Selection Of Aptamers Research Articles

Selection of streptococcal glucan-binding protein C specific DNA aptamers to inhibit biofilm formation.

Streptococcus mutans is a commensal oral bacterium, yet its capacity for extensive biofilm formation is a major contributor to dental caries. This study presents a novel biofilm inhibition strategy by targeting GbpC, a cornerstone protein in S. mutans biofilm architecture, with specific DNA aptamers. Using SELEX (Systematic Evolution of Ligands by EXponential enrichment), we selectively targeted the extracellular domain of GbpC while incorporating structurally similar antigen I/II protein and a GbpC-deficient S. mutans strain as counter-targets to ensure high specificity. Aptamer selection was further refined through a panning method that combined primer-blocked asymmetric PCR with AlphaScreen technology. Detailed binding analyses via biolayer interferometry and microscale thermophoresis confirmed the interaction between top aptamer candidates and GbpC. Functional assays demonstrated that two lead aptamers evidently inhibited biofilm formation in wild-type S. mutans without affecting the GbpC-deficient strain, highlighting the aptamers' specificity. These results confirm that the selected aptamers retain specificity even in the complex bacterial culture matrix, validating the efficacy of our selection approach. Notably, these aptamers represent the first instance of using DNA aptamers to inhibit S. mutans biofilm formation by disrupting glucan binding. These aptamers hold promise as lead molecules for the development of biofilm-targeting therapies in dental care.

Aptamer-conjugated liposome system for targeting MUC1-positive cancer: an in silico screening approach

This study aims to overcome the adverse effects of conventional cytotoxic chemotherapy on healthy organs by developing a target-specific approach utilizing Doxorubicin (DOX)-encapsulated liposome conjugated with aptamer which has high binding affinity to the overexpressed Mucin-1 (MUC1) protein in various cancer types. To ensure optimal aptamer selection, we conducted a comprehensive in silico comparison of several MUC1-targeting aptamers, including 5TR1, MA3, S1.6, S2.2, and STRG2. As a results, the S1.6 aptamer was selected as a targeting ligand by comparing the thermodynamic stability, docking score, confidence score, and binding affinity. Also, spectrophotometer, gel electrophoresis, and Dynamic light scattering (DLS) confirm the size, zeta potential, DOX encapsulation rate, stability, and aptamer conjugation of liposomes. In addition, flow cytometry results validate MUC1 expression in MCF7 cells while not in MDA-MB-231 cells, while confocal microscopy confirmed the specific cellular uptake of the lipo-aptamer complex. Consequently, this aptamer-mediated liposomal drug delivery approach shows potential for enhancing the specificity and reducing the side effects of conventional chemotherapy, while the use of in silico methods provides an efficient and cost-effective strategy for screening and optimizing aptamer candidates, simplifying the overall development process. Further in vivo evaluation for therapeutic efficacy and clinical applications is warranted.Graphical

Selection of High-Affinity and Selectivity AFB1 Circular Aptamer for Biosensor Application.

Detecting trace amounts of aflatoxin B1 (AFB1), one of the most toxic food contaminants, is crucial for efficiently preventing potential health risks. Circular aptamers are promising candidates for bioanalytical applications due to their enhanced biological and structural stability as well as their compatibility with rolling circle amplification (RCA). Herein, we employed a high-efficiency magnetic chain graphene oxide-based SELEX to generate circular aptamers that bind AFB1 with high affinity and selectivity. Notably, the selected circular aptamer, CAFB1-A-2, exhibited a significant sequence similarity to the classical AFB1 linear aptamer but demonstrated a distinct thermodynamic mechanism for binding. The binding of CAFB1-A-2 was driven by both enthalpy and entropy, whereas the classical linear aptamer exhibited a notable entropy loss that required a greater enthalpy compensation. Utilizing this circular aptamer, we developed a sensitive AFB1 detection system featuring an RCA-assisted allosteric DNAzyme biosensor with a detection limit (LOD) of 265 pM. Furthermore, the constructed aptasensor successfully detected AFB1 in spiked rice and corn, achieving LODs of 11 and 9 μg/kg, respectively, within approximately 4 h. This study provides a sensitive and reliable alternative to the development of an aptasensor for AFB1, holding great potential in the field of food safety detection.

