Nucleic acid aptamers are single-stranded DNA or RNA molecules, for example, Broccoli aptamer [3] or Spiegelmer / L-RNA aptamer (with L-ribonucleic acid ) [4], which bind to organic or inorganic molecules and are characterized by high specificity to the target molecule [5]. Due to the three-dimensional structure of their molecules, aptamers can bind to the targets via hydrophobic and van der Waals bonds. There is a large variety of target molecules for aptamers, including ions, nucleotides, peptides, antibodies and cell membrane compartments. Aptamers are developed as an alternative to antibodies in medical and basic research technologies.

Aptamers have certain advantages over antibodies [6]. The stability of aptamers is higher. In terms of manufacturing, the generation of aptamers is a cheaper, easier and faster process. Besides, from the ethical point of view, animals are not required for their production.

Moreover, the specificity and thus variability of results are significantly lower, since aptamers are chemically synthesized. Also, conjugation linkers and detection labels can be inserted at specific positions of the nucleotide chain [7]. Fica SM et al cloned three copies of MS2 coat protein aptamer at the 3´-end of MINX pre-mRNA and utilized it to affinity-purify P complex [8]. Aptamers are resistant to high temperatures and their regeneration can be easily performed and repeated. Besides, complete automatization of the synthesis and selection process is easy to achieve [9]. With regard to the size, aptamers are smaller than antibodies and therefore can be easily transported and penetrate tissues better than antibodies. DNA aptamers, upon the binding of small molecules, are resistant against exonuclease III and I, and can be detected via SYBR Gold, or when multiplexed, by sequence-specific molecular beacons [10]. Litke JL et al designed the Tornado (Twister-optimized RNA for durable overexpression) expression system which enables rapid RNA circularization and renders RNA aptamers with high stability inside cells [11].

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Figure 1. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. After incubation with the target, the DNA or RNA sequences with the highest specificity are selected, followed by the PCR amplification for DNA molecules or transcription into DNA and then PCR for RNA molecules. Normally, 12-15 selection cycles are applied to receive the aptamers which will be cloned and sequenced [1].

There are two general methods for aptamer selection. Aptamers are made in the technique called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) invented in 1990 [12, 13]. Target-specific sequences are obtained by the cycles of selection and replication. The library containing sequences with a particular length is incubated with target molecules. This incubation leads to binding of aptamers with the highest affinity with the target molecule. During the process, only nucleic acids bound with target compound are selected and undergo replication, while the other nucleotides are removed from the sequence library (Figure 1). To reduce the selection period, the SELEX method can be modified by combination with surface plasmon resonance or capillary electrophoresis [14, 15]. Specific aptamers can be generated for molecules, bacteria, viruses and cells. The binding to the target molecules is dependent on the secondary and tertiary conformation of the aptamers.

Besides, aptamers can target whole cells. The process generating aptamers against cells of interest is named cell-SELEX. This method can be used to distinguish tumor cells from normally differentiated cells [16, 17]. In this case, aptamers are generated against the proteins located on the cell surface. Thus, the cell-SELEX method creates a pool of aptamers generated against cell surface proteins in their normal conformation. The selection step of this method includes incubation of the aptamer with normal and target cells. Initial incubation with normal control cells removes the aptamers binding to the cell surface proteins on control cells. During the next step, the remained aptamers are screened against target cells. The selected aptamer library can be evaluated using a fluorescence-labeled DNA library and the cells are assessed by fluorescence microscopy or flow cytometry. The final step includes cloning, selection and synthesis of detected sequences. The cell-SELEX approach was used to develop aptamers against tumor cells and different tissues [18].

To achieve simultaneous measurement of affinity and specificity for many candidate aptamers, Cho et al. developed Quantitative Parallel Aptamer Selection System (QPASS) [19]. This method combines the SELEX method and next-generation sequencing and allows identification of specific aptamers followed by in situ synthesis. Importantly, the time used for binding evaluation does not depend on the number of analyzed potential aptamers.

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Figure 2. Bead-based selection process. The method includes incubation of the bead-conjugated library with a fluorescently labeled target protein molecule, separation of the protein-aptamer complexes, removal of the target protein and PCR amplification and sequencing of bead-conjugated aptamers. The bottom figures demonstrate the screening of bead-conjugated aptamer library: (a) analysis of bead-conjugated aptamer library using light microscopy; (b) identification of the aptamer linked to the target protein under a fluorescent microscope; (c) separation of a positive bead under a fluorescent microscope [2].

