, incubated with, injected into, flowed over, etc.

[0102] In various embodiments, one or more steps are included prior to the contacting step to render the target more susceptible or accessible to binding by the aptamers. This pretreatment may result in a change in the following non-limiting examples of reactions conditions: pH, salt concentration, concentration of metal ion(s), temperature, detergent concentration, and sulfhydryl agents. The pretreatment steps may include a step that results in a change in the secondary and/or tertiary structure of the target including the unwinding or denaturation of the target in a sample. The pretreatment may further include a renaturation step, before or after one or more aptamers have been brought into contact with the sample. In yet another further embodiment, one or more steps are added prior to the contacting step in order to remove undesirable substances (e.g., cells, cell debris, organic/inorganic particulate matters) and molecules (e.g., soluble and/or insoluble contaminants) from the sample. [01031 1° certain embodiments, the excess aptamers or unbound aptamers are separated from the aptamers that are bound to certain entities present in the sample prior to the detection/measurement step. The method can be a homogenous method or a heterogenous method. In various embodiments, the binding of each of the aptamers to the molecular entities in the sample can be determined separately or concurrently. In a preferred embodiment, the plurality of aptamers are allowed to contact the sample and bind to the target at the same time. In another embodiment, the plurality of aptamers is allowed to contact the sample and bind to the target under the same conditions, hi yet another embodiment, the binding of the plurality of aptamers to the respective epitopes on the target are detected or measured at the same time. Depending on the type of label or reporter used in the detection method for each of the aptamers, the binding of the aptamers to a target can be measured separately or concurrently. [0104] To aid in detection and quantitation of bound aptamers (hybridization assays) or reporting nucleic acids amplified (nucleic acid amplification) using methods well known in the art, labels can be directly incorporated into aptamers, probes, or amplified reporter nucleic acids. Labels can also be chemically coupled to aptamers or probes.

[0105] A nucleic acid probe as used herein refers to an oligonucleotide which binds through complementary base pairing to a subsequence on the aptamer, a reporter template or a reporter nucleic acid. A nucleic acid probe is complementary to a subsequence when it will anneal only to a single desired position on that aptamer, reporter template, or reporter nucleic acid under conditions determined as described below. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. It will be understood by those of skill that minor mismatches can be accommodated by reducing the stringency of the hybridization media. Hybridization assays are well known in the art and include but is not limited to Southern blotting, Northern blotting, dot blotting, etc., wherein a labeled nucleic acid probe is brought into contact with the aptamer, reporter template or reporter nucleic acid that is immobilized on a solid phase. The methods of the invention contemplate the use of hybridization assays for detecting the presence of an aptamer, a reporter template or a reporter nucleic acid. For discussions of nucleic acid probe design and annealing conditions, see, e.g., Sambrook, et at., Molecular Cloning: A Laboratory Manual (2nd Ed., VoIs. 1-3, Cold Spring Harbor Laboratory (1989)), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques (Berger and Kimmel (eds.), San Diego: Academic Press, Inc. (1987)), or Current Protocols in Molecular Biology, (Ausubel, et al. (eds.), Greene Publishing and Wiley-Interscience, New York (1987), all of which are incorporated herein by reference.

[0106] As used herein, a label is any molecule that can be associated with an aptamer, a probe, or amplified reporter nucleic acid, directly or indirectly, and which results in a measurable, detectable signal, either directly or indirectly. Many such labels for incorporation into nucleic acids or coupling to nucleic acid or antibody probes are known to those of skill in the art. Examples of labels suitable for use in the detection means of the invention are radioactive isotopes, fluorescent molecules, phosphorescent molecules, enzymes, antibodies, and ligands. Methods for detecting and measuring signals generated by labels are also known to those of skill in the art. For example, radioactive isotopes can be detected by scintillation counting or direct visualization; fluorescent molecules can be detected with fluorescent spectrophotometers; phosphorescent molecules can be detected with a spectrophotometer or directly visualized with a camera; enzymes can be detected by detection or visualization of the product of a reaction catalyzed by the enzyme; antibodies can be detected by detecting a secondary label coupled to the antibody. Such methods can be used directly in the disclosed method of detection and amplification. Accordingly, detection or measurement can be performed by autoradiography, phosphoimager analysis, fluorometry, spectrofluorometry, luminescence measurement, colorimetric procedures, or absorbance measurement.

[0107] Examples of suitable fluorescent labels include fluorescein, 5,6- carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3-diazol-4-yl (NBD), coumarin, dansyl chloride, and rhodamine. Preferred fluorescent labels are fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester) and rhodamine (5,-tetramethyl rhodamine). These can be obtained from a variety of commercial sources, including Molecular Probes, Eugene, OR and Research Organics, Cleveland, Ohio. [0108] Speicher et al. (1996, Nature Genet. 12:368-375, which is incorporated herein by reference in its entirety) describes a set of fluors and corresponding optical filters spaced across the spectral interval 350-770 run that give a high degree of discrimination between all possible fluor pairs. This fluor set, which is preferred for the methods of the invention, consists of 4'-6-diamidino-2-phenylinodole (DAPI), fluorescein (FITC), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Any subset of this preferred set can also be used where fewer aptamers are required. The absorption and emission maxima, respectively, for these fluors are: DAPI (350 run; 456 mm), FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 mm), Cy5 (652 nm; 672 mm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm). The excitation and emission spectra, extinction coefficients and quantum yield of these fluors are described by Ernst et al., Cytometry 10:3-10 (1989), Mujumdar et al., Cytometry 10:11-19 (1989), Yu, Nucleic Acids Res. 22:3226-3232 (1994), and Waggoner, Meth. Enzymology 246:362- 373 (1995). These fluors can all be excited with a 75 W Xenon arc. [0109] Labeled nucleotides are preferred forms of label since they can be directly incorporated into the products of nucleic acid amplification during synthesis. Examples of labels that can be incorporated into amplified DNA or RNA include nucleotide analogs such as BrdUrd (Hoy and Schimke, Mutation Research 290:217-230 (1993)), BrUTP (Wansick et al., J. Cell Biology 122:283-293 (1993)) and nucleotides modified with biotin (Langer et al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or with suitable haptens such as digoxygenin (Kerkhof, Anal. Biochem. 205:359-364 (1992)). Suitable fluorescence-labeled nucleotides are fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP (Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred nucleotide analog label for DNA is BrdUrd (BUDR triphosphate, Sigma), and a preferred nucleotide analog label for RNA is Biotin- 16-uridine-5 '-triphosphate (Biotin- 16-dUTP, Boehringher Mannheim).

[0110] Labels that are incorporated into amplified nucleic acid, such as biotin, can be subsequently detected using sensitive methods well-known in the art. For example, biotin can be detected using streptavidin-alkaline phosphatase conjugate (Tropix, Inc.), which is bound to the biotin and subsequently detected by chemiluminescence of suitable substrates (for example, chemiluminescent substrate CSPD, Tropix, Inc.). A preferred label for use in detection of amplified RNA is acridinium-ester-labeled DNA probe (GenProbe, Inc., as described by Arnold et al., Clinical Chemistry 35:1588-1594 (1989)). An acridinium-ester-labeled detection probe permits the detection of amplified RNA without washing because unhybridized probe can be destroyed with alkali.

[0111] A solid phase can be used in the detection methods of the invention. In one embodiment, one or more aptamer(s) from the set of aptamers that bind specifically to oligopeptides present on the target, are associated a solid phase. A solid phase may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. A preferred form of solid phase is a microtiter plate, such as a standard 96- well microtiter plate or a high throughput type 384- well plate. Another preferred form of solid phase is an array or a microarray to which one or more different aptamers have been immobilized in an array, grid, or other organized pattern.

[0112] Solid phase for use in the methods can include any solid material to which oligonucleotides can be coupled and that enables aptamer-target interaction. This includes materials such as acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids. A solid phase can have any useful form including thin films or membranes, beads, bottles, dishes, wells, fibers, woven fibers, shaped polymers, particles, microparticles and nanoparticles. A preferred form for a solid phase is a microtiter plate. Those skilled in the art will know many other suitable solid phases, or will be able to ascertain the same by use of routine experimentation.

[0113] An aptamer that binds specifically an epitope of a target can be immobilized on a solid phase to allow capture of the target on the solid phase. Such capture provides a convenient means of washing away reaction components that might interfere with subsequent detection steps. By attaching different aptamers to different predetermined addressable regions of a solid phase, different targets can be captured at the predetermiend locations on the solid phase. For example, in a microtiter plate multiplex assay, aptamers specific for up to 384 different oligopeptide segments can be immobilized on a 384-well microtiter plate, each in a different well. Capture will occur only in those wells where one or more protein(s) comprise the corresponding oligopeptide segment to which the respective aptamer recognize and bind. Once the target is captured, binding of the other aptamers of the set can be carried out and the binding of the aptamers detected. Optionally, the immobilized aptamer can be used in combination in the detection. Alternatively, prior to contacting the sample with the solid phase (with an immobilized aptamer), a target can be brought first into contact with the other aptamers in the set, and then allowed to interact with the aptamer on the solid phase. Those skilled in the art will recognize that the methodology described herein is similar to a sandwich-type immunoassay, and those techniques, manipulations and variations thereof in binding, washing, and detecting the binding can be adopted in the methods of the invention.