Binding-Site Directed Selection and Large-Scale Click-Synthesis of a Coagulation Factor XIa-Inhibiting Circular DNA Aptamer.

Factor XIa (FXIa) is a plasma protease thatplays a crucial role in the intrinsic pathway of blood coagulation, making it a promising target for antithrombotic therapy.Circular DNA aptamers, with their dramatically enhanced biological and structural stability, hold great potential as new-generation DNA-based anticoagulants. However, the functional selection and large-scale synthesis of them remains a substantial challenge. Here, we explored a selection strategy to enrich circular DNA aptamers that can target the catalytic domain of FXIa and inhibit its function. An asymmetrical dumbbell structured DNA library featuring a hairpin-like primer-binding site was utilized for selection. This approach has resulted in the identification of a circular DNA aptamer, named FICAPT1, which exhibits high binding affinity (Kd= 20 nM), notable stability (half-life of 16 hoursin human plasma) and outstanding anticoagulationactivity (significantly prolonged aPTT), showing great promise as a novel anticoagulant. The hairpin-like constant region further served as a scaffold for the large-scale click-synthesis (~ 2 mg/mL) of the refined circular aptamer. The approach presented in this study enables the efficient generation of functional circular DNA aptamers and their synthesis at a satisfactory scale, making it adaptable for the production of various circular aptamers for therapeutic applications.

Serum assisted PD-L1 aptamer screening for improving its stability

Aptamers have shown potential for diagnosing clinical markers and targeted treatment of diseases. However, their limited stability and short half-life hinder their broader applications. Here, a real sample assisted capture-SELEX strategy is proposed to enhance the aptamer stability, using the selection of specific aptamer towards PD-L1 as an example. Through this developed selection strategy, the aptamer Apt-S1 with higher binding affinity and specificity towards PD-L1 was obtained as compared to the aptamer Apt-A2 which was screened by the traditional capture-SELEX strategy. Moreover, Apt-S1 exhibited a greater PD-L1 binding associated conformational change than Apt-A2, indicating its suitability as a biorecognition element. These findings highlight the potential of Apt-S1 in clinical applications requiring robust and specific targeting of PD-L1. Significantly, Apt-S1 exhibited a lower degradation rate in 10% diluted serum or pure human serum, under the physiological temperature and pH value, compared to Apt-A2. This observation suggested that Apt-S1 possesses higher stability and is more resistant to damage caused by the serum environmental factors, highlighting the superior stability of Apt-S1 over Apt-A2. Furthermore, defatted and deproteinized serum were used to investigate the potential reasons for the improved stability of Apt-S1. The results hinted that the pre-adaptation to nucleases present in serum during the selection process might have contributed to its higher stability. With its improved stability, higher affinity and specificity, Apt-S1 holds great potential for applications in PD-L1 assisted cancer diagnosis and treatment. Meanwhile, the results obtained in this work provide further evidence of the advantages of the real capture-SELEX strategy in improving aptamer stability compared to the traditional strategy.

Aptamer selection via versatile microfluidic platforms and their diverse applications.

Aptamers are synthetic oligonucleotides that bind with high affinity and specificity to various targets, making them invaluable for diagnostics, therapeutics, and biosensing. Microfluidic platforms can improve the efficiency and scalability of aptamer selection, especially through advancements in systematic evolution of ligands by exponential enrichment (SELEX) methods. Microfluidic SELEX methods are less time-consuming and labor-intensive and include critical steps like library preparation, binding, partitioning, and amplification. This review examines the contributions of microfluidic technology to SELEX-based aptamer identification, with alternative methods like conditional SELEX, in vivo-like SELEX and Non-SELEX for selecting aptamers and also discusses critical SELEX steps over the past decade. This work also examined the integrated microfluidic systems for SELEX, highlighting innovations such as conditional SELEX and in vivo-like SELEX. These advancements provide potential solutions to existing challenges in aptamer selection using conventional SELEX, especially concerning biological samples. A trend toward non-SELEX methods was also reviewed and discussed, wherein nucleic acid amplification was eliminated to improve aptamer selection. Microfluidic platforms have demonstrated versatility not only in aptamer selection but also in various detection applications; they allow for precise control of liquid flow and have been essential in the advancement of therapeutic aptamers, facilitating accurate screening, enhancing drug delivery systems, and enabling targeted therapeutic interventions. Although advances in microfluidic technology are expected to enhance aptamer-based diagnostics, therapeutics, and biosensing, challenges still persist, especially in up-scaling microfluidic systems for various clinical applications. The advantages and limitations of integrating microfluidic platforms with aptamer development are further addressed, emphasizing areas for future research. We also present a perspective on the future of microfluidic systems and aptamer technologies, highlighting their increasing significance in healthcare and diagnostics.