Since it may be difficult to amplify some of the oligonucleotides using PCR, another method of aptamer called a bead-based selection was developed (Figure 2). In this method, a bead-based oligonucleotide library is created using noncleavable beads. This step is followed by incubation with the fluorescently labeled protein and protein-linked aptamer beads can be selected by a fluorescence microscope or flow cytometry [20].

The Slow Off-rate Modified Aptamers (SOMAmers) are short deoxyoligonucleotides, which bind to peptides or proteins and can be isolated in vitro. SOMAmers are generated using libraries of modified dUTPs inserted into the DNA sequence to produce high specificity and binding affinity and specific secondary structures [21]. SOMAmers are designed to detect protein targets with high affinity and can be efficiently used in proteomics. This technique applies synthetic nucleotides structurally similar to amino acid chains and SELEX methodology to isolate aptamers with slow off-rates. Frottin F et al incubated HEK293T with GFP-SOMAmer from SomaLogic (which became a public company in 2021) to enable DNA-PAINT under super-resolution microscopy to establish nucleolus as a phase-separated protein quality control entity [22].

A multiplex proteomic assay using SOMAmers was used as a proteomics platform for biomarker identification [23]. The assay was performed to quantify proteins in proteomics samples in solution due to better kinetic characteristics of binding and dissociation [24]. The specimens, selected for the assay, undergo incubation with SOMAmers linked to both biotin and fluorescent marker, followed by binding of SOMAmer-protein complexes to streptavidin beads. The complexes are separated from the beads by exposure to UV light, followed by binding to the second type of beads, Catch-2. Finally, the SOMAmers are released from the beads and can be hybridized to specific nucleotide probes using a DNA microarray technology. The detection of SOMAmers by microarray allows quantification of proteins in biological samples.

A multiplex affinity assay, called SOMAscan, is also based on SOMAmers, which bind initial tertiary structures of proteins [25]. SOMAscan was applied to identify biomarkers for diseases such as lung cancer, mesothelioma, chronic kidney disease, tuberculosis and Alzheimer’s disease [26] or serum proteins [27, 28]. In addition, exosomal proteins associated with prostate cancer were identified using SOMAscan [29]. SOMAscan may be applied to different methods of laboratory diagnostics due to the moderate size, structural stability, easy production and low lot-to-lot variability of SOMAmers. These characteristics make SOMAmers suitable for analytical platforms and laboratory methods, including histochemistry [30], molecular imaging [31] and affinity purification [32].

Proteomics techniques using antibodies are considered to be specific and reliable. However, cross-reactivity of antibodies is the reason for the notable limitations of multiplex proteomics platforms [33]. Due to their constraints, antibodies can be replaced by aptamers in Western blotting [34] and Enzyme Linked Oligonucleotide Assay (ELONA) [35, 36] and protein chips for the identification of unknown proteins from biological samples [37]. In particular, aptamers were applied for the identification of potential biomarkers for chronic kidney disease [38] and non-small cell lung cancer [23].

Recently developed aptamer sensors include microarray slides and flow cells using quartz crystal detection systems [39, 40]. Interestingly, following measurement, immobilized on sensors aptamers may be denatured to strip off bound analyzed molecules and after that refolded into active conformations by washing in specific buffers. Moreover, it is relatively easy to add some linkers between the aptamers and the chip surface without changing the functional regions of the aptamers [41].

To determine biomarkers of cardiac allograft vasculopathy, serum proteins were screened using Slow Off-rate Modified Aptamer (SOMAscan) assay [42]. Bioinformatics studies have identified 14 vasculopathy biomarkers, which are involved in several crucial mechanisms, such as inflammation, thrombosis and apoptosis. The observed biomarkers demonstrated significant discriminative ability for cardiac vasculopathy. In addition, Nayor et al have applied DNA aptamer-based proteomic platform to detect circulating markers of cardiac remodeling and heart failure [43]. N-terminal proB-type natriuretic peptide, thrombospondin-2 and mannose-binding lectin were found to be associated with high risk of cardiac failure.

Another recent study has shown that aptamer-based proteomics can be used to analyze urine protein levels for patients with urinary tract infections [44]. The authors have applied machine learning to select the specific urine biomarkers, such as B-cell lymphoma protein, cathepsin S, heat shock 70kDA protein 1A, mitogen activated protein kinase and transgelin.