[0114] In one specific embodiment, the capturing of a target on a solid phase with one aptamer that binds an epitope on the target, followed by the detection of one or more signal (s) or reporter molecule(s) after unbound materials are washed away, allows the specific detection of the target. The invention also provides detection means that involves the use of a combination of labels that either fluoresce at different wavelengths or are colored differently. One of the advantages of fluorescence for the detection of hybridization probes is that several aptamers can be visualized simultaneously. The presence of all the labels on the solid phase indicates that all the aptamers that are labeled are bound to a molecular entity that is present on the solid phase. If the solid phase is a particle, the particles can be separated from the solution phase by a variety of methods, such as centrifugation, filtration, magnetization, etc. In a non-limiting example, a set of four aptamers are used to bind specifically to oligopeptides present in a target; one of the four aptamers is immobilized onto a solid phase, while the other three aptamers are labeled and allowed to bind the target in solution phase. Upon capture of the target on the solid phase by the immobilized aptamer, aptamers that are not bound to the target as well as non-target molecules that happen to bind the labeled aptamers but do not bind the immobilized aptamer are washed away. Non-target molecules that are bound non-specifically to the immobilized aptamer do not bind the other labeled aptamers and are thus not detectable even they remain on the solid phase. Only the target that can bind to the immobilized aptamer and bind to the other three labeled aptamers are visualized or detected on the solid phase. The detection of all three labels in a location indicate the presence of the target. Depending on the complexity of the sample and the desired level of specificity, this method can be carried out using a minimum of two aptamers, one for immobilization and one labeled for detection of the target.

[0115] Methods for immobilization of oligonucleotides to solid phase substrates are well established. Oligonucleotides can be coupled to substrates using established coupling methods. For example, suitable attachment methods are described by Pease et al., Proc. Natl. Acad. Sci. USA 91(11):5022-5026 (1994), and Khrapko et al., MoI Biol (Mosk) (USSR) 25:718-730 (1991). A method for immobilization of 3'-amine oligonucleotides on casein-coated slides is described by Stimpson et al., Proc. Natl. Acad. Sci. USA 92:6379-6383 (1995). A preferred method of attaching oligonucleotides to solid-state substrates is described by Guo et al., Nucleic Acids Res. 22:5456-5465 (1994).

[0116] The invention also provides a multiplex assay based on the method described above wherein the solid phase is an addressable location on a substrate. In a multiplexed assay, each target is detected by its own set of aptamers. In each set, one aptamer is immobilized to an addressable location, and the other aptamers are each labeled by a different dye. Thus, each addressable location contains an immobilized aptamer that can bind specifically to an oligopeptide segment present on a particular target, wherein the oligopeptide segment serves as a unique identifier for the target in the sample. The addressable location is dedicated to the detection of one target. The other aptamers in the set, each labeled by a different dye, are in solution phase and are allowed to bind to the target. Based on the same principle, the presence of all the labels at a location on the solid phase indicates that all the aptamers that are labeled are bound to the target that is present in the location. Although there is a limited number of dyes available for labeling, the aptamers in solution phase that belong to different sets for different targets can be labeled by the same set of dyes. The spatial separation of different locations allow separate detection and identification of different targets even the same set of dyes are used. The address of the location corresponds to the identity of the target to be detected or measured.

[0117] In one embodiment, the invention provides detection methods that exploits the simultaneous and proximate binding of a set of aptamers to oligopeptide segments that are present on a target. This aspect of the invention includes molecular interactions that are dependent on the proximity of the aptamers bound concurrently to a target. The interactions result in the formation of a reporter template that can be amplified by any nucleic acid amplification methods known in the art. In various embodiments, the methods include ligation reactions during the formation of a reporter template, see, for example, Fredriksson et al., Nature Biotechnol. 2002, 20:473-477, which is incorporated herein by reference in its entirety.

[0118] In various embodiments, the ends of the aptamers are extended and comprise a linking region which comprises a nucleotide sequence that is complementary to and can thus hybridize with its counterpart sequence that is present on another aptamer or a connector oligonucleotide. Each aptamer in a set has a neighboring aptamer which can be (i) directly connected by hybridization of the complementary linking regions, or (ii) indirectly connected via a connector oligonucleotide which comprises complementary sequences to each of the linking regions of a pair of neighboring aptamers. When each of the aptamers in the set for a target binds to the target, the free ends of the aptamers which comprise the linking regions and that are not involved in binding the respective epitopes are brought sufficiently close to each other and hybridize (i) together to form a portion of a reporter template or (ii) to the regions of a connector which comprises sequences that are complementary to the respective linking regions of the neighboring aptamers. The connector is an oligonucleotide that comprises separate regions, wherein each region comprises a nucleotide sequence that is complementary to the linking region of an aptamer. In one specific embodiment, one of the two complementary linking regions of a connector is located at the 5' end or 3' end of the connector. In another specific embodiment, the two complementary linking regions of a connector are located one at the 5' end and the other at the 3' end of a connector. In another specific embodiment, the connector comprises 5' and 3' end sequences adjacent to the complementary linking regions, wherein these end sequences do not form base-pairing with the aptamers, thus reducing ligation-independent amplification products which may arise from spurious priming. A connector is required for each pair of neighboring aptamers. In a specific embodiment, each pair of neighboring aptamers is linked by a connector with different nucleotide sequences. A skilled person in the art would know the procedures that can be used, with routine experimentation, (i) to design a connector that will minimize unintended cross- hybridization to other nucleic acid molecules in the reaction; (ii) to design a connector that will minimize unintended self-hybridization; and (iii) to titrate the optimal concentration of connectors in a ligation reaction.

[0119] According to the invention, the target in a sample thus acts to promote the joining of the different aptamers that are bound to the target to form a reporter template. In one non-limiting example, to form a linear reporter template using a set of aptamers comprising four different aptamers, aptamerl and aptamer2 can be joined by connector 1; aptamer2 and aptamer3 can be joined by connector2; and aptamer3 and aptamer4 can be joined by connector3. In another non-limiting example, to form a circular reporter template using a set of aptamers comprising five different aptamers, aptamerl and aptamer2 can be joined by connectorl; aptamer2 and aptamer3 can be joined by connector2; aptamer3 and aptamer4 can be joined by connector3; aptamer4 and aptamer5 can be joined by connector4; and aptamer5 and aptamerl can be joined by connector5. The joining of the aptamers and/or the joining of the aptamers with the connectors can be mediated by one or more ligation reactions. The ligation of each neighboring pairs of aptamers with or without using a connector can be carried out sequentially, in groups of pairs, or simultaneously in the same reaction. [0120] Any DNA ligase is suitable for use in the methods described above.

Preferred ligases are those that preferentially form phosphodiester bonds at nicks in double-stranded DNA. That is, ligases that fail to ligate the free ends of single-stranded DNA at a significant rate are preferred. Thermostable ligases are especially preferred. Many suitable ligases are known, such as T4 DNA ligase (Davis et al., Advanced Bacterial Genetics— A Manual for Genetic Engineering (Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1980)), E. coli DNA ligase (Panasnko et al., J. Biol. Chem. 253:4590-4592 (1978)), AMPLIGASE™ (Kalin et al., Mutat. Res., 283(2): 119-123 (1992); Winn-Deen et al., MoI Cell Probes (England) 7(3):179-186 (1993)), Taq DNA ligase (Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)), Thermus thermophilus DNA ligase (Abbott Laboratories), Thermus scotoductus DNA ligase and Rhodothermus marinus DNA ligase (Thorbjarnardottir et al., Gene 151:177-180 (1995)). T4 DNA ligase is preferred for ligations involving RNA target sequences due to its ability to ligate DNA ends involved in DNA:RNA hybrids (Hsuih et al., Quantitative detection of HCV RNA using novel ligation-dependent polymerase chain reaction, American Association for the Study of Liver Diseases (Chicago, 111., Nov. 3-7, 1995)). [0121] In one embodiment, the reporter template is a linear molecule. In another embodiment, the reporter template is a circular molecule which can be formed by the hybridization and/or ligation of the free ends of a linear template. In yet another embodiment, the reporter template is a circular molecule which has been cleaved by a restriction enzyme to form a linear reporter template. In various embodiments, single stranded portions (including gaps) of a reporter template can be converted into double stranded form by treatment with a nucleic acid polymerase. In various embodiments, a DNA reporter template can be used to make a RNA reporter template or RNA reporters by a RNA polymerase; a RNA reporter template can be used to make a DNA reporter templete or DNA reporters by a reverse transcriptase. A description of the various polymerases that can be used is provided below. According to the topology and chemistry (i.e., DNA or RNA, with or without modified nucleotides) of the reporter template, the skilled person in the art would recognize that different techniques of nucleic acid amplification known in the art can be applied to generate a detectable signal. After amplification, the resulting reporter nucleic acids can be detected or measured by any techniques known in the art, which include but is not limited to nucleic acid staining, and fluorescence. Optionally, the reporter nucleic acids may be digested with a restriction enzyme prior to analysis.

[0122] One of the best known methods of nucleic acid amplification is the polymerase chain reaction (PCR; see Mullis, K.B., 1987, U.S. Patent No. 4,683,195 and 4,683,202). Many variations of these techniques are known in the art and can be applied in the methods of the invention, such as but not limited to self sustained sequence replication (Guatelli et al.,1990, Proc Natl Acad Sci. 87:1874 1878), transcriptional amplification system (Kwoh, et al., 1989, Proc Natl Acad Sci. 86:1173 1177), Q Beta Replicase (Lizardi et al., 1988, BioTechnology 6:1197). In one embodiment, a linear reporter template is amplified using a pair of primers: forward primer and reverse primers that hybridize specifically to regions of the reporter template and initiate nucleic acid synthesis on opposite strands of the template. The preferred lengths of such single- stranded nucleic acid primers are at least 9 to 30 nucleotides. For detection of the amplified product, the nucleic acid amplification may be performed using radioactively, fluorescently, luminescently, bioluminescently-labeled nucleotides. [0123] In a specific embodiment, the assays of the invention use quantitative PCR

(QPCR) technology, see, for example, Bustin, S. A. (2002). "Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems." J Molec Endocrin 29: 23-39; "Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream", Ginzinger DG. Exp Hematol 2002 Jun;30(6):503-12; "Quantitative RT-PCR: pitfalls and potential", Freeman et al., (1999) Biotechniques 26, 112-122, which are incorporated herein by reference in their entirety. [0124] In one embodiment, after a nucleic acid reporter template is formed, a template specific primer extension or reverse transcription (RT) reaction is performed, with a template-specific primer, followed by nucleic acid amplification. The amount of amplified reporter nucleic acid is linked to fluorescence intensity using a fluorescent reporter molecule. The point at which the fluorescent signal is measured in order to calculate the initial template quantity can either be at the end of the reaction (endpoint QPCR) or while the amplification is still progressing (real-time QPCR). In endpoint QPCR, fluorescence data are collected after the amplification reaction has been completed, usually after 30—40 cycles, and this final fluorescence is used to back- calculate the amount of template present prior to PCR.

