Updates on Aptamer Research

For many years, different probing techniques have mainly relied on antibodies for molecular recognition. However, with the discovery of aptamers, this has changed. The science community is currently considering using aptamers in molecular targeting studies because of the many potential advantages they have over traditional antibodies. Some of these possible advantages are their specificity, higher binding affinity, better target discrimination, minimized batch-to-batch variation, and reduced side effects. Overall, these characteristics of aptamers have attracted scholars to use them as molecular probes in place of antibodies, with some aptamer-based targeting products being now available in the market. The present review is aimed at discussing the potential of aptamers as probes in molecular biology and in super-resolution microscopy.


Introduction
Nucleic acids (NAs) were for a long time considered compounds whose major functions were related to the storage of inherited information (DNA) and its transfer from gene to protein through RNA [1,2]. However, studies have over time confirmed that NAs perform other functions such as enzymatic catalysis and the regulation of transcription. The discovery of these additional functions of NAs has compelled the scholarly community to reconsider its original position concerning the functions of NAs [1]. Consequently, this change has led the scientific community to propose the "RNA world theory" [3][4][5]. In particular, this theory posits that NAs possess the ability to perform multiple functions and have presumably facilitated all the catalytic reactions that started life on earth [4,6]. The discovery of aptamers is indeed a valuable contribution towards the empirical determination of the multifunctional nature of NAs [1,7]. Essentially, aptamers are small, often ranging between 20 to 60 nucleotides, single-stranded DNA or RNA oligonucleotides that are capable of binding target molecules not only with high specificity but also with high affinity [8][9][10][11][12].
Aptamers, the output of systematic evolution of ligands by exponential enrichment (SELEX) process, have been available for about three decades. In particular, the development of aptamers is attributed to the work of Craig Tuerk and Larry Gold in 1990 [13]. Gold and Tuerk focused on investigating the nature of the "translational operator" within the bacteriophage T4 gene 43 mRNA. This research led to performing the first SELEX experiment that produced two winning hairpins, the wild-type T4 sequence as well as another containing quadruple mutations, both bound to the gene 43 protein with the same affinities [13,14]. Although, Tuerk's experiments pioneered the SELEX process, the process was not named SELEX until Szostak and Ellington developed the same method and coined the term SELEX [15]. The term refers to a process that entails progressive separation of single-stranded DNAs or RNAs (ssDNA/RNA) combinatorial single-stranded oligonucleotide library via repeated rounds of binding, partitioning, and amplification [16][17][18]. It is noted that following the development of this novel technology, different modified SELEX techniques have emerged over the past three decades. Table 1 summarizes the different SELEX methods, as described in their original context. Some of these developments tackled the ssDNA/RNA library improvement, while many others addressed the development of the SELEX process. In the contemporary research environment, cell-SELEX (illustrated in Figure 1) has been extensively used to identify and select aptamers that can help in the diagnosis as well as the development of treatments for different diseases, particularly for cancer [18][19][20]. Thus, cell-SELEX technology has become increasingly important in medical research. 1992 [21] In vivo SELEX The selection and amplification steps occur inside the living cell using retroviral-based replication system Produce aptamers that are functional in vivo Provided a method to transfer aptamer selection and amplification from in vitro to in vivo 1993 [22,23] Counter SELEX Uses a second elution step against a molecule of similar target structure (e.g., caffeine, which differs from theophylline at the N-7 position) Increase aptamer's specificity and affinity towards target molecules Isolated aptamer of high-specificity to theophylline 1994 [24] cDNA-SELEX Uses a preselected natural oligonucleotide pool that binds to the protein of interest rather than using a synthetic library     Currently, a significant number of generated aptamers are indeed capable of binding different targets, including large protein complexes, simple inorganic molecules, and whole cells [10]. From this prospective, aptamers are simply considered nucleotide analogues of antibodies [11,106,107]. However, compared to antibodies, the generation of aptamers is significantly easier and cheaper. Thus if appropriately developed, these characteristics make aptamers ideal candidates for a wide variety of applications such as disease diagnosis, biosensor design, nanotherapy, molecular imaging, and the purification of particular target molecules from complex mixtures [9,10,108].