Selection of biofilm-inhibiting ssDNA aptamers against antibiotic-resistant Edwardsiella tarda by inhibition-SELEX and interaction with their binding proteins.

Biofilms can increase bacterial resistance to antibiotic therapies. Edwardsiella tarda with biofilm is highly resistant to antibacterial treatment, especially for the antibiotic-resistant strain. In this study, we obtained biofilm-inhibiting aptamers against antibiotic-resistant E. tarda via a novel systematic evolution of ligands by exponential enrichment (SELEX) technique, called inhibition-SELEX. After four rounds of screening and validation, we identified aptamers IB1, IB2, and IB3, which demonstrated biofilm-inhibition and biofilm-degradation rates of 69%, 75%, and 62% and 51%, 63%, and 45% at 2μmol/L, respectively, against antibiotic-resistant E. tarda. Magnetic separation, SDS-PAGE, and mass spectrometry analyses revealed that all three aptamers could bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), while IB2 could also bind to formate C-acetyltransferase (FA). Through molecular docking and molecular dynamics simulations, it was found that the four complexes primarily interact through hydrogen bonding. Among them, IB1-GAPDH exhibited the strongest stability, followed by IB2-FA, then IB2-GAPDH, and IB3-GAPDH was the least stable. Our results suggest that IB1, IB2, and IB3 may inhibit and degrade E. tarda biofilm by interfering with the synthesis, secretion, and transportation of its extracellular polysaccharides and proteins by interacting with GAPDH and FA.

Glycosylation-Targeting Aptamer for the Feasible Construction of a Dual Aptamer-Based Plasmonic Immunosandwich Assay in Cancer Diagnostics.

Fibroblast activation protein (FAP) is an important antigen in the tumor microenvironment, which plays a crucial role in promoting extracellular matrix remodeling and tumor cell metastasis. A circulating form of soluble FAP has also been identified in the serum, becoming a biomarker for pan-cancer diagnosis and prognosis. However, the current peptide substrate-based enzymatic activity detection or antibody-dependent detection methods have been hindered by insufficient selectivity and complex operations, so it is valuable to develop effective nucleic acid aptamers as FAP affinity ligands. In order to deeply explore the biomimetic recognition technology, this study proposed an elaborate aptamer screening strategy for targeting the protein characteristic structure. Taking the glycosylation of the FAP protein as a target, four FAP-specific aptamers with high performance were successfully generated. Further, using the champion aptamer as a recognition tool and combining it with ultrasensitive detection technology-surface enhanced Raman scattering (SERS), a novel dual aptamer-based sandwich sensor was constructed for the rapid determination of FAP. Due to the dual-specific recognition of the orthogonal aptamer pair, the sandwich method obviously improved the selectivity to FAP protein, with a maximum cross-reactivity of less than 8% and a quantitation limit of 100 pg/mL. It was conveniently applied in high-sensitive and high-selective detection of serum FAP in cancer patient samples. Therefore, the research of this study not only opens new access for the selection of antiglycan aptamers but also boosts the application of the FAP aptamer as a recognition tool in cancer diagnostics.

Dimeric DNA Aptamers for the Spike Protein of SARS-CoV-2 Derived from a Structured Library with Dual Random Domains.