[0125] More preferably, the more sensitive and reproducible method of realtime

QPCR is used to measure the fluorescence at each cycle as the amplification progresses. This allows quantification of the template to be based on the fluorescent signal during the exponential phase of amplification. A fluorescent reporter molecule (such as a double stranded DNA binding dye, or a dye labeled probe) is used to monitor the progress of the amplification reaction. The fluorescence intensity increases proportionally with each amplification cycle in response to the increased amplicon concentration, with QPCR instrument systems collecting data for each sample during each PCR cycle. The reporter molecule used in real-time reactions can be (1) a template- specific probe composed of an oligonucleotide labeled with a fluorescent dye plus a quencher or (2) a non-specific DNA binding dye such as but not limited to SYBR®Green I that fluoresces when bound to double stranded DNA. [0126] A higher level of detection specificity is provided by using an internal probe with primers to detect the QPCR product of interest. In the absence of a specific target sequence in the reaction, the fluorescent probe is not hybridized, remains quenched, and does not fluoresce. When the marker probe hybridizes to the target marker sequence, the reporter dye is no longer quenched, and fluorescence will be detected. The level of fluorescence detected is directly related to the amount of amplified target in each PCR cycle. A significant advantage of using probe chemistry compared to using DNA binding dyes is that multiple marker probes can be labeled with different reporter dyes and combined to allow detection of more than one target marker polynucleotide in a single reaction (multiplex QPCR). A preferred approach for analyzing quantitative data is to use a standard curve that is prepared from a dilution series of control reporter nucleic acid of known concentration.

[0127] In another embodiment, the invention provides the use of rolling circle amplification (RCA) to generate detectable reporter nucleic acids which constitute the signal for the presence of a target. RCA is the prolonged extension of an oligonucleotide primer annealed to a circular nucleic acid template, wherein a continuous sequence of tandem copies of the circular template is synthesized. RCA has the advantage of an isothermal process. See, for example, Fire and Xu 1995, Proc. Natl. Acad. Sci. 92:4641-4645, which is incorporated herein by reference in its entirety. In one embodiment, random hexamers are used with phi29 polymerase to amplify a circular reporter template with up to 10,000 fold amplification. Variations of RCA that use more than one primer (such as but not limited to hyperbranched RCA) are well known and can be used which results in exponential amplification, see, for example, Lizardi et al., Nature Genet. 1998: 19:225-232; Dean et al., 2001, Genome Res. 11:1095-1099; Baner et al., 1998, Nucleic Acids Res. 26:5073-5078; U.S. patent no. 5,854,033; 6,210,884; 6,921,642; which are incorporated herein by reference in its entirety.

[0128] DNA polymerases useful in the rolling circle amplification step are referred to herein as rolling circle DNA polymerases. For rolling circle replication, it is preferred that a DNA polymerase be capable of displacing the strand complementary to the template strand, termed strand displacement, and lack a 5' to 3' exonuclease activity. Strand displacement is necessary to result in synthesis of multiple tandem copies of the ligated aptamers. A 5' to 3' exonuclease activity, if present, might result in the destruction of the synthesized strand. It is also preferred that DNA polymerases for use in the disclosed method are highly processive. The suitability of a DNA polymerase for use in the disclosed method can be readily determined by assessing its ability to carry out rolling circle replication. Preferred rolling circle DNA polymerases are bacteriophage phi29 DNA polymerase (U.S. patent nos. 5,198,543 and 5,001,050), phage M2 DNA polymerase (Matsumoto et al., Gene 84:247 (1989)), phage phiPRDl DNA polymerase (Jung et al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)), VENT™ DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)), Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J. Biochem. 45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19 (1991)), PRDl DNA polymerase (Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276 (1994)), and T4 DNA polymerase holoenzyme (Kaboord and Benkovic, Curr. Biol. 5:149-157 (1995)). Phi29 DNA polymerase is most preferred. The ability of a polymerase to carry out rolling circle replication can be determined by using the polymerase in a rolling circle replication assay such as those described in Fire and Xu, Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995).

[0129] Strand displacement can be facilitated through the use of a strand displacement factor, such as helicase. It is considered that any DNA polymerase that can perform rolling circle replication in the presence of a strand displacement factor is suitable for use in the disclosed method, even if the DNA polymerase does not perform rolling circle replication in the absence of such a factor. Strand displacement factors useful in the methods of the reaction include BMRFl polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91 (22): 10665-10669 (1994)), single-stranded DNA binding proteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)). [0130] Another type of DNA polymerase can be used if a gap-filling synthesis step is used. When using a DNA polymerase to fill gaps, strand displacement by the DNA polymerase is undesirable. Such DNA polymerases are referred to herein as gap- filling DNA polymerases. Unless otherwise indicated, a DNA polymerase referred to herein without specifying it as a rolling circle DNA polymerase or a gap-filling DNA polymerase, is understood to be a rolling circle DNA polymerase and not a gap-filling DNA polymerase. Preferred gap-filling DNA polymerases are T7 DNA polymerase (Studier et al., Methods Enzymol. 185:60-89 (1990)), DEEP VENT™ DNA polymerase (New England Biolabs, Beverly, Mass.), and T4 DNA polymerase (Kunkel et al., Methods Enzymol. 154:367-382 (1987)). An especially preferred type of gap-filling DNA polymerase is the Thermus flavus DNA polymerase (MBR, Milwaukee, Wis.). The most preferred gap-filling DNA polymerase is the Stoffel fragment of Taq DNA polymerase (Lawyer et al., PCR Methods Appl. 2(4):275-287 (1993), King et al., J. Biol. Chem. 269(18): 13061-13064 (1994)).

[0131] Any RNA polymerase which can carry out transcription in vitro and for which promoter sequences have been identified can be used in the disclosed rolling circle transcription method. Stable RNA polymerases without complex requirements are preferred. Most preferred are T7 RNA polymerase (Davanloo et al., Proc. Natl. Acad. Sci. USA 81:2035-2039 (1984)) and SP6 RNA polymerase (Butler and Chamberlin, J. Biol. Chem. 257:5772-5778 (1982)) which is highly specific for particular promoter sequences (Schenborn and Meirendorf, Nucleic Acids Research 13:6223-6236 (1985)). Other RNA polymerases with this characteristic are also preferred. Because promoter sequences are generally recognized by specific RNA polymerases, the aptamers and/or linkers should contain a promoter sequence recognized by the RNA polymerase that is used. Numerous promoter sequences are known and any suitable RNA polymerase having an identified promoter sequence can be used. Promoter sequences for RNA polymerases can be identified using established techniques.

[0132] The materials described above can be packaged together in any suitable combination as a kit useful for performing the disclosed methods of detection. It is expected that by using different combinations of aptamers and detection means, an array of different diagnostic assays can be created for the same target, and that these assays can be compared and optimized for use under different medical or environmental conditions.

5.4 TARGETS OF THE INVENTION

[0133] According to the invention, a target may include but is not limited to, a homopolymeric protein, a heteropolymeric protein, a multiprotein complex, a phosphorylated protein, a glycoprotein, a lipoprotein, an acylated protein, a prenylated protein, an ubiquinated protein, a methylated protein, a sulfated protein, a membrane- bound protein, a DNA-protein complex, or a RNA-protein complex. [0134] A protein detectable by the methods of the invention comprises at least the same number of amino acid residues as and usually many more than the sum of the numbers of amino acid residues that are bound by the aptamers used to detect the protein. Typically, the protein has more than 15 amino acid residues, and can have at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acid residues. [0135] Particularly useful targets are proteins whose presence or levels correlate with a disease or disorder. The presence or levels of such a target may correlate with the risk, onset, progression, amelioration and/or remission of a disease or disorder. The target can be a biomarker, wherein the presence, absence, or amount correlates with the prognosis of a disease in a subject who is under treatment. In one embodiment, the target is a protein having a modification such as, but not limited to, phosphorylation, glycosylation, methylation, ubiquination, or acylation. In another embodiment, the analyte is a synthetic protein. [0136] In a particular embodiment, the target protein is a human-derived hormone such as, but not limited to, gastrin, secretin, cholecystokinin, insulin, glucagon, thyroxin, triiodothyronine, calcitonin, parathyroid hormone, thymosin, releasing hormones, oxytocin, vasopressin, growth hormone, prolactin, melanophore-stimulating hormone, thyrotrophic hormone, adrenocorticotrophic hormone, follicle-stimulating hormone, luteinizing hormone, or melatonin.

[0137] In one embodiment, the target is a marker for a disease or disorder. Such disease or disorder can be, without limitation, an allergy, anxiety disorder, autoimmune disease, behavioral disorder, birth defect, blood disorder, bone disease, cancer, circulatory disease, tooth disease, depressive disorder, ear condition, eating disorder, eye condition, food allergy, food-borne illness, gastrointestinal disease, genetic disorder, heart disease, hormonal disorder, immune deficiency, infectious disease, inflammatory disease or disorder, insect-transmitted disease, nutritional disorder, kidney disease, leukodystrophy, liver disease, mental health disorder, metabolic disease, mood disorder, musculodegenerative disorder, neurological disorder, neurodegenerative disorder, neuromuscular disorder, personality disorder, phobia, pregnancy complication, prion disease, prostate disease, psychological disorder, psychiatric disorder, respiratory disease, sexual disorder, skin condition, sleep disorder, tropical disease, vestibular disorder or wasting disease.