Aptamer-Facilitated Biomarker Discovery (AptaBID) applies aptamers for biomarker discovery. In this technology, the aptamers are produced for cell surface markers considering their native conformation. The specific characteristics of AptaBiD include multiple rounds of aptamer production, which reduce possible variations in cellular populations. The AptaBID method includes three main steps: 1) multiple rounds of aptamer selection, 2) separation of biomarkers from target cells, 3) identification of cellular biomarkers using mass spectrometry. Besides the identification of biomarkers, these aptamers can be applied to isolate or visualize the cells by flow cytometry and fluorescent microscopy and to regulate activities of cell surface receptors [45, 46]. Moreover, the aptamers made in AptaBID can be used as delivery vehicles for transporting therapeutic compounds to damaged tissues [47].

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Figure 3. ELASA assays. (a) Direct ELASA applies biotin-conjugated aptamer and streptavidin–HRP. (b) Indirect ELASA applies the aptamer acts as the detection agent, while antibody connects the aptamer and the target protein. (c) In aptamer–target–antibody, the aptamer functions as the capturing agent and the antibody functions as the detecting agent. (d) In antibody–target–aptamer, the antibody functions as the capturing agent and the aptamer functions as the detecting agent [1].

Aptamers can be effectively used in both biotechnology and clinical medicine. They can be beneficial for diagnostic methods in clinical oncology and ophthalmology. Aptamers can replace antibodies in different detection methods. In particular, aptamers are used in an ELISA-based detection assay called ELASA (Figure 3).

In addition, aptamers bound to gold electrodes were used to identify cancer cells by the highly sensitive electrical method of detection [48]. The aptamers were applied to detect the epidermal growth factor receptor (EGFR) expressed on the surface of many cancer cells. This technique has a lower cost and higher sensitivity compared to other methods of EGFR detection.

Recent studies show potential applications of aptamers in cancer therapy. Aptamers can affect signaling pathways and transcription factors, block specific receptors and facilitate immune surveillance. In particular, aptamers binding cytotoxic gelonin were applied to specifically target prostate cancer cells [47]. In addition, an aptamer conjugated with complement system member C1q was applied to target breast adenocarcinoma cells [49]. Using an electron microscope, the authors demonstrated osmotic tumor cell damage caused by fixation of aptamer-C1q complex on the cellular membrane. The study showed the ability of aptamers to activate an anti-tumor immune response. Interestingly, McNamara et al. synthesized the 4-1 BB protein-binding aptamers which co-stimulate CD8+ T-lymphocytes to suppress the growth of tumor cells [46]. Also, aptamers targeting nucleolin and EpCAM on cancer cells were recently developed [50].

Besides, a complex of a DNA aptamer with doxorubicin was demonstrated to bind HER2-expressing breast cancer cells [51]. Since HER2 is highly expressed by breast and lung tumor cells, this method may generate new therapeutic approaches for these malignancies.

Several DNA aptamers with increased structural diversity were developed to probe the conformation of recombinant human erythropoietin products [52]. The generated aptamers were shown to identify erythropoietin products by differences in binding and equilibrium affinity parameters. In addition, SOMAmers are actively applied for proteomics studies. Daniels et al. have reported application of the SOMAscan assay for synchronous evaluation of various plasma proteins with solid reproducibility [53].

Macugen® is one of the most effective applications of aptamers in ophthalmology. Aptamer pegaptanib-containing Macugen® is used in ophthalmology for treatment of choroidal neovascularization, which is characterized by the elevated levels of the vascular endothelial growth factor (VEGF). Binding of pegaptanib to VEGF prevents VEGF/ VEGF receptor interactions and following signaling events [54]. Importantly, pegaptanib selectively targets only pathological vascular growth-dependent neovascularization but does not affect normal physiological mechanisms of VEGF-mediated formation of blood vessels. Moreover, aptamer pegaptanib does not induce any side effects including hemorrhage and increased blood pressure, which are common for other VEGF inhibiting drugs [55].

A recent study investigated whether AS1411, a nucleolin-binding DNA aptamer can affect corneal neovascularization. The authors have reported that AS1411 significantly suppressed neovascularization [56]. Moreover, AS1411 aptamer inhibited the induction of vasculogenic miR-21 and miR-221.

Aptamers may be applied to detect viruses or cells infected with viruses [57]. Moreover, they can interrupt different stages of viral replication and inhibit binding of the viruses to the target cells [58]. Several studies suggested a potential application of aptamers in both diagnostics and therapy of viral infections, such as hepatitis B and C viruses [59, 60], human immunodeficiency virus [61] and H5N1 avian influenza [62]. In addition, human papillomavirus is also a target virus for aptamers. In particular, an RNA aptamer binding papillomavirus EG7 oncoprotein was obtained for diagnostic purposes [63]. Thus, aptamers may be effective as both therapeutic compounds blocking viral amplification and diagnostic reagents.

