Abstract

I. Introduction

Nucleic acid aptamers are synthetic single-stranded oligonucleotides that bind specifically to their target molecules when the aptamers are folded into unique tertiary conformations depending on their sequences. The binding affinities of aptamers to target molecules are the results of various binding forces such as hydrophobic interactions, hydrogen bonding, van der Waals interactions, aromatic ring stacking, and shape complementary fitting.¹

Emerging as an alternative to antibodies in diagnostics and therapeutics,² the aptamers are selected from a pool of 10¹⁴–10¹⁶ randomized nucleic acid library, through an in vitro molecular evolution method called Systematic Evolution of Ligands by EXponential enrichment (SELEX).^3,4

Dubbed as nucleic acid antibodies, the best-elected aptamers possess notable advantages over protein-based antibodies, such as better stability, high affinity and specificity, more structural flexibility for various types of chemical modifications, and being small in size (approximately 10 to 30 kDa).^5,6 These properties allow aptamers to bind stably to more specific epitopes, especially at low antigen density.⁶ Remarkably, aptamers also demonstrate a cell-internalizing property in native living cells, which means that they could directly enter the cells without the need for permeabilization, via a receptor-dependent system.⁷

Despite their distinct advantages, however, applications of aptamers are still less explored in comparison with antibodies (antibodies have been studied for more than a century). Antibodies have been largely exploited as labelling and imaging agents, in addition to being popularly used as a carriage in targeted drug delivery via antibody–drug conjugates (ADCs).⁸ While antibodies are proven to be powerful tools in diagnostic and therapeutic applications, they are not without limitations. For instance, most monoclonal antibodies can only be produced biologically in animals with high manufacturing cost, in addition to complicated quality control. Antibodies also suffer from poor cell/tissue penetration due to their large size (~150 kDa), which then makes intracellular target labelling under native conditions not feasible. Consequently, in recent years, researchers have been attracted to the utilization of cell-internalizing aptamers as pharmacological agents in diagnostics and therapeutics.

Internalization of aptamers is particularly crucial for in vivo applications in targeted delivery or therapeutics. Several comprehensive reviews on modification strategies and applications of modified intracellular aptamers have recently been published. Hirao's group reported developments in the creation of artificial extra base pairs for enhanced properties of DNA aptamers.⁹ In early 2021, Hollenstein's group discussed non-native modifications along with the challenges faced in the design and synthesis of novel modified oligonucleotides.¹⁰ Rossi's and Obika's groups reported nice reviews on versatile intracellular applications of aptamers and biological applications of nucleic acid analogues, respectively.^11,12 In this review, hence, we will focus our discussion on the various methods for generating cell-internalizing aptamers particularly via cell-based SELEX. Importantly, we will highlight the internalization property of the aptamers, along with their uptake mechanisms and subcellular localization in cells.

II. Aptamer generation by cell-SELEX

Aptamers are largely generated in vitro through the SELEX technique, which provides a tunable process in a controlled evolutionary manner, by increasing the stringent conditions to select high-affinity and high-specificity aptamers to targets of interest. A randomized oligonucleotide library with 10¹⁴–10¹⁶ diversity is used to isolate oligonucleotides that bind to the selected targets, such as small molecules, sugars, peptides or proteins.^3,4,13,14 Once aptamer sequences are obtained by SELEX, the winning aptamers are synthesized chemically in a large scale production.

In aptamer isolation against proteins, the SELEX process requires high-quality purified recombinant proteins to eliminate off-target binders. However, some pharmacologically relevant proteins, such as G protein-coupled receptors (GCPRs) and serine/threonine kinase receptors, cannot be fully purified in their intact forms, due to their complexity in the purification process.¹⁵ Additionally, when a protein-binding aptamer is obtained via SELEX using recombinant proteins, more often than not, the aptamer would bind to the region of the proteins involving their membrane-embedded and/or intracellular domains, not exclusively to the preferable extracellular cell-surface domain. Consequently, this method is not preferable for cell-surface protein targets.