The science community has in the recent years developed super-resolution imaging techniques that offer three-dimensional imaging capabilities that overcome the diffraction restrictions of conventional light microscopy especially in viewing lateral dimensions [109,110]. For example, techniques such as single-molecule localization or stimulated emission depletion microscopy (STED) and secondary ion mass spectrometry microscopy have enabled scientists to visualize whether objects are in fact localized [111,112]. However, the advancement of these techniques requires additional improvements in the fluorescent labels to harness the power of super resolution as the image resolution is also restricted by the effectiveness of the labelling probes' size, specificity, stability, and density [113]. Indeed, the development of these novel techniques has encouraged researchers to reconsider labelling in super resolution. Aptamers' favourable characteristics have attracted scientists to consider using them in place of antibodies in super resolution. The focus of this review is to demonstrate the potential of aptamers as probing tools that can be used in super resolution.

Antibodies and Aptamers
Antibodies, as pointed out in the preceding section, are a popular class of compounds that have long been used in research to provide molecular identification for a broad array of applications, including disease diagnosis and therapy. Consequently, antibodies have made significant contributions towards the improvement of diagnostic assays [114,115]. In fact, it can be argued that antibodies have become increasingly indispensable in most of the clinical diagnostic tests in the modern medicine. However, the emergence of SELEX (outlined in Table 1) has made it possible for researchers to progressively isolate oligonucleotide sequences capable of recognizing nearly any class of target molecules not only with high affinity but with high specificity as well [115]. Although completely distinct from antibodies, aptamers are thought to emerge as a class of compounds that rival antibodies in terms of both diagnostic and therapeutic applications [116]. Indeed, the available body of knowledge has confirmed that this class of molecules mimics the properties of antibodies in several formats. Mairal et al. contended that the high demand for diagnostic assays that can effectively help in the management of present and emerging diseases has soared in recent years and it is suggested that aptamers could potentially satisfy various molecular recognition needs in these diagnostic assays [114]. Although the research on aptamers is still in its infancy, there is compelling evidence that this field of research is progressing at a fast pace [117].
The discovery of aptamers has already helped circumvent some of the key limitations associated with antibodies [118]. A critical evaluation of the present body of knowledge on antibodies and aptamers shows that these classes both have advantages and disadvantages (summarized in Table 2) over each other with multiple pieces of empirical evidence reveal that aptamers provide a broad array of clear-cut merits over conventional antibodies [115,[119][120][121].
Some of the key advantages of aptamers include their inertness towards the surrounding cells [114,122,123]. Specific aptamers are capable of binding to a target molecule with high precision and, thus, it is thought that this can facilitate a broad array of diagnostic and therapeutic applications without the fear of potential non-specific binding [114]. Another advantage is that they often have a higher binding affinity [123,124]. It is indeed acknowledged that in cases where several ligands can bind to the same receptor site, aptamers have been found to offer a relatively higher binding affinity. It is worth noting that, in practice, the higher the affinity level of a biological molecule used in molecular recognition, the less quantity is needed in molecular identification, which may reduce the costs of performing molecular identification studies [122,125,126]. Aptamers also often exhibit excellent target molecule specificity compared to antibodies [123]. According to Kedzierski et al., there are aptamers that exhibit >10,000-fold binding affinity for theophylline over caffeine [123]. There is no doubt that this excellent target specificity makes aptamers ideal bio-markers as they increase the accuracy of molecular recognition. Another advantage is that aptamers facilitate the discovery of unknown biomarkers [123,127]. Kedzierski et al. explained that different SELEX techniques are capable of identifying unknown biomarkers and consequently, this has in the recent years hastened the discovery of diseases and therapeutics [123]. Some other advantages of aptamers over the conventional antibodies include discovery time savings, minimized batch-to-batch variation, in vitro vs. in vivo testing advantages, increased stability, and reduced side effects [123].