Multimeric aptamer strategies are often adopted to improve the binding affinity of an aptamer toward its target molecules. In most cases, multimeric aptamers are constructed by connecting pre-identified monomeric aptamers derived from in vitro selection. Although multimerization provides an added benefit of enhanced binding avidity, the characterization of different aptamer pairings adds more steps to an already lengthy procedure. Therefore, an aptamer engineering strategy that directly selects for multimeric aptamers is highly desirable. Here, an in vitro selection strategy is reported on using a pre-structured DNA library that forms dimeric aptamers. Rather than using a library containing a single random region, which is nearly ubiquitous in existing aptamer selections, the library contains two random regions separated by a flexible poly-thymidine linker. Following sixteen rounds of selection against the SARS-CoV-2 spike protein, a relevant model target protein due to the COVID-19 pandemic, the top aptamers displayed superb affinity with KD values as low as 150 pM. Further analysis reveals that each random region functions as a distinct binding moiety and works together to achieve higher affinity. The demonstrated strategy provides an accelerated method to obtain high-affinity aptamers, which may prove useful in future aptamer diagnostic and therapeutic applications.

PH‐Directed Capture‐SELEX for Nanomolar Affinity Aptamers for Kanamycin Detection

AbstractKanamycin A is a widely used antibiotic, although it has a narrow therapeutic window demanding highly accurate monitoring. The sensing of kanamycin A using aptamers is of great interest since aptamers can be used for continuous monitoring with a rapid response. While kanamycin has been the target for at least four previous aptamer selections, the binding affinities of the reported DNA aptamers are still sub‐optimal. All the previous aptamer selections were performed at pH 7.5 or higher. Given that kanamycin A has four amino groups with pKa values close to 7, we herein selected DNA aptamers for kanamycin A at both pH 6 and pH 8. The selection at pH 6 enriched aptamers although the pH 8 selection library remained highly diverse in the end. The best aptamer named KAN6‐1 showed a dissociation constant of around 320 nM measured using isothermal titration calorimetry in the selection buffer. In buffers without salt, binding can happen from pH 6 to 8. Specific binding was confirmed using mutation studies. A strand displacement assay was developed with a limit of detection (LOD) of 100 nM in buffer. Similar LOD values were also obtained in lake water and in 10 % human serum. Comparisons were also made with some previously reported DNA aptamers. This study shows the importance of pH value on the selection of aptamers and provides a new aptamer for kanamycin A detection.

Post-Selection Design of Aptamers: Comparative Study of Affinity of the DNA Aptamers to Recombinant Extracellular Domain of Human Epidermal Growth Factor Receptors.

The current work presents comparative assessment of affinity of the designed DNA aptamers for extracellular domain of the human epidermal growth factor receptor (EGFR*). The affinity data of the 20 previously published aptamers are summarized. Diversity of the aptamer selection methods and techniques requires unification of the comparison algorithms, which is also necessary for designing aptamers used in the post-selection fitting to the target EGFR* protein. In this study affinities of the DNA aptamers from two families, U31 and U2, previously obtained by Wuetal. from the same selection [Wuetal. (2014) PLoS One, 9, e90752] and their derivatives- GR20, U2s, and Gol1 obtained by us through rational design, were compared. Affinity of the aptamers to EGFR* was measured by two different methods: a solution-phase technique- fluorescence polarization of FAM-labeled aptamers, and by a kinetic method using biolayer interferometry technique with aptamers immobilized on the surface. Unlike the values of equilibrium dissociation constants obtained through titration and expressed in units of protein concentration, analysis of the titration curve profiles themselves and kinetics of interaction proved to be more informative. This allowed us to identify how even subtle changes in the aptamers and their structures affect affinity. Hypotheses regarding the "structure-function" relationships and recognition mechanisms were formulated. The data obtained for the set of aptamer constructs are critical for moving forward to examination of aptamer interactions with EGFR on the cell surface.

Selection of Plastic-Binding DNA Aptamers for Microplastics Detection.

Plastics are critical materials for modern technological applications, yet environmental contamination by microplastics has become a growing concern. In this study, DNA aptamers were isolated for two of the most abundant plastic materials: polyvinylchloride (PVC) and polystyrene (PS). These aptamers contain approximately 90 % cytosine and thymine but only 10 % purine content. Among them, the PVC-1 aptamer binds to PVC with a six-fold higher capacity than a random sequenced DNA. Among the tested plastic materials, PVC and PS exhibited the highest specific binding capacity. Using fluorophore-labeled PVC-1 aptamer, PS/PVC microplastics as low as 1 mg were detected, and the aptamer was selective for microplastics over other environmentally relevant materials, such as silica. Molecular dynamics simulations indicated that the aptamer attempted to maximize contact with the plastic surface for adsorption. This plastic-binding aptamer is expected to find applications in environmental monitoring and has fundamental implications for surface-binding aptamers.