To this end, the SELEX method against whole living cells (termed as cell-SELEX) has emerged. Cell-SELEX allows the generation of aptamers which bind to the cell-surface areas of target molecules in their native state without prior knowledge of the target identity.^16,17 Since the aptamer recognizes its targets in their native conformations on the target cells, it also leads to the discovery of novel biomarkers and unexpected cellular events.¹⁸ In addition, target recognition for biological applications in in vivo settings is more likely. Toward practical use in the required settings, high affinities to the targets are also one of the key challenges to be attained.

Similar to SELEX targeting simple molecules, the process for cell-SELEX involves repeated cycles of selection and amplification (Fig. 1). The main key process can be divided into three steps for each cycle:

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Fig. 1

Simplified schematic representation of the cell-SELEX process. Each cycle starts with incubation of the oligonucleotide library with the target cells, followed by removal of the unbound oligonucleotides by washing to collect the cell-bound oligonucleotides, and finally ends with amplification of the cell-bound oligonucleotides by PCR.

i) incubation of an oligonucleotide library with target cells;

ii) removal of unbound oligonucleotides and collection of the cell-bound oligonucleotides;

iii) amplification of the cell-bound oligonucleotides by PCR for preparation of the next selection–amplification cycle.

The cycle is repeated for several rounds until the oligonucleotides that specifically and tightly bind to the target cells are sufficiently enriched, and they are subsequently sequenced to identify the isolated aptamer candidates. Usually, a pre- and/or post-counter selection step by using control cells with no potential targets of interest is implemented in the selection cycles, to eliminate nonspecific binders. This step will then enhance the target specificity of the enriched oligonucleotides.

A. Challenges in aptamer generation using cell-SELEX

The cell-SELEX technique is a rather complex process and has some technical challenges. The protocol depends on the quality and viability of the cells used during the selection process. Hence, proper cell culture maintenance is crucial. This includes appropriate growth media conditions and consistent cell confluency and cell passage number used in the procedure.¹⁹ Poor cell culture conditions and prolonged cell growth would not only affect the cell morphology, but also perturb the native conformation and expression level of target proteins on the cell surface, which consequently reduce the efficiency of aptamer generation by cell-SELEX.^1,16

Interference from dead cells is highly undesirable during the selection process. The presence of dead cell population would lead to nonspecific binding of the aptamers which causes false positive sequences (due to enrichment of off-target or unwanted sequences), resulting in selection failure.^19,20 Mayer's group reported the implementation of the fluorescence-activated cell sorting (FACS) technique in the selection procedure to separate certain subpopulations of cells from other subpopulations, such as dead-cell/live-cell populations, within cultured cell mixtures.¹⁹ This technique has been shown to serve as a more efficient route for reducing false positives in cell-SELEX.

False positives can also arise due to nonspecific internalization of DNA aptamers into target cells when incubation is performed at physiological temperature. For this reason, the aptamer–target cell incubation process is commonly performed at 4 °C, despite the concern that aptamers selected at low temperatures may display dissimilar binding affinity and specificity to those selected at physiological temperatures.¹⁶

Some other approaches emerged to eliminate false positives in aptamer selection. For instance, Tan's group recently developed a strategy to remove nonspecific and weak binders from the living cell surface via aptamer displacement reaction using complementary DNA (cDNA), toehold-mediated cDNA (tcDNA), and single-stranded binding protein (SSB).²¹ In the study, they demonstrated that nonspecific cell surface-bound ssDNAs interact tightly with SSB, while the winning aptamer sequence binds to its target cells firmly (Fig. 2A). On this basis, non-specific binders with low affinity can be separated from target cells by displacement reaction, and eventually could be eliminated from the selection process by washing or centrifugation. This hence improves the efficiency of cell-SELEX. Note that fast kinetics to minimize the internalization of nonspecific aptamers is essential for the success of the displacement reaction.²¹

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Fig. 2

A) Schematic representation of SSB-assisted cell-SELEX and conventional cell-SELEX. B) Schematic illustration of the cell-internalization SELEX.

B. Cell-SELEX and its variety

Cell-SELEX technology has become increasingly popular. Many aptamers are generated through this technique on more than 80 different cell lines targeting various proteins.¹⁸Table 1 summarizes some examples of aptamers obtained by cell-SELEX and their identified target proteins.