It is worth noting that nanobodies, antibody-mimicking binders that are a single variable domain of an antibody, are also capable of specific binding and are also used in molecular targeting [128]. Previous studies have suggested that the use of affinity probes such as aptamers and nanobodies has many advantages in light microscopy, particularly in the field of super resolution [129,130]. De Castro et al. explained that steric hindrance causes less impairment on these small molecular probes and, thus, facilitates their penetration into biological samples and their binding to epitopes that are traditionally inaccessible to larger antibodies [129]. Indeed, traditional light microscopy suffers from limited resolution capabilities due to light diffraction [113,131]. Many methodologies that are capable of overcoming this limitation were developed in the recent years [113]. In fact, it has been reported that diffraction-unlimited microscopes are improving rapidly and, as a result, it is now practical to attain excellent resolutions of small elements of less than 10 nm [132]. Nevertheless, the enhancement of sample preparation and methodologies used in staining is still lagging behind [39]. The conventional immunostaining technique largely depends on affinity tools that at times are larger than the protein of interest making it impossible to fully exploit the potential of contemporary imaging techniques [129,133]. However, it is anticipated that small probes such as nanobodies and aptamers would have a significant impact on the precision of staining biological samples [129]. Recent studies have in fact suggested that when compared to traditional antibody techniques, nanobodies are capable of positioning the fluorescent molecules closer to the intended target, and thereby resulting in enhanced localization precisions using super-resolution microscopy [134,135]. Likewise, in a comparative study of aptamers vs. antibodies that involved STED, it was concluded that in super-resolution microscopy, aptamers offer superior staining of different cellular receptors. Since aptamers are cheap to select and nanobodies usually have higher affinities, it is thought that aptamers and nanobodies complement each other and together they should help advance the effectiveness of super-resolution imaging [136].
Although the use of aptamers as molecular identification compounds has emerged as a viable approach for diagnostics, therapeutics, and biosensing [124], aptamers do present a set of challenges that have inadvertently hampered both their research and commercialization. Firstly, relatively few aptamers bind small molecules [124]. It is argued that small molecules are crucial targets for research because of their clinical and commercial uses as well as diverse biological functions. Therefore, the availability of relatively few aptamers binding to small molecules restricts research on their biological functions, clinical use, and commercial use. Secondly, aptamers are likely to encounter rapid clearance rate from the circulation because of their small size as well as degradation by nucleases especially for unmodified aptamers [123]. Further, there are examples where developing a selective aptamer still represents a challenge. For example, aptamers that were selected against monomeric/oligomeric forms of amyloid peptide had very low affinity to these forms, although they exhibited a strong non-specific affinity to amyloid high-molecular weight fibrils [137,138]. It is thought, however, that aptamers still hold promise for the treatment of Alzheimer's disease because current amyloid inhibitors and antibodies are not effective at the membrane surface, where inhibition of amyloid aggregate and channel formation is needed to stop disease progression [139]. Janas et al. 2019 shows that functional exosomes containing the selected pool of aptamers can inhibit the formation of amyloid aggregates and channel activity at the membrane surface [139].
Nevertheless, aptamer microarray technology has recently been developed in an effort to overcome the drawbacks and limitations of antibody microarrays, especially when targeting small molecules with one epitope [140]. Indeed, aptamers that target small organics, peptides, proteins, viruses, cells, and bacteria are now commercially available from a number of emerging companies such as Aptagen and AMSBIO. Other companies have been established focusing on aptamers-based drug discovery such as OSI Pharmaceuticals, Nascacell Technologies in collaboration with Discovery Partners International, and Aptanomics. In addition, aptamer-based clinical trials are currently being run by different companies such as Baxter Biosciences, Corgentech, Bristol-Myers Squibb, Gilead Sciences, NOXXON Pharma, Oxford Cancer Research, Pfizer, and Regado Biosciences (reviewed in [141][142][143]), while others are developing aptamer-based diagnostics or therapeutics including Aptamer Group, Base Pair Biotechnologies, and Ophthotech Corporation [144].