[0138] In another embodiment, the target is a marker for an infection or infectious disease such as, but not limited to, acquired immunodeficiency syndrome (AIDS/HIV) or HTV-related disorders, Alpers syndrome, anthrax, bovine spongiform encephalopathy, (BSE), chicken pox, cholera, conjunctivitis, Creutzfeldt-Jakob disease (CJD), dengue fever, ebola, elephantiasis, encephalitis, fatal familial insomnia, Fifth's disease, Gerstmann-Straussler-Scheinker syndrome, hantavirus, helicobacter pylori, hepatitis (hepatitis A, hepatitis B, hepatitis C), herpes, influenza, avian influenza, Kuru, leprosy, lyme disease, malaria, hemorrhagic fever (e.g. , Rift Valley fever, Crimean-Congo hemorrhagic fever, Lassa fever, Marburg virus disease, and Ebola hemorrhagic fever), measles, meningitis (viral, bacterial), mononucleosis, nosocomial infections, otitis media, pelvic inflammatory disease (PID), plague, pneumonia, polio, prion disease, rabies, rheumatic fever, roseola, Ross River virus infection, rubella, SARS, salmonellosis, septic arthritis, sexually transmitted diseases (STDs), shingles, smallpox, strep throat, tetanus, toxic shock syndrome, toxoplasmosis, trachoma, tuberculosis, tularemia, typhoid fever, valley fever, whooping cough or yellow fever. In most cases, the target is from the pathogen or the target protein is encoded by genetic materials from the pathogen.

[0139] In another embodiment, the target is a marker for a blood disorder such as, but not limited to, anemia, gout, hemophilia A, hemophilia B, leukemia, myeloproliferative disorders, sepsis, sickle cell disease or thalassemia. In another embodiment, the analyte is a marker for a metabolic disease such as, but not limited to, acid maltase deficiency, diabetes, galactosemia, hypoglycemia, Lesch-Nyhan syndrome, maple syrup urine disease (MSUD), Niemann-Pick disease, phenylketonuria or urea cycle disorder.

[0140] In another embodiment, the target can be a marker for a heart disease such as, but not limited to, arrhythmogenic right ventricular dysplasia, atherosclerosis/arteriosclerosis, cardiomyopathy, congenital heart disease, endocarditis, enlarged heart, heart attack, heart failure, heart murmur, heart palpitations, high cholesterol, high tryglycerides, hypertension, long QT syndrome, mitral valve prolapse, postural orthostatic tachycardia syndrome, tetralogy of fallots or thrombosis. [0141] In another embodiment, the target is a marker for cancer such as, but not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma), colon carcinoma, rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatic cancer , bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system; and other tumor types and subtypes (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma), heavy chain disease, metastases, or any disease or disorder characterized by uncontrolled or abnormal cell growth.

[0142] Also, target proteins that are markers for other conditions can be assayed such as, but not limited to, pregnancy, alcoholism, drug abuse, allergy, poisoning, secondary effects of, or responses to, treatments or secondary effects of diseases. [0143] In another embodiment, the present invention provides for a method of diagnosing a disease or disorder in a subject comprising the steps of contacting at least two aptamers with a sample from the subject that might or might not contain the target, wherein said at least two aptamers each binds to a different epitope on the target protein under the appropriate conditions; detecting or measuring binding of the at least two aptamers to said target protein; and detecting or measuring binding of the at least two aptamers to the target, wherein detection or measurement of binding indicates presence or amount, respectively, of the target; and wherein the disease or disorder is determined to be present when the absence, presence or amount of the target differs from a control value representing the amount of target present in an analogous sample from a subject not having the disease or disorder. Preferably, a set of four or five different aptamers are used in the diagnostic methods of the invention.

[0144] The present invention also encompasses methods for determining a prognosis for a disease, disorder or other condition. Prognostic biomarkers for the response (toxic or ameliorative) can be assayed to provide information important for treatment course and dosages. Many such biomarkers are under evaluation for use as companion diagnostics to a particular drug or course of treatment. Prognosis of a disease or determination of possible response to a therapeutic treatment generally involves staging of the disease or disorder. For example, a baseline can be determined prior to manifestation of any symptoms, at a point in the progression of the disease or disorder, or before, during or after therapeutic intervention. Staging refers to the grouping of patients according to the extent of their disease. Staging is useful in choosing treatment for individual patients, estimating prognosis, and comparing the results of different treatment programs. Staging of many cancers is performed initially on a clinical basis, according to the physical examination and laboratory radiologic evaluation. The most widely used clinical staging system is the one adopted by the International Union against Cancer (UICC) and the American Joint Committee on Cancer (AJCC) Staging and End Results Reporting. [0145J Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons (see, e.g., Linder, 1997, Clin Chem. 43:254-266). In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as "altered drug action." Genetic conditions transmitted as single factors altering the way the body acts on drugs are referred to as "altered drug metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. The methods of the invention can be used to determine or monitor the interactions of a subject's genetic factors (biomarkers) and the metabolism of a drug, further enabling the practice of personalized medicine. [0146] In yet another embodiment, the present invention provides for a method of staging a disease or disorder in a subject comprising the steps of contacting at least two aptamers with a sample from the subject that might or might not contain the target, wherein said at least two aptamers each binds to a different epitope on the target protein under the appropriate conditions; detecting or measuring binding of the at least two aptamers to said target protein; and detecting or measuring binding of the at least two aptamers to the target, wherein detection or measurement of binding indicates presence or amount, respectively, of the target; and wherein the stage of a disease or disorder is determined when the absence, presence or amount of the target is compared with the amount of target present in an analogous sample from a subject having no disease and/or disorder or having a particular stage of the disease or disorder. Preferably, a set of four or five different aptamers are used in the disease staging methods of the invention. [0147] In a particular embodiment, the invention can be used in the screening and diagnosis of prostate cancer. Several markers that correlate with prostate cancer are known in the art such as, for example, prostate specific antigen (PSA), human kallikrein 2, BPSA, pro PSA, prostate specific membrane antigen (PSMA), hepsin (a transmembrane serine protease), pirn 1 (a serine/threonine kinase) (See, e.g., Dhanasekaran et al., 2001, "Delineation of prognostic biomarkers in prostate cancer", Nature. 412:822 826). Such markers can be useful for the diagnosis, prognosis, staging, response to treatment and/or management of prostate cancer. PSA (also known as human glandular kallikrein 3), a kallikrein-like serine protease, is recognized as a valuable tumor marker for the screening, diagnosis and management of human prostate cancer. Levels of serum PSA levels have clinical significance in prostate disease management, such as evaluating risk for prostate cancer, determining pretreatment staging, monitoring treatment efficacy and detecting recurrence of disease (Gao et al., 1997, "Diagnostic and prognostic markers for human prostate cancer", Prostate. 31(4):264-281). In the following non limiting example, the targets of interest, for exemplary purposes, are a molecular complex of human prostate specific antigen and alpha-1-antichymotrypsin ("PSA-ACT") and free uncomplexed human prostate specific antigen. PSA-ACT is a cancer-associated biomarker which can be used for early detection of prostate cancer.

[0148] The following is an exemplary amino acid sequence of human prostate specific antigen (hPSA, Accession no. NP_001639.1) and the preferred tripeptides which can be used for aptamer selection and binding (the tripeptides are underlined): MWVPWFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWOVLVASRGRAVCGGV LVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK NRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIE PEEFLTPKKLOCVDLHVISNDVCAOVHPOKVTKFMLCAGRWTGGKSTCSGDSG GPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIV ANP (SEQ ID NO: 1)

[0149] Aptamers that bind specifically to any one of EKH, PQW, RHS, LKN,

QKV, or KST can bind to PSA. A combination of two or more of such aptamers, preferably four or five, can be used to specifically identify PSA and/or determine the amount of PSA in a sample. The tripeptides identified herein are non-limiting examples and it is expected that other tripeptides can also be used to detect PSA. [0150] The following is the amino acid sequence of human alpha- 1 antichymotrypsin (ACT, Accession no. NP_001076.2) and the preferred tripeptides which can be used for aptamer selection and binding (the tripeptides are underlined):

MERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASAN VDFAFSLYKQLVLKAPDKNVIFSPLSISTALAFLSLGAHNTTLTEILKGLKFNLTE TSEAEIHQSFQHLLRTLNQSSDELQLSMGNAMFVKEOLSLLDRFTEDAKRLYGS EAFATDFQDSAAAKKLINDYVKNGTRGKITDLIKDLDSQTMMVLVNYIFFKAK WEMPFDPQDTHQSRFYLSKKKWVMVPMMSLHHLTIPYFRDEELSCTWELKYT GNASALFILPDQDKMEEVEAMLLPETLKRWRDSLEFREIGELYLPKFSISRDYNL NDILLQLGIEEAFTSKADLSGITGARNLAVSQVVHKAVLDVFEEGTEASAATAV KITLLSALVETRTIVRFNRPFLMIIVPTDTQNIFFMSKVTNPKQA (SEQ ID NO: 2)

[0151] Aptamers that bind specifically to any one of LTE, EQL, TRT, and KQA, can bind to ACT. A combination of two or more of such aptamers, preferably four, can be used to specifically identify ACT and/or determine the amount of ACT in a sample. The tripeptides identified herein are non-limiting examples and it is expected that other tripeptides can also be used to detect PSA.

[0152] Moreover, as PSA and ACT form a complex in vivo, the aptamers that bind PSA and the aptamers that bind ACT can be used in combination to detect or measure PSA-ACT complex. In this embodiment, detection that is based on proximity- dependent ligation and nucleic acid amplification can be advantageously used to distinguish the complexed form of PSA and free PSA. The skilled person will recognize that only routine experimentation is required to optimize the combination of PSA aptamers and ACT aptamers for detecting and measuring the complex. In an non- limiting example, the aptamers that bind EKH, PQW, RHS, and LKN on PSA can be used in combination with an aptamer that binds KQA on ACT. [0153] Table 2 provides an exemplary list of targets of interest.