Cell-specific aptamers obtained by cell-SELEX, and their target proteins. The aptamers' names along with their types and sequence/nucleotide lengths are included

Target cell	Target protein	Aptamer name	Aptamer type	Aptamer length	Ref.
T cell acute lymphoblastic leukemia CCRF-CEM cells	Protein tyrosine kinase-7 (PTK7)	sgc8	DNA	88	45
Acute myelogenous leukemia NB4 cells	Sialic-acid-binding immunoglobulin-like lectins (siglec-5)	K19	DNA	285	46
Burkitt's lymphoma Ramos B cell lines	Immunoglobulin heavy mu	TD05	DNA	48	47
Glioblastoma U251 cells	Tenascin-C	GBI-10	DNA	69	48
Differentiated neuroblastoma SH-SY5Y cells	Neural cell adhesion molecule L1 (L1CAM)	yly12	DNA	51	49
Lung adenocarcinoma cells	Cathepsin D	LC-183	DNA	80	50
Breast cancer MCF-7 cells	Annexin A2	ACE4	RNA	95	51
Pancreatic adenocarcinoma PL45 cells	CD71	XQ-2d	DNA	56	52
Ovarian cancer TOV-21G cells	Stress-induced phosphoprotein 1 (STIP1)	TOV6	DNA	84	53
Glioblastoma U-87 MG cells	Integrin α5β1	H02	RNA	75	54

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While the cell-SELEX strategy has successfully generated aptamers as promising tools in cell detection and therapy,^22,23 there are two major issues that may limit their usefulness for greater applications as delivery and therapeutic agents: 1) susceptibility of aptamers to degradation by serum or nuclease, and 2) inefficient internalization by living cells.²⁴

Several research groups have attempted to address these problems by improving the selection techniques. Giangrande's group modified the cell-based selection process, termed cell-internalization SELEX, to optimize the cellular uptake by incubating the oligonucleotide library at 37 °C for a period of time and subsequently removing the unbound and surface-bound aptamers by stringent 0.5 M NaCl salt washing (Fig. 2B).^25,26 This approach enables the recovery of rapidly-internalized aptamers while eliminating non- and slowly-internalized binders. Thiel et al. generated aptamers that are efficiently internalized into target cells upon binding with their target protein, human epidermal growth factor receptor 2 (HER2). The aptamers were further applied to deliver chemo-sensitizing siRNAs targeting anti-apoptotic gene Bcl-2 in breast cancer cells.²⁵ In another study, insertion of G-rich sequences at the 3′-terminus was largely found to increase cellular uptake, especially for standard phosphodiester oligonucleotides.²⁷

Adding functionality to natural nucleotides could enhance their binding affinity and/or specificity, which enlarges their structural properties and expands their physicochemical properties targeting the cells. The addition can be by introducing either modified nucleotides or unnatural bases as the fifth- and sixth-letters.^9,10,28–39 Common strategies to modify nucleotides include modifications on the sugar ring and/or phosphodiester backbone for resisting nuclease degradation, and on the nucleobase for improving binding affinity and specificity.^40–44

Our group developed a new cell-SELEX method by introducing an additional highly hydrophobic unnatural base, 7-(2-thienyl)imidazo[4,5-]pyridine (Ds), on top of the four natural nucleotides (A, C, G, T), in a random sequence library.^35,55–58 This method using genetic alphabet Expansion SELEX is called cell-ExSELEX^34,58 and the bound oligonucleotides are subjected to PCR amplification involving the third base pair, Ds, and its pairing partner, Px (2-nitro-4-propynylpyrrole).⁵⁷ Contrasting the hydrophilic nature of nucleic acids, the unnatural hydrophobic bases strengthen the interactions between the aptamers and hydrophobic parts of target proteins, resulting in efficient aptamer generation with superior affinity.^{32,35,59–61} By using the cell-ExSELEX system, our group successfully identified several high-affinity DNA aptamers with excellent stability targeting specifically breast cancer cells, such as MCF-7, MDA-MB-231 and T-47D.⁵⁸ All of the aptamers are internalized within the bound cells. It was found that one of the aptamers, named 05-MB231, binds to all of the cancer cells that were tested but not to noncancerous cells. Interestingly, the 05-MB231 sequence contains one Ds base that is crucial for its binding affinity. This aptamer also showed outstandingly fast uptake into T-47D cells, followed by efficient internalization and distinct accumulation in the perinuclear region (unpublished data). This shows a promising property for cancer therapy.