Super-Resolution Imaging
For many years, light microscopy has significantly improved the scientific understanding of how cells function [113]. In fact, the different spheres of biology are believed to have developed from images that have been acquired under light microscopes. Nonetheless, studies that utilize conventional light microscopy techniques have been limited to the resolution of about 200 nm [113]. The science community has for many years focused on developing several imaging techniques to address the diffraction of light [145]. Some of these imaging techniques include multiphoton fluorescence microscopy and confocal microscopy. Fundamentally, these novel imaging techniques have not only significantly enhanced the resolution of cellular images, but have also reduced the out-of-focus fluorescence background [113]. Consequently, this has facilitated optical sectioning and three-dimensional imaging. Although these techniques have significantly improved resolution, the approaches are still limited by light diffraction [146]. In practice, these advanced techniques have often achieved resolutions of~100 nm in all three dimensions [113,146].
Recently, this limitation in light microscopy has been overcome by many improved microscopy techniques, with some of the techniques achieving resolutions of~10 nm [130]. The capability of these novel advanced microscopy techniques to circumvent light diffraction has significantly improved imaging precision [113]. Consequently, this has enabled researchers to identify important cellular details that could not be isolated using the traditional diffraction-limited instruments. STED, single-molecule localization techniques (SMLM), positron-emission tomography (PET), single-photon emission computed tomography (SPECT), and cryo-electron microscopy (cryo-EM) are some of the common diffraction-unlimited techniques that have made a significant impact in biological sciences [147][148][149][150][151]. For example, stochastic optical reconstruction microscopy (STORM), an SMLM technique, offers a high spatial resolution for the position of the fluorophore [152]. It is worth noting that large labels, for example, antibodies, can provide a misleading position of the fluorophore from the target molecule [153]. However, the discovery and subsequent use of different SMLM techniques made it possible to accurately pinpoint the position of the fluorophore from the target molecule.
In principle, STED utilizes pairs of synchronized laser pulses, which play a critical role in overcoming the challenge of light diffraction [154]. The basic functioning mechanism of this super-resolution microscopy technique is that it creates images through selective deactivation of fluorophores [155]. This deactivation reduces the illumination area at the focal point and thus improves the achievable resolution for a specific system.
PET has also remarkably revolutionized the field of super resolution, particularly in biomedical research [91,93,94]. This advanced imaging technique utilizes small amounts of radiotracers, a unique camera, and a computer to help in the evaluation of tissue and organ functions in patients [156]. It is actually possible to detect early onset of diseases by identifying changes happening at the cellular level using PET [130,[157][158][159].
Finally, cryo-EM is another super-resolution technique that is used to image frozen hydrated specimens at cryogenic temperatures [160,161]. This imaging technique allows the specimens used in super-resolution studies to remain in their native state [161]. Thus, this enables the study of fine cellular structures, protein complexes, and viruses at molecular resolution [162].

Aptamers for Super-Resolution Imaging
The enhanced imaging precision that resulted from the development of the previously discussed imaging techniques has also revealed that traditional staining using large affinity tags, for example, antibodies, are not sufficiently accurate [130]. It is argued that since aptamers are significantly smaller than antibodies, these molecules might provide a practical advantage in super-resolution imaging [129,130,163].
In their study, de Castro et al. particularly compared the live staining of transferrin receptors acquired with varied fluorescently labelled affinity probes, including the natural receptor ligand transferrin, specific monoclonal antibodies, or aptamers [130]. Analysis of the collected data showed that there were insignificant variations between the three mentioned staining strategies when imaging is carried out with traditional laser scanning confocal microscopy. Nevertheless, the aptamer tag showed clear superiority over antibodies in the super-resolved images acquired with STED microscopy. Thus it was concluded that compared to the natural receptor ligand transferrin and specific monoclonal antibodies, aptamer staining is ideal in super-resolution microscopy [130]. It is worth noting that similar observations were reported when aptamers were compared to antibodies targeting different epitopes including prostate-specific membrane antigen and EGFR [18,129,136,164].