5.5 KITS OF THE INVENTION

[0154] The invention also relates to kits comprising one or more aptamers for detecting proteins, and/or reagents useful for generating a signal for detection or measurement.

[0155] In one embodiment, a kit comprises in a first container, a first aptamer that binds specifically to an oligopeptide and one or more additional containers that comprise other aptamers that bind specifically to an oligopeptide. In another embodiment, a kit of the invention comprises (a) in a first container at least one aptamer, wherein the aptamer binds specifically to a tripeptide; and (b) a detection means to detect said aptamer when bound to said tripeptide.

[0156] In a specific embodiment, the aptamers in different containers in the kit have different nucleotide sequences and all bind specifically to the same oligopeptide. It is expected that different aptamers can bind to the same oligopeptide under different environmental conditions. The aptamers in the kit can be members of an aptamer family or members of different aptamer families. Such kits comprising oligopeptide-specific aptamers are the basic components of the detection system of the invention. [0157] In other embodiments, the aptamers in different containers in the kit bind specifically to different oligopeptides. Typically, the aptamers in such a kit can be used to detect a specific target that has the oligopeptide epitopes in its amino acid sequence. Also included in the kit can be a sample that contains the target which can be used as a control.

[0158] In specific embodiments, a kit comprises (a) in a first container, a first aptamer that binds a first oligopeptide; (b) in a second container, a second aptamer that binds a second oligopeptide; (c) in a third container, a third aptamer that binds a third oligopeptide; and (d) in a fourth container, a fourth aptamer that binds a fourth oligopeptide. The first oligopeptide, second oligopeptide, third oligopeptide, fourth oligopeptide can all be present in the amino acid sequence of a target protein. Optionally, the kit may comprise a fifth aptamer that binds a fifth oligopeptide. The fifth oligopeptide can also be present in the amino acid sequence of a target protein. In yet another embodiment, the kit can comprises one or more containers which comprise a mixture of aptamers of different oligopeptide specificities, i.e., some or all of the aptamers in a set can be premixed in one or more of these containers in specified ratios. [0159] The kits of the invention can further comprise a detection means to detect the binding of the aptamers to its individual oligopeptides. In another embodiment, the kit comprises a detection means to detect the concurrent binding of the aptamers to a target. Such detection means are described in details in an earlier section. In various embodiments, the kit further comprises (a) one or more containers comprising different oligonucleotide primers and/or linkers that facilitate nucleic acid ligation and/or amplification, and/or (b) a ligase and/or a polymerase. The kits may also comprise nucleotides and buffers for nucleic acid ligase and/or polymerase reactions. The containers in the kits can be made specially to fit the automated reagent delivery systems used by clinical diagnostic instruments.

[0160] The kits of the invention can further comprise instructions for carrying out the detection method generally or specifically for a target. The kits can be configured for different purposes including but not limited to disease diagnosis, disease staging, public health screening, companion diagnostics for a drug or a course of treatment, protein isolation, detection of protein toxins or pathogens in the environment such as food, water, and air, proteomic research consumables, clinical laboratory reagents, quality control reagents. The kit may comprise information for assessing computer databases that contains amino acid sequences and/or secondary structure analysis. 6. EXAMPLES

[0161] In this example, aptamers that bind four tripeptides GEL, DGI, KAI and

LAS were isolated. These four tripeptides are present in mouse cathepsin D. Using a structure-switch method, it is shown that the aptamers interact with mouse cathepsin D. The example further demonstrates that the combination of four aptamers can specifically detect the protein using a multiple aptamer-based proximity-dependent ligation assay. The results prove that the invention may be applied generally to detect specifically a protein of interest based on its amino acid sequence.

6.1. METHODS

[0162] Preparation of tripeptide-affinity column. The tripeptides Ac-LAS- amide, Ac-DGI-amide, Ac-GEL-amide, and Ac-KAI-amide, were synthesized by New England Peptide, Inc. HiTrap NHS-activated columns were purchased from Amersham Pharmacia Biotech (Cat. # 17-0716-01) and used to make the peptide affinity chromatography. The peptides were dissolved in standard coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8) to a final concentration of 0.5 mM. The column was washed with 6 ml of ice cold 1 mM HCl at the flow rate of 1 ml/min. After column wash, 1 ml of the tripeptide solution was added and incubated at 25°C for 30 min. The column was washed three times with 2 ml of 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3, and another three times with 0.1 M acetate, 0.5 M NaCl, pH 4. The wash procedure was repeated two more times. Finally, 2 ml of buffer A containing 20 mM Tris-HCl, pH 7.3; 140 mM NaCl; 5 mM KCl; 5 mM MgCl; 1 mM CaCl₂; and 0.02% Triton X-100 was added onto the column. The column was stored at 4°C before use. [0163] DNA aptamer library. DNA pool (APTl-L) containing ₆o random nucleotides with the sequence of GCA GTC TCG TCG ACA CCC (N)₆O GTG CTG GAT CCG ACG CAG (SEQ ID NO: 3), where N represents A, T, G or C, was synthesized and purified by polyacrylamide gel electrophoresis (PAGE). Sense primer (APT1-5) GCA GTC TCG TCG ACA CCC (SEQ ID NO: 4), antisense primer (APTl- 3) CTG CGT CGG ATC CAG CAC (SEQ ID NO: 5) and antisense oligonucleotide of APTl -3 (APTl -3A) GTG CTG GAT CCG ACG CAG (SEQ ID NO: 6) were synthesized and PAGE purified. PCR reaction was carried out in 50 μl of solution containing 1 μl of 4.4 μM APTl-L, 1 μl of 10 μM APT1-5, 1 μl of 10 mM APT1-3, 2 μl of water, and 45 μl of PCR SuperMix (Life Technology 10790-020). The reaction was incubated at 94°C for 5 minutes, repeated 22 cycles with 94°C for 30 seconds, 60⁰C for 30 seconds and 72°C for 30 seconds, and finally 72°C for 7 minutes. The PCR product was purified using PCR purification kit (Qiagen 28106).

[0164] Single stranded DNA generation. The purified PCR product was used as the template for the generation of single stranded DNA. PCR reaction was carried out in a total volume of 50 μl with 3 μl of the PCR product, 1 μl of 10 μM APT 1-5, 1 μl of 10 μM APTl -3 A for inactivating the remaining APT 1-3 primer, 45 μl of PCR SuperMix (Life Technology 10790-020). The reaction was incubated at 94°C for 5 minutes, repeated 22 cycles with 94°C for 30 seconds, 60⁰C for 30 seconds and 72°C for 30 seconds, and finally 72°C for 7 minutes. For each tripeptide affinity column, PCR products from 6 tubes (300 μl) were pooled together into a 1.5 ml tube, add 30 μl of 3 M Na Acetate, 0.75 ml of 100% ethanol and centrifuged to precipitate the PCR product. The single stranded DNA was dissolved in 1 ml of buffer A and passed through a 0.2 μm filter, incubated at 70⁰C for 10 min, room temperature for 30 min, and then put on ice. The samples were ready to load onto the HiTrap NHS-activated tripeptide affinity column.

[0165] In vitro selection. The HiTrap NHS-activated tripeptide columns were washed three times with 2 ml of buffer A. The single stranded DNA samples were loaded onto the columns. After 30 min at room temperature, the columns were washed one time with 11 ml of buffer A. DNA samples were eluted with three loadings of 0.8 ml of 0.5 mM tripeptide in buffer A. The samples were collected in 6 tubes, each tube contained about 400 μl of the eluted sample. DNA sample was precipitated by adding 1 ml of 100% ethanol. The precipitated DNA was dissolved in 6 μl OfH₂O. Three μl of the DNA sample was used for PCR reaction containing 1 μl of APT 1-5 and 1 μl of APT 1-3 primers, 45 μl of the SuperMix. The PCR reaction was carried out with 22 cycles as described before. The PCR products were combined from the 6 tubes and purified by PCR purification kit. Three μl of the PCR sample was used for the single stranded PCR reaction. The PCR reaction condition was the same as before using primers Apt 1-5 and Apt 1-3 A. The single stranded PCR products were pooled, ethanol precipitated and used for the second round selection. This in vitro selection was repeated twelve times and the final PCR product was cloned into a TA vector and sequenced.

[0166] Binding assay. One μl of the affinity purified DNA aptamers were labeled with ³²P-ATP by single stranded PCR using primer Apt 1-5 for 5 cycles. The samples were purified using the PCR purification kit and counted. Ten thousands cpm samples were loaded onto the tripeptide affinity column. After 30 min incubation at room temperature, the columns were washed with 11 ml of buffer A. The remaining bound DNA was eluted with 3 loadings of 1 ml of 3 mM tripeptide in the same buffer. Each fraction was analyzed with Cerenkov counting. The number of counts from the eluted fractions was divided by the total counts to give the fraction of bounded DNA. [0167] Isolation of DNA aptamers against four tripeptides. Four tripeptides were randomly selected GEL, DGI, KAI and LAS, from mouse cathepsin D (CatD) protein for the isolation of specific aptamers. The tripeptides were synthesized and coupled to HiTrap NHS-activated column. The starting DNA pool was a mixture of 96- mer single-stranded DNA (ssDNA) molecules containing randomized 60-nucleotide inserts. The ssDNA molecules that bound to the tripeptides were eluted and amplified by PCR. The PCR products were further selected using the tripeptide affinity columns for sub-library generation. The selection procedure was repeated twelve times and the final products were cloned and sequenced. The nucleotide sequences of the cloned aptamers that bind the tripeptides are shown in Table 3. Although some of the clones have identical nucleotide sequences, different aptamer families are isolated for each of tripeptides.