Tan's and Benner's group reported an artificially expanded genetic information system (AEGIS) in which they introduced two nonstandard Z and P nucleotides to make a six-letter GACTZP DNA library.^36,62 The nucleotide Z contains a nitro functionality not found in the four natural nucleotides. By using the AEGIS cell-SELEX technique, they reported aptamer ZAP-2012 that binds to breast cancer cell line MDA-MB-231 with a dissociation constant value of 30 nM. Importantly, the binding affinity is reduced significantly when Z and/or P is replaced by natural nucleotides, suggesting substantial contributions of the nonstandard nucleotides to the aptamer–target binding interactions.³⁶

Obika et al. utilized cell-internalization SELEX with an artificial DNA library to generate aptamers with high cell-internalizing ability.⁶³ The library contains a hydrophobic uracil derivative (5-((3-indolyl)propionamide-N-allyl)-2′-deoxyuridine, U^trp). The study showed that all U^trp-containing aptamers are internalized into cells more easily compared to the unmodified DNA random library. The internalization ability of the aptamers is reduced when U^trp is substituted with deoxythymidine, signifying the important role of U^trp in the cell-penetrating function of aptamers. The authors then demonstrated a successful application of the base-modified aptamers to deliver antisense oligonucleotides (ASOs) into A549 cells.⁶³

The chemical structures of the modified hydrophobic bases are summarized in Fig. 3.

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Fig. 3

Chemical structures of the natural (A, T, G, C) and modified bases (Ds, Px, Z, P, U^trp) discussed in the text. R: functional groups.

III. Mechanisms of aptamer internalization and subcellular localization

Internalization of aptamers into a specific cell is one of the important properties for the development of aptamer-based therapeutics, especially in cancer research. To this end, exploration and investigation of their cellular uptake mechanism are of important interest.

Aptamers are widely generated against cell surface targets. Ideally, once they bind to the receptor targets, they would enter the cells via endocytic pathways. Endocytosis is an energy-requiring process, in which internal membranes are formed from the invagination of the plasma membrane. By this means, extracellular materials (macromolecules, liquid, and so on) hence can be internalized into the cells. One protein that largely participates in this fission of membrane process is dynamin.⁶⁴

A high rate of endocytosis is particularly critical; however, the specific mechanisms underlying aptamer movement into and within target cells have not been widely investigated. There are well-defined endocytic pathways that are involved in the internalization mechanism of aptamers upon binding with their target receptors on the surface of the cell membrane. In general, they include receptor-mediated endocytosis and macropinocytosis.^65,66 Receptor-mediated endocytosis can be divided into two types: clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CvME). CME and CvME are regulated by the activities of cargo molecules and other proteins. As aptamer internalization may involve multiple and interchangeable endocytic pathways,⁶⁷ elucidating the aptamer uptake and cellular fate is essential for a deeper understanding of its potential therapeutic and delivery activities.

Once molecules like aptamers are internalized into mammalian cells, they go through complex intracellular pathways leading to various destinations. Most, if not all, of them are transported into endocytic vessels in the cytoplasmic space and sorted at two major sorting sites, namely the early and late endosomes.⁶⁸ At the early endosomes, the molecules are either initially sorted to the route back to the cell surface through recycling endosomes or trafficked to lysosomes for degradation.⁶⁹ This intracellular journey is regulated by intricate protein machinery,⁶⁹ such as the Rab GTPase family,⁷⁰ the ESCRT complex⁷¹ and the retromer complex.⁷² Generally, most aptamers are subsequently destined to localize in the endosomes, lysosomes, Golgi apparatus, endoplasmic reticulum, and rarely in the mitochondria.¹¹

A. Clathrin-mediated endocytosis (CME)

CME is the uptake of materials bound to the cellular surface into the cells through the aid of clathrin-coated vesicles. This endocytic portal is fundamental to various plasma membrane activities and crucial to a wide range of eukaryotic cells. The soluble major coat protein, clathrin, forms a triskelion structure composed of three clathrin heavy chains, with tightly associated clathrin light chains. It then polymerizes into a lattice-like coat, which is then also described as “vesicles in a basket”.⁶⁶