Aptamer and DNAzyme Based Colorimetric Biosensors for Pathogen Detection.

The detection of pathogens is critical for preventing and controlling health hazards across clinical, environmental, and food safety sectors. Functional nucleic acids (FNAs), such as aptamers and DNAzymes, have emerged as versatile molecular tools for pathogen detection due to their high specificity and affinity. This review focuses on the in vitro selection of FNAs for pathogens, with emphasis on the selection of aptamers for specific biomarkers and intact pathogens, including bacteria and viruses. Additionally, the selection of DNAzymes for bacterial detection is discussed. The integration of these FNAs into colorimetric biosensors has enabled the development of simple, cost-effective diagnostic platforms. Both non-catalytic and catalytic colorimetric biosensors are explored, including those based on gold nanoparticles, polydiacetylenes, protein enzymes, G-quadruplexes, and nanozymes. These biosensors offer visible detection through color changes, making them ideal for point-of-care diagnostics. The review concludes by highlighting current challenges and future perspectives for advancing FNA-based colorimetric biosensing technologies for pathogen detection.

Aptamer and DNAzyme Based Colorimetric Biosensors for Pathogen Detection

AbstractThe detection of pathogens is critical for preventing and controlling health hazards across clinical, environmental, and food safety sectors. Functional nucleic acids (FNAs), such as aptamers and DNAzymes, have emerged as versatile molecular tools for pathogen detection due to their high specificity and affinity. This review focuses on the in vitro selection of FNAs for pathogens, with emphasis on the selection of aptamers for specific biomarkers and intact pathogens, including bacteria and viruses. Additionally, the selection of DNAzymes for bacterial detection is discussed. The integration of these FNAs into colorimetric biosensors has enabled the development of simple, cost‐effective diagnostic platforms. Both non‐catalytic and catalytic colorimetric biosensors are explored, including those based on gold nanoparticles, polydiacetylenes, protein enzymes, G‐quadruplexes, and nanozymes. These biosensors offer visible detection through color changes, making them ideal for point‐of‐care diagnostics. The review concludes by highlighting current challenges and future perspectives for advancing FNA‐based colorimetric biosensing technologies for pathogen detection.

SERS-based CRISPR/Cas12a assays for protein biomarker prostate-specific antigen detection.

Sensitive and accurate detection of protein biomarkers is crucial for disease diagnosis, especially for early diagnosis. Here, we describe surface-enhanced Raman scattering (SERS)-based CRISPR/Cas12a assays (S-CRISPR) for protein biomarker detection. Firstly, an S-CRISPR-driven enzyme-linked immunosorbent assay (S-CasLISA) was developed utilizing a capture antibody coated on a microplate to recognize the target and a detection antibody labeled with active DNA to trigger the activity of CRISPR/Cas12a. With this assay, we achieved detection of prostate-specific antigen (PSA) as models at thepicogram level. The limit of detection (LoD) of S-CasLISA was 0.17pgmL-1 and in the range of 0.1pgmL-1 to 10ngmL-1. Further, we applied aptamer to S-CRISPR (S-Apt-CRISPR), combining the high sensitivity of SERS with the high selectivity of aptamers, while simplifying the operation process of CRISPR detection of protein biomarkers. The proposed S-Apt-CRISPR also could detect picogram-level PSA and without repeated washing steps. The LoD of S-Apt-CRISPR was 0.35pgmL-1 and in the range of 0.1pgmL-1 to 10ngmL-1.Both SERS-based CRISPR/Cas12a assays were validated with clinical samples and demonstrated accuracy consistent with the chemiluminescence immunoassay. The introduction of the CRISPR/Cas12a system with SERS has the effect of improving the analytical capabilities of the system, thereby broadening and facilitating its application in the analysis of sensitive and accurate protein biomarkers.

Efficient Correction of DNA Hetero–