[0168] Table 3: Nucleotide sequences of the cloned aptamers with different tripeptide binding specificities.

[0169] A number of aptamers were selected for each tripeptide and used for binding assay. Four aptamers with high binding ability (Fig. 1), GEL-aptamer, KAI- aptamer, DGI-aptamer and LAS-aptamer, were selected for the protein detection experiments (Table 4).

[0170] Table 4: Sequences of aptamers, FDNA and QDNA for structure- switching assay. The sequences of aptamers are underlined, FDNA and its antisense sequences are indicated in italics and the QDNA and its antisense sequences are shown in bold.

Name Sequence

MAP-KAI CCTGCCACGCTCCGCCCTGCTCACTGGCGCAGCGGGTGGAGT

GTTAAGATGAATTGCGGTGTGGGCCGGCCTCTATTGGC (SEQ ID

NO: 36) MAP-GEL CCTGCCACGCTCCGCCCTGCTCACΥGGCGAAGCGGGCTGAAGT

GCACACAGCTGGAGGAGTATTGTTGGGTGCTC (SEQ ID NO:37) MAP-LAS CCrGCC4CgCrCCCCCCrCCTCACTGACGAAGTGGGTGTATAG

CGAATAATCATTAAGAAAGGGCGCTGTGTTGTG (SEQ ID NO:

38)

MAP-DGi CCTGCCACGCTCCGCCCTGCTCAGΎGAGCCΎ AAAATATTGCTT

AGTAAGGGTGGTCTGGCTCCGAGAGGGGT (SEQ ID NO: 39) FDNA F AM-GCAGGGCGGAGCGTGGCAGG (SEQ ID NO: 40)

QDNA CCCGCTGCGCCAGTG-DABCYL (SEQ ID NO: 41)

DGI-QDNA ATTTT AGGCTCAGTG— DABCYL (SEQ ID NO: 42)

LAS-QDNA CCCACTTCGTCAGTG-DABCYL (SEQ ID NO: 43)

[0171] Generation and purification of recombinant mouse Cat D and E. coli

Dahp proteins. Mouse Cat D was cloned from mouse brain cDNA (Invitrogen) and Dahp was cloned from E. coli genomic DNA using PCR method. The primers used for mouse Cat D were: 5'-CCT GAA TTC ATG AAG ACT CCC GGC GT-3' (SEQ ID NO: 44) and 5'-ATC AAG CTT GAG TAC GAC AGC ATT GGC-3' (SEQ ID NO: 45), and for Dahp were 5'-GAA TTC ATG AAT TAT CAG AAC GAC GAT TTA CG- 3' (SEQ ID NO: 46) and 5'-AAG CTT CCC GCG ACG CGC TTT TAC T-3' (SEQ ID NO: 47). The coding regions of Cat D and Dahp were inserted into pET 24a(+) vector (Novagen) and grown in E.coli BL21(DE3) (Novagen). Cells from 100 ml of broth were collected after incubation with ImM IPTG for 4 hours at 37°C. The Dahp protein was purified as described by Ni-NTA manual (Novagen ). The recombinant mouse Cat D was purified using a precipitation method as described in (Shou et al., Basic Medical Sciences and Clinics 7 (1997) 227). Briefly, cells were incubated in 30 ml PBS containing ImM PMSF and 1 g/L lysozyme for 20 minutes on ice. After the incubation, 0.3 ml Triton X-IOO was added to the cell suspension and incubated on ice for 10 minutes. The suspension was sonicated for 30 seconds and centrifuged at 15000 rpm using the Beckman JA- 14 rotor for 15 minutes at 4°C. The pellet was resuspended in 20 ml of 10 mM EDTA solution, pH 8.0, sonicated for 30 seconds and then collected by centrifugation in a Beckman JA-14 rotor at 5000 rpm for 15 minutes at 4°C. The EDTA precipitation procedure was repeated three times. The pellet was then resuspended in 15 ml of 2 mM EDTA solution, pH 8.0. The suspension was sonicated until the sample became white. Fifteen ml of cold 40 mM NaOH was added to the sample and sonicated again until the sample turned to clear solution. Finally, 7.5 ml of 40% glycerol solution containing 5mM PMSF and 5% Leupeptin was added and the sample was stored at minus 70⁰C.

[0172] Structure-switching assay. The structure-switching assay was performed as previously reported (Nutiu et al., J Am Chem Soc 125 (2003) 4771-4778.). Briefly, the DNA aptamer was modified with an addition of a short oligonucleotide sequence at the 5 '-end (MAP). Fluorescent group labeled oligonucleotide (FDNA) and quench group labeled oligonucleotide were synthesized (Sangon). The sequences were listed in Table 3. The assay was carried out with 160 nM MAP, 320 nM FDNA, 480 nM QDNA and different concentrations of proteins in 20 μl of buffer A. The reaction was incubated at 37°C for 60 minutes. DNA engine OPT1CON2 continuous fluorescence detector (MJ Research) was used to measure the fluorescence signals generated by the interaction between the aptamers and proteins.

[0173] Proximity-dependent ligation assay. The aptamers (LA-GEL, LA-KAI,

LA-LAS and LA-DGI) for the ligation assay (LA) were synthesized and the sequences were listed in Table 5. The connectors and primers used were connectorl, connector2, connector3, connector4, primerl and primer2 (Table 5). In the experiment with five aptamers (LA-GEL, LA-KAI, LA-LAS, LA-DGI and LA-GELl), the connectors and primers were connector5, connector6, primer2f, primer3f, primer4f, primer5r and primerδr (Table 5). The mouse Cat D protein, truncated Cat D protein, E.coli lysis and BSA were diluted with 1% BSA. One μl of aptamer at the concentration of 10 pM was incubated with 5.0 μl of proteins at room temperature for Ih. Seven μl of distilled water, 1.4 μl of 5 x T4 DNA ligase buffer, 0.2 μl of T4 DNA ligase (Invitrogen), 0.4 μl of connectors (25 μM) and 1.0 μl of the pre-incubated protein mixture were added to a tube, mixed and incubated at room temperature for 5-7 h. After the ligation, IuI of each sample was subjected to hyperbranched rolling circle amplification (Zhang et al., Gene 274 (2001) 209-216; Zhang et al., Gene 211 (1998) 277-285; and Dean et al., Genome Res 11 (2001) 1095-1099), in a total volume of 10 μl in the presence of 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 10 mM (NH-O₂SO₄, 4 mM dithiothreitol, 200 μg/mL BSA, 10 U of phi29 DNA polymerase (New England Biolabs), 1.0 μM each primer and 0.5 mM dNTPs. The reaction was incubated for 8-24 h at 30⁰C followed by 10 min at 65°C to inactivate the polymerase. The amplified products were detected with 1% agarose gel electrophoresis.

[0174] Table 5: Sequences of aptamers, connectors and primers for proximity- dependent ligation assay. The sequences of aptamers were underlined, the arms (linking regions) and their complementary sequences in the aptamers were indicated in bold. Name Sequence

LA-GEL ACTTCAGCCCTTTTTTTTAATCACTTATGCGAAGCGGGCTGAA GTGCACACAGCTGGAGGAGTATTGTTGGGTGCTCTTTCTCCTC CAGC (SEQ ID NO: 48) LA-KAI TTAACACTCCTTTTTTTTATCTTGCGCAGCGGGTGGAGTGTTAA

GATGAATTGCGGTGTGGGCCGGCCTCTATTGGCTTTCCCACAC

CGC (SEQ ID NO: 49) LA-LAS CGCTATACACTTTTTTATCACTTATACGAAGTGGGTGTATAGC

GAATAATCATTAAGAAAGGGCGCTGTGTTGTGTTTTTCCTTTCT

TAA (SEQ ID NO: 50) LA-DGI TAAGCAATATTTTATCACTTATCCATAGCCTAAAATATTGCTTA

GTAAGGGTGGTCTGGCTCCGAGAGGGGTTGAATTCTGAGCCA

GACC (SEQ ID NO: 51) LA-GELl AACATCTACGTTTTTTTTAATCACTTATAGAGGGCCGTAGATGT

TATACTGTGGGTAGTATAGGCTTGGTTTTTATACTACCC (SEQ

ID NO: 52) connectorl 5'-AAAGGGCTGAAGTGGTCTGGCTCTTT-S' (SEQ ID NO: 53) connector2 5'-AAAGGAGTGTTAAGCTGGAGGAGTTT-S' (SEQ ID NO: 54) connector3 5'-AAAGTGTATAGCGGCGGTGTGGGTTT-S' (SEQ ID NO: 55) connector4 5'-AAAATATTGCTTATTAAGAAAGGTTT-S' (SEQ ID NO: 56) primerl 5'-CGAAGCGGGCTGAAGTGCA-S' (SEQ ID NO: 57) primer2 5'-TCTTAACACTCCACCCGCT-S' (SEQ ID NO: 58) connector5 5'-TTTCGTAGATGTTGGTCTGGCTCTTT-S' (SEQ ID NO: 59) connectorό 5'-TTTGGGCTGAAGTGGGTAGTATATTT-S' (SEQ ID NO: 60) primer2f 5'-GGAGTGTTAAGCTGGAGGAG-S' (SEQ ID NO: 61) primer3f 5'-GTGTATAGCGGCGGTGTGGG-S' (SEQ ID NO: 62) primer4f 5'-ATATTGCTTATTAAGAAAGG-S' (SEQ ID NO: 63) primer5r 5'-GAGCCAGACCAACATCTACG-S' (SEQ ID NO: 64) primerδr 5'-TATACTACCCACTTCAGCCC-S' (SEQ ID NO: 65)

[0175] Database Search. A pattern search was conducted in the PIR database ( http://pir.georgetown.edu/pirwΛvw/search/pattern.shtnil ) to identify proteins containing the tripeptides. First, a search was conducted for proteins containing the sequence of LASX(l,300)DGIX(l,300)GELX(l,300)KAI, where X(l,300) represents that there are 1 to 300 amino acids between the two tripeptides. The permutations of tripeptides were then used to conduct the pattern search one by one (Table 6). The searches covered the summed occurrences of all tripeptides. We searched the PIR NREF database to identify proteins containing either three (GEL, DGI, KAI) or four tripeptides (GEL, DGI, KAI and LAS) sequences. It was found that more than one thousand proteins that contain three of these tripeptide sequences in humans. There were only 2, 17 and 35 proteins that contained all four tripeptides in E. coli, mouse and human, respectively (Table 6). [0176] Table 6: Number of proteins containing four tripeptides in E. coli, mouse and human. All permutations of four tripeptides were listed and used for search in the PIR-NREF database ( http://pir.georgetown.edu/pirwww/ ). One to three hundred random amino acids were inserted between each pair of tripeptides.