Also, Song et al have described aptamers, which had high-binding affinity to SARS-CoV-2 RBD [64]. The Kd parameters for CoV2-RBD aptamers were 5.8 nM and 19.9 nM. The study has suggested that those aptamers might have the same binding sites at ACE2 on SARS-CoV-2 RBD. In addition, Woo et al have developed a fluorescence-based assay, which applied aptamers for detection of SARS-CoV-2 RNA. In this assay called sensitive splint-based one-pot isothermal RNA detection, the aptamer is used to detect viral RNA. The technique includes a ligation reaction and transcription by T7 RNA polymerase. The produced transcript generates an RNA aptamer, which binds to a dye and induces fluorescence only if the target RNA is present [65].

Another study has developed ssDNA aptamers aiming to detect Newcastle disease virus (NDV) [66]. The selection of aptamers was performed by the systematic evolution of ligands by exponential enrichment (SELEX) combined with DNA sequencing. The selected aptamer sequences were applied to generate a sandwich enzymatic linked aptamer assay (ELAA). The results of the assay were confirmed by quantitative real-time PCR, showing significant accuracy.

With regard to bacterial infections, aptamers might be used for the development of screening tests for the diagnosis of methicillin-resistant Staphylococcus aureus. Fan et al. have reported the selection and analysis of DNA aptamers, which bind to the penicillin binding proteins [67]. In addition, Zhong et al. have suggested that the dual-aptamers labeled polydopamine-polyethyleneimine dots would be effective to detect Pseudomonas aeruginosa [68]. This novel biosensor showed a linear response to Pseudomonas aeruginosa and was suggested to be used to detect the pathogen in various food products, including milk and juice.

Peptide aptamers are small stable proteins with a high-affinity binding surface for a specific protein. Affimers are an important alternative to antibodies and nucleic acid aptamers. Dissociation constants of peptide aptamers are similar to or better than those of antibodies. Peptide aptamers are usually applied as disrupters of protein-protein interactions. Peptide aptamer libraries can be based on yeasts, yeast two-hybrids, bacteria and retroviruses. Peptide aptamer techniques aim to screen for peptide aptamers that can interact with a protein of interest and generate a specific phenotype.

Peptide aptamers against different protein targets were used for both in vitro and in vivo biomedical studies. Affimers against K6- and K33-linkage-specific ubiquitins have been generated and verified through their crystal structures, remedying the lack of unique antibodies against these two linkage types in ubiquitin research [69]. Several affimers were shown to inhibit the viral capsid formation, DNA replication and virion production of hepatitis B virus [70]. In addition, peptide aptamers were generated to bind CDK2, a vital cell cycle factor for progression from G1 into S phase. These peptide aptamers were demonstrated to increase the number of cells in the G1 phase [71]. Affimers displayed higher specificity in purifying IL-37b and proinsulin from human plasma than corresponding monoclonal antibodies [72].

Moreover, affimers were used to influence the activity of the E2F transcription factors which play an essential role in cell cycle regulation and regulation of the switch between division and differentiation. In particular, affimers synthesized by Fabbrizio et al. were shown to prevent E2F binding to DNA and inhibit fibroblasts growth in vitro [73]. Also, several new affimers targeting GTP-binding proteins Ras [74] and Rho [75] and the BCR oncogenic protein [76] have recently been generated.

With regard to the forward genetics, several studies identified peptide aptamers targeting a pheromone pathway of Saccharomyces cerevisiae [77, 78]. These studies suggested possible applications of affimers in dissecting signaling pathways. In addition, peptide aptamers were identified in a screen for resistance to trimethoprim in cells dependent on thymidine [79]. The specificity of peptide aptamers was confirmed by yeast two-hybrid method. Besides, both bacteriostatic and bacteriocidal peptide aptamers were isolated by screening for affimer toxicity to Escherichia coli [79].

Although affimers act similarly to antibodies with regard to binding to proteins, they were found to be more stable at low pH and with heat. Recent studies suggested potential applications of affimers, including modulation of extracellular receptors, targeting specific proteins in vivo, improving super-resolution microscopy, targeting small organic compounds and different in vitro assays.

With regard to the suppression of the activity of extracellular receptors, affimers were demonstrated to bind VEGFR2 molecules involved in the regulation of angiogenesis [80]. Importantly, affimers showed higher sensitivity to the target molecules compared to antibodies and suppressed activation of VEGFR-regulated pathways. In addition, affimers can be used for in vivo imaging of tumor tissues due to their fast clearance characteristics [80]. Moreover, new methods potentiating the binding of affimers in vivo have recently been suggested [81].