In another study that targeted three membrane receptors that are relevant to human health and cycle between the intracellular space and the plasma membrane, de Castro et al. observed that, compared to the majority of antibodies, aptamers were capable of revealing more epitopes and thus, offered denser labelling of stained structures, which enhanced the quality of the information obtained from the images [129]. In this study aptamer labelling was advantageous under both super-resolution imaging and light microscopy imaging.
EGFR aptamer labelling also achieved a better quality in dSTORM imaging. In a recent study, aptamer labelling achieved detailed and precise structural analysis of the active EGFR that forms large clusters compared to EGFR at rest. This active-to-rest EGFR structure difference has not been detected using traditional antibodies [163]. The ability of aptamers to detect small structural changes makes them promising candidates for studying the morphological changes occur for the membrane proteins during different biological activities [163].
Additionally, in vivo PET scanning of BT474 mice showed higher uptake of 18 F-labelled aptamers that specifically bind to breast cancer expressing HER2 [165]. The confocal images also confirmed the specificity of 18 F-labelled aptamers to HER2-positive cells [165]. For strategies employed in the development of aptamer-based SPECT and PET imaging we refer the readers to an informative review by Hassanzadeh et al. [166].
Aptamers are particularly appropriate structure-changing molecules that are capable of combining highly selective biorecognition as well as signal transduction [167]. It is possible to select aptamers towards nearly any ligand molecules through SELEX and can readily be incorporated into the functional devices that boost new applications that range from DNA-machines in sensors to the bioseparations. It has been reported that binding of ligands often affects the stability of the nucleotides that the ligand directly contacts [168]. Ligand binding also causes distal rearrangements in the structure of aptamers. These structural changes can easily be observed by evaluating the cleavage patterns using cryo-EM. Aptamer-based labelling need to be done before the freezing step [162]. In their study, Stanlis et al. showed that ssDNA aptamers could be a useful tool for achieving accurate localization of specific proteins, under freeze-substitution fixation conditions, during studying the cellular fine structures using cryo-EM [169]. They indicated that aptamers are sufficiently soluble probes to be used along with the organic solvents used in freeze-substitution conditions; however, it failed to achieve high-affinity binding [169].
Apart from possessing unique physical properties that make them superior to the conventional antibodies in super-resolution imaging, there are several particular modifications that allow aptamers to achieve the highest possible resolution. Choosing an acceptable photostable probe is one of the key features dictating the super-resolution imaging quality [170][171][172]. Using stable tagged-aptamer-based probes in super-resolution microscopy allows obtaining favourable signal-to-noise ratio and also localizing the probes to specific targets without being degraded by the cellular machinery. One particular approach to achieve acceptable aptamer stability is the use of Spiegelmer technology. For example, instead of endonuclease-sensitive RNA aptamers (D-form), researchers select endonuclease-resistant RNA aptamers (L-form) that result from binding to the mirror-image of the intended target molecule [173][174][175][176]. The mirror-image aptamer (L-form) subsequently is expected to bind the natural target molecule [173,177]. Additionally, the substitution of the natural D-ribose with L-ribose renders the mirror-image aptamer completely stable. Another strategy that can be used to enable aptamers to achieve better resolution is to optimize the density of the target molecule and similarly the concentration of the library, that would increase the signal (target-aptamer interaction)-to-noise (interfering species) ratio [174,178,179].
The discovery of aptamers presents an opportunity for biomedical researchers to develop novel molecular targeting probes and effective diagnostic/treatment tools for different diseases. Undoubtedly as well, these molecules are promising tools for super-resolution microscopy and have already been shown superior to conventional antibodies in some applications.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
NAs nucleic acids SELEX systematic evolution of ligands by exponential enrichment STED stimulated emission depletion microscopy SMLM single-molecule localization techniques PET positron-emission tomography Cryo-EM cryo-electron microscopy STORM stochastic optical reconstruction microscopy