Permutation of Tripeptides E. coli Mus Homo

LASX(1,300)DGIX(1,300)GELX(1,300)KAI i 7 3

LASX( 1 ,300)DGIX( 1 ,30O)KAIX(1 ,30O)GEL 0 1 0

LASX(l,300)GELX(l,300)DGIX(l,300)KAI 0 0 0

LASX(l,300)GELX(l,300)KAIX(l,300)DGI 0 0 2

LASX(1 ,30O)KALX(1 ,30O)GELX(1 ,30O)DGI 1 0 5

LASX(1 ,30O)KALX(1 ,30O)DGIX(1 ,30O)GEL 0 0 3

DGIX(l,300)LASX(l,3OO)GELX(l,3OO)KAI 0 0 2

DGIX(1 ,30O)LASX(1 ,30O)KAIX(1 ,30O)GEL 0 0 0

DGIX( 1 ,30O)GELX(1 ,300)LASX( 1 ,30O)KAI 0 0 0

DGIX(l,300)GELX(l,300)KAIX(l,300)LAS 0 1 2

DGIX(1 ,30O)KAIX(1 ,30O)GELX(1 ,30O)LAS 0 0 3

DGIX(1, 30O)KAIX(1, 30O)LASX(1, 30O)GEL 0 0 0

GELX(l,30O)DGLX(l,300)LASX(l,3OO)KAI 0 0 3

GELX(l,300)DGIX(l,300)KAIX(l,300)LAS 0 0 0

GELX(1, 30O)LASX(1, 30O)DGIX(1, 30O)KAI 0 0 1

GELX(1,300)LASX(1,300)KAIX(1,300)DGI 0 0 2

GELX(1 ,3OO)KA1X( 1 ,300)LASX( 1 ,30O)DGI 0 2 5

GELX(l,300)KAIX(l,300)DGIX(l,300)LAS 0 4 3

KAIX(1 ,30O)DGIX(1 ,30O)GELX(1 ,30O)LAS 0 0 0

KAIX(1, 30O)DGLX(1, 30O)LASX(1, 30O)GEL 0 0 0

KAIX(1 ,30O)GELX(1 ,30O)DGIX(1 ,30O)LAS 0 0 0

KAIX(1 ,30O)GELX(1, 30O)LASX(1, 30O)DGI 0 0 0

KAIX(l,300)LASX(l,300)GELX(l,300)DGI 0 0 1 KAIX(1,300)LASX(1,300)DGIX(1, 30O)GEL 0 2 0

Total number 2 17 35^~

6.3. RESULTS

[0177] Detection of aptamer-protein interaction. The structure-switching technology of Nutiu et al. (J Am Chem Soc 125 (2003) 4771-4778) was modified to detect the aptamer-protein interactions. The advantage for using this method is that that the aptamers can be labeled with fluorescent groups without affecting the secondary structure of aptamer (Nutiu et al., Chemistry 10 (2004) 1868-1876 and Nutiu et al., J Am Chem Soc 125 (2003) 4771-4778). Furthermore, the fluorescence signals can be easily detected as soon as the aptamers interact with proteins (Nutiu et al., Chemistry 10 (2004) 1868-1876 and Nutiu et al., J Am Chem Soc 125 (2003) 4771-4778). The modified aptamers (MAP) contain about thirty nucleoside bases at the 5'-end followed by the full length aptamer sequences (Table 4). Two other oligonucleotides were used for this assay (Table 4), one labeled with a fluorophore (FDNA) while another labeled with a quencher (QDNA) (Nutiu et al., Chemistry 10 (2004) 1868-1876 and Nutiu et al., J Am Chem Soc 125 (2003) 4771-4778). The FDNA was designed to anneal to the 5 '-end of MAP while most of the QDNA should interact with the motif of aptamer. This design should bring the fluorophore and the quencher together and therefore no fluorescence signals should be generated (Nutiu et al., Chemistry 10 (2004) 1868-1876 and Nutiu et al., J Am Chem Soc 125 (2003) 4771-4778). For additional details, see WO 03/062422 which is incorporated herein by reference in its entirety. When the aptamer interacts with the target protein, the QDNA is disassociated from the aptamer and the fluorescence signals can be generated from the FDNA (Nutiu et al., Chemistry 10 (2004) 1868-1876 and Nutiu et al., J Am Chem Soc 125 (2003) 4771-4778). Different concentrations of partially purified recombinant mouse Cat D protein were used in the system. The results demonstrated that addition of the protein resulted in the elevation of fluorescence signals in a dose response manner (Fig. 2). Bovine serum albumin (BSA), a protein without the tripeptides sequences, was used as a control. It was found that addition of BSA did not induce any fluorescence signal even at very high concentrations (Fig. 2). This result showed that there was no interaction between BSA and the aptamers. [0178] To further test the specificity of the aptamer-protein interaction, we used

E. coli Dahp protein, which contains two KAI sequences for the structure-switching experiment. We found that Dahp induced the fluorescence emission when MAP-KAI was present in the assay, indicating an interaction between Dahp and KAI specific aptamer (Fig. 2). As expected, higher concentrations of Dahp protein generated more fluorescence signals. Surprisingly, the addition of BSA at high concentrations slightly increased the signal. It is possible that BSA in the solution might affect the stability of the structure-switching aptamers and therefore slightly increased the signals. Taken together, these results suggest that aptamers targeting tripepetides can bind to proteins containing the tripeptide sequences and the interactions between the aptamers and proteins are specific.

[0179] Detection of Cat D protein based on the amino acid sequence. The combination of several aptamers was tested for specific detection of the mouse Cat D protein. Proximity-dependent ligation assay (Zhu et al., Biol Chem 387 (2006) 769-772; Gullberg et al., Proc Natl Acad Sci USA 101 (2004) 8420-8424; and Fredriksson et al., Nat Biotechnol 20 (2002) 473-477) was modified and adapted for this purpose (Fig. 3). In the assay, much effort has been taken to optimize the ligation system to reduce background ligation (Fredriksson et al.,Nat Biotechnol 20 (2002) 473-477). In this experiment, two arms (linking regions) with ten nucleotides were added to both ends of the aptamers in such a way that the arms could anneal to the binding portion of the aptamer. This intramolecular interaction could block the annealing reaction between the connectors and the aptamers before target binding, thereby reducing the background of the proximity-dependent ligation (Fredriksson et al., Nat. Biotechnol 20 (2002) 473- 477). The target protein may interact with the aptamers, leading to the conformation change of the aptamers and then resulting in the release of the arms. The connectors were able to anneal to the aptamers for the proximity-dependent ligation. Four different aptamers were added to the reaction that contained partially purified recombinant mouse Cat D protein. In the reaction, the interaction between aptamers and Cat D protein led to the release of the arms (linking regions) which hybridize to the connectors, followed by succcessive ligations leading to the formation of a ring, i.e., a circular reporter template. The single stranded DNA ring was amplified by phi29 DNA polymerase in a rolling circle mechanism (Baner et al., Nucleic Acids Res 26 (1998) 5073-5078 and Lizardi et al., Nat Genet 19 (1998) 225-232) ( Fig. 3 ). The amplified products were detected in 1% agarose gel. The results showed that the protein could be detected in a dose response manner (Fig. 4a). Importantly, the technology could detect the protein at the concentration of as low as 100 amol.

[0180] To test the specificity of the aptamer-protein interaction, a truncated mouse Cat D protein lacking the LAS tripeptide at the N-terminal were used. While interacting with the truncated protein, these four aptamers could not form a ring for rolling circle amplification. Indeed, the result showed that no amplified product could be detected even when 10 nM of the truncated protein was used in the assay system (Fig. 4a).

[0181] To further test the robustness of the assay, recombinant Cat D protein was detected in a cell extract that contained a large number of other proteins using the proximity-dependent ligation assay. To increase the specificity, five aptamers were used in the assay (Table 5). Since Cat D protein contains two GEL tripeptide sequences, two GEL, one LAS, one DGI, and one KAI aptamers were added to the reaction containing an extract of the Cat D vector- transformed E. coli. Mock vector-transformed E. coli extract was included as the control. The results demonstrate that these five aptamers could effectively detect Cat D protein with high specificity and sensitivity, while no signal was detected from the mock transformed cell extract (Fig. 4b).

6.4. DISCUSSION

[0182] To perform a proof of principle experiment, four tripeptides based on the amino acid sequence of mouse cathepsin D (Cat D) was used for the generation of specific DNA aptamers. It has been shown that the tripeptides adopt stable structures in water, which reflects to some extent the intrinsic structural property of the respective amino acids in proteins (Eker et al., J Am Chem Soc 124 (2002) 14330-14341 and Eker et al., J Am Chem Soc 125 (2003) 8178-8185.). Aptamers targeted to short peptide should dominate the structure of the complex (Ye et al., Chem Biol 6 (1999) 657-669). The data presented herein shows that DNA aptamers targeting to tripeptides were able to interact with proteins containing the tripeptide sequences.