Upon aptamer binding onto the receptor, CME proceeds through several stages: 1) initiation of clathrin-coated pits on the aptamer–receptor binding zone, 2) curvature and invagination of the membrane, 3) fission of the membrane that is catalyzed by dynamin, and 4) the release of clathrin-coated vesicles that are rapidly uncoated and fused with endosomes (Fig. 4A).⁷³

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Fig. 4

Mechanisms of aptamer internalization. Aptamers can be internalized into cells via several endocytic pathways: A) clathrin-mediated endocytosis (CME), B) caveolae-mediated endocytosis (CvME), and C) macropinocytosis. Most internalized aptamers accumulate in the endosomes, while some are sorted back to the cell surface or other cellular compartments such as lysosomes for degradation.

The common method to investigate the aptamer uptake by CME is by using internalization inhibitors, such as dynasore (a dynamin inhibitor) and chlorpromazine.^11,74 DNA aptamer R13, which targets ovarian cancer cells, was reported to possess good targeting ability in tissue sections. The time-dependent internalization of R13 indicated an active uptake process and its uptake occurs through the CME pathway. In the study, it was found that the internalization of R13 decreased significantly in target cells pre-treated with dynasore (inhibitor of CME), but no change upon treatment with amiloride (inhibitor of macropinocytosis).⁷⁵

The application of aptamers as targeted delivery tools has now become more apparent.⁷⁶ Aptamers with defined structures would allow assembly with drug molecules, fluorescent dyes or nanoparticles, without loss of function.⁷⁷ RNA aptamer A10, which has 2′-fluoro modification in pyrimidines, has extensively been studied as a delivery tool. It recognizes prostate-specific membrane-antigen (PSMA) on prostate cancer cells.⁷ Aptamer A10 was shown to deliver drug-encapsulated nanoparticles to treat tumors in mice and also to carry siRNA molecules into tumor sites, thus possessing mRNA knockdown activity.^78,79 This cargo delivery occurs upon the binding of A10 onto cell surface PSMA through a CME mechanism.

The CME mechanism of aptamers is also examined by colocalization studies with fluorescently labeled transferrin. A G-rich DNA aptamer C10.36 (36-nucleotide length) was reported to recognize Burkitt's lymphoma cells selectively. Aptamer C10.36 was folded into a G-quadruplex structure and taken up by the cells with a relatively fast kinetics (a maximum uptake was reached after 15 minutes).⁸⁰ The colocalization study revealed high staining correlation of C10.36 with transferrin, in which its cellular uptake is mainly characterized via clathrin-mediated endocytosis.⁸¹ Moreover, when the cells were pre-incubated with dynasore, the C10.36 uptake was greatly reduced.⁸⁰

IV. Conclusions and outlook

Many studies have demonstrated the high potential of nucleic acid aptamers for their use in diagnostic and therapeutic fields. They exhibit target specificity, in addition to being relatively small in size, non-immunogenic and easily stabilized through chemical modifications. Since its first discovery about three decades ago, aptamer technology has shown great potential in producing bioactive and biostable pharmacological agents, as evidenced by the number of aptamers that successfully entered clinical trials and by the rapidly growing aptamer industry.^5,103,104

Internalization of cell-binding aptamers is an important aspect for in vivo practical applications in targeted delivery or as drug candidates. In this review, we have discussed methods to generate cell-internalizing aptamers, along with their challenges and improvement strategies. Importantly, we have highlighted the cellular fate and uptake mechanism of the aptamers to better understand their mechanism of action.

Although significant achievement has been accomplished to date, there is still only one therapeutic aptamer that has been approved by the US FDA for clinical use (aptamer pegaptanib sodium/Macugen for patients with age-related macular degeneration).¹⁰⁵ For practical use, high affinity and stability of aptamers would be required. With the vast growing improvement tactics to tackle major issues of aptamer utilization in vivo (such as susceptibility to degradation by serum or nuclease, and inefficient cellular internalization), it is anticipated that aptamer technology, especially cell-internalizing aptamers, will progressively find use as a new class of diagnostic and therapeutic practical agents.

Author contributions

S. H. A., M. K., and I. H. conceived and planned this review. S. H. A. took the lead in writing the manuscript and prepared the first draft including all the figures. All authors provided critical feedback and helped finalize the manuscript.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

Acknowledgments

References