Affimers are applied to high-resolution microscopic techniques. In particular, labeling of affimer molecules in a site-dependent way allows the closest location of the fluorophore to the target molecule and improves the results of super-resolution imaging. For instance, nanobodies were used for the detection of nuclear pore proteins and other target molecules [82, 83]. Moreover, small organic molecules can also be targets for affimers. Successful isolation of affimers specifically targeting 2,4,6-trinitrotoluene was recently reported [80]. In addition, affimers may replace antibodies in different in vitro assays. For instance, an ELISA applying affimers was developed for the determination of fibrotic markers. With regard to gene expression analysis techniques, affimer-based microarrays were also designed and may be used in clinical diagnosis [84].

Designed ankyrin repeat proteins (DARPins) are a group of non-immunoglobulin binding molecules, which are made of closely packed repeats of amino acid residues. Ankyrin repeat domains have solenoid conformation with a hydrophobic core. There are multiple applications of DARPins as tools in biochemistry, diagnostics, and therapies since they show high robustness and extreme stability.

Basic research applications include fluorescent binding molecules, crystallography, and biosensors. As to new binders against fluorescent markers, the generation of DARPins against the monomeric teal fluorescent protein 1 (mTFP1) has recently been reported. These anti-mTFP1 DARPins have been applied to translocate Rab proteins to the nuclei [85]. Another powerful application of DARPins is the generation of diffraction-quality crystals for X-ray crystallography. For instance, binding of DARPins to maltose binding protein (MBP) leads to the crystallization of an MBP fusion protein. Three crystals of MBP fused with human phosphatase 1 bound to DARPins have been described [86]. Meksiriporn B et al. devised a survival selection strategy to design DARPins against post-translationally phosphorylated proteins [87]. Radom F et al. developed software to predict the complexing of DARPins with its cognate targets [88].

In addition to basic research applications, DARPins have been used for diagnostic purposes. Breast tumor tissues have been analyzed by immunohistochemistry and immunofluorescence for HER2 receptor expression using HER2 receptor-specific DARPins [89]. Also, DARPin G3 has been labeled with 111In and used for positron emission tomography for detection of HER2-positive tumor cells [90]. Also, an anti-CUB domain-containing protein 1, a protein upregulated in tumor cells, has been targeted by several DARPins in lung carcinoma cells [91]. Besides, cathepsin B, a valuable diagnostic marker in tumors and inflammatory disorders, has been detected by optical imaging using the selective DARPin 8h6 in mouse models [92]. Notably, DARPin 8h6 has been shown to bind cathepsin B with picomolar affinity.

Several therapeutic applications of DARPins have recently been reported. With regard to allergy, neutralizing DARPins against IgE have been generated to decrease IgE levels [93]. Also, the epidermal growth factor receptor (EGFR), which is known to be involved in tumorigenesis, has been targeted by DARPins [94]. In addition, DARPins have been shown to act as antigen-binding regions in chimeric antigen receptors (CARs). For example, CAR-engineered T cells employing HER2-targeting DARPins G3 and 929 bind to HER2/expressing tumor cells and inhibit their growth [95]. Some of the DARPins are presently being evaluated in clinical trials. In particular, MP0250, a potential anti-tumor DARPin-containing drug, has binding specificities for vascular endothelial growth factor-A (VEGF-A) and one anti-hepatocyte growth factor (HGF) [96]. Also, Abicipar Pegol, an anti-VEGF DARPin, has been effectively used to treat polypoidal choroidal vasculopathy [97]. In addition, a group of DARPins have been generated to inhibit MET kinase activity and downstream signaling [98]. PASylation and XTENylation can extend the serum half life of DARPins [99].

Non-antibody protein binding/capture reagents are effectively applied in areas of biotechnological research and therapy (Table I). For instance, nucleic acid aptamers are used in laboratory research, clinical diagnostics and therapy of cancer and cardiovascular and viral diseases. Generation of aptamers is a cheap process, and they have advantages over antibodies. Therapeutic compounds may be conjugated with aptamers and used for target therapies in oncology. Moreover, recently generated aptamer-conjugated compounds can be used for both imaging and treatment. Besides, peptide aptamers are effectively used for both in vitro and in vivo biomedical studies. Transport nutrients, such as RBDs, are applied to analyze cell metabolism and characterize drug efficacy. These advantages show that non-antibody protein binding/capture reagents can be used for new diagnostic methodologies and therapeutic strategies.