[0183] Aptamers against tetrapeptide, pentapeptide or hexapeptide could be used in the invention. Tripeptides were chosen for the proof of principle experiment on a consideration of the size of the universal library. The maximum number of aptamers for tripeptides needed to cover all possible combinations of twenty amino acids is 8000 (20 x 20 x 20), while the number reaches 160,000 for tetrapeptides and 20 times more for pentapeptides and so on. A database search demonstrated that there were only 2, 17 and 35 proteins that contain these four tripeptide sequences in E. coli, mouse and human, respectively (Table 6). It is expected that systematic analysis of protein sequences using bioinformatϊcs tools may significantly reduce the number of new aptamers needed to recognize and detect all the proteins in the proteomes. These results prove that when aptamers with tripeptide specificities were chosen on the basis of amino acid sequence analysis, it is possible to use four aptamers, or at most five aptamers, to detect a specific protein.

7. EXAMPLES

[0184] In this example, aptamers that bind four tripeptides GEL, DGI, KAI and

LAS were used to capture the mouse cathepsin D protein.

7.1. METHODS

[0185] Aptamers were synthesized and labeled with biotin (Sangon). The DNA sequences of the aptamers were: Con-biotin:5'-Biotin-atc act tat ate cat-3' (SEQ ID NO: 66); GEL-biotin: 5 '-Biotin- aat cac tta tGC GAA GCG GGC TGA AGT GCA CAC AGC TGG AGG AGT ATT GTT GGG TGC TC-3' (SEQ ID NO: 67); KAI-biotin: 5'- Biotin- ate ttG CGC AGC GGG TGG AGT GTT AAG ATG AAT TGC GGT GTG GGC CGG CCT CTA TTG GC-3' (SEQ ID NO: 68); LAS-biotin: 5'-Biotin- ate act tat ACG AAG TGG GTG TAT AGC GAA TAA TCA TTA AGA AAG GGC GCT GTG TTG TG-3' (SEQ ID NO: 69); DGI-biotin: 5'-Biotin- ate act tat cca tAG CCT AAA ATA TTG CTT AGT AAG GGT GGT CTG GCT CCG AGA GGG GT-3' (SEQ ID NO: 70). A 240 μl aliquot of streptavidin magnetic beads ( New England Biolab ) was washed three times with 400 μl buffer A. In a total volume of 120 μl, twenty mM of biotin-labeled aptamers were incubated with the washed streptavidin magnetic beads in buffer A for 30 minutes at room temperature. The beads were washed three times with buffer A. A 220 μl aliquot of the buffer A containing different concentrations of recombinant mouse Cat D, BSA or clear extract of bacteria were added to the beads and incubated for 30 minutes at room temperature. The sample was washed three times with buffer A containing 140 mM to 190 mM NaCl and the absorbed protein was eluted with 100 μl 0.02M NaOH at room temperature. The eluted protein was analyzed in 4-20% SDS-PAGE and stained with Coomassie brilliant blue G250. The protein bands were quantitatively analyzed with UVP bioimaging systems using the software labworks 4.0 (UVP).

7.2. RESULTS

[0186] Different numbers of DNA aptamers were used in this assay. One aptamer Biotin-DGI, two aptamers Biotin-KAI/ Biotin-GEL, three aptamers Biotin-KAI/ Biotin-GEL/ Biotin-LAS and four aptamers Biotin-KAI/ Biotin-GEL/ Biotin-DGI/ Biotin-LAS were added to each streptavidin magnetic bead tube. A biotin labeled control oligonucleotide of twelve bases with the same sequence of 5 '-end of other biotin labeled aptamers was added to make the DNA at the same concentration for each tube. It was postulated that the combination of four aptamers could interact with all four tripeptide sequences of Cat D, and therefore had the highest affinity for the protein. Similarly, three aptamers should have higher affinity than two aptamers and so on. The partially purified recombinant mouse Cat D protein in a solution containing excess amount of BSA and lysozyme was applied to the magnetic beads. The beads were washed with increasing concentrations of salt and finally the protein was eluted with dilute NaOH. The Cat D protein was eluted from the beads containing one, two or three aptamers with increasing wash stringency. The wash condition was optimized so that only the combination of four aptamers could capture the mouse Cat D protein (Fig. 5). Although large amount of BSA and lysozyme were added to the sample containing the mouse Cat D protein and the beads, the aptamers specifically captured the mouse Cat D protein.

AU references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED:

1. A method for detecting or measuring a target that comprises a protein, said method comprising the steps of :

(a) contacting at least four aptamers with a sample comprising the target, wherein said at least four aptamers each binds to a different oligopeptide epitope on the protein under the appropriate conditions; and

(b) detecting or measuring binding of said at least four aptamers to the target; wherein detection or measurement of the binding of said at least four aptamers to the target collectively indicates the presence or amount, respectively of the target.

2. A method of diagnosing a disease or disorder in a subject comprising the steps of : (a) contacting at least four aptamers with a sample from the subject that might or might not contain a target that comprises a protein, wherein said at least four aptamers each binds to a different oligopeptide epitope on the protein under the appropriate conditions; and

(b) detecting or measuring binding of said at least four aptamers to the target; wherein detection or measurement of the binding of said at least four aptamers to the target collectively indicates presence or amount, respectively, of the target; and wherein the disease or disorder is determined to be present when the absence, presence or amount of the target differs from a control value representing the amount of target present in an analogous sample from a subject not having the disease or disorder.

3. A method of staging a disease or disorder in a subject comprising the steps of

(a) contacting at least four aptamers with a sample from the subject that might or might not contain a target that comprises a protein, wherein said at least four aptamers each binds to a different oligopeptide epitope on the protein under the appropriate conditions; and

(b) detecting or measuring binding of said at least four aptamers to the target; wherein detection or measurement of the binding of said at least four aptamers to the target collectively indicates presence or amount, respectively, of the target; and wherein the stage of a disease or disorder is determined when the absence, presence or amount of the target is compared with the amount of target present in an analogous sample from a subject having no disease and/or disorder or having a particular stage of the disease or disorder.

4. The method of claim 1, 2, or 3, wherein said at least four aptamers consist of a total of four or five aptamers, and wherein each said oligopeptide epitope consists of a tripeptide.

5. The method of claim 1, 2, or 3, wherein step (b) comprises detecting or measuring the concurrent binding of each one of said at least four aptamers to the target.

6. The method of claim 1, 2, or 3, wherein step (b) comprises (c) ligating of said at least four aptamers to each other via oligonucleotide connectors while said at least four aptamers are bound to the target, to form a reporter template; (d) amplification of the reporter template to produce reporter nucleic acids; and (e) detecting or measuring the reporter nucleic acids.

7. The method of claim 1, 2, or 3, wherein said detecting or measuring is performed by autoradiography, phosphoimager analysis, fluorometry, spectrofluorometry, luminescence measurement, colorimetric procedures, or absorbance measurement.

8. The method of claim 6, wherein said amplification of the reporter template comprises hybridizing at least one primer to the reporter template and extending the primer by rolling circle amplification in the presence of detectably-labeled nucleotides.

9. The method of claim 1, 2, or 3, which further comprises prior to step (b), the step of removing unbound aptamers and/or sample materials not bound to one or more of said at least four aptamers.

10. The method of claim 1, 2, or 3, wherein one of said at least four aptamers is present on a solid phase.

11. The method of claim 1 , 2, or 3, wherein the target is a protein, a glycoprotein, a lipoprotein, a multiprotein complex, a phosphoprotein, a methylated protein, a prenylated protein, a ubiquinated protein, an acylated protein, or a sulfated protein.

12. The method of claim 1, 2, or 3, wherein one of said at least four aptamers does not bind to the protein when the protein has undergone a post-translational modification, or is complexed with another molecule.

13. The method of claim 2 or 3, wherein said disease or disorder is an allergy, autoimmune disease, birth defect, blood disorder, bone disease, cancer, circulatory disease, food borne illness, gastrointestinal disease, genetic disorder, heart disease, hormonal disorder, infectious disease, metabolic disease, neurological disorder, neurodegenerative disorder, pregnancy complication, prostate disease, respiratory disease, sexual disorder, skin condition.

14. The method of claim 2 or 3, wherein said disease is prostate cancer.

15. The method of claim 1 , 2, or 3, wherein the target is from a pathogen.

16. The method of claim 1, 2, or 3, wherein the target is a biomarker, wherein the presence, absence, or amount correlates with a prognosis of a disease in a subject who is under treatment/

17. A composition comprising an aptamer that binds specifically to a tripeptide.

18. The composition of claim 17, wherein the tripeptide is selected from the group consisting of LTE, EQL, TRT, KQA, EKH, PQW, RHS, LKN, QKV, KST, GEL, DGI, KAI and LAS.

19. A kit comprising (a) in a first container at least one aptamer, wherein the aptamer binds specifically to a tripeptide; and (b) a detection means to detect said aptamer when bound to said tripeptide.

20. A method for selection of an aptamer that binds specifically to a tripeptide, said method comprising:

(a) incubating a tripeptide with a mixture of oligonucleotides under conditions wherein members of the oligonucleotide mixture form complexes with the tripeptide;

(b) separating the resulting complexes of the tripeptide and oligonucleotides from the uncomplexed oligonucleotides, wherein the complexed oligonucleotides constitute aptamers; (c) eluting the aptamers from the complexes of step (b) and optionally amplifying the aptamers; and

(d) repeating step (a) and (b) with the aptamers of step (c).

21. A method for isolating a target, said method comprising:

(a) contacting at least four aptamers with a sample comprising the target, wherein said at least four aptamers each binds to a different oligopeptide epitope on the protein under the appropriate conditions;

(b) isolating said at least four aptamers that are bound to the target; and

(c) eluting the aptamers from the target and recovering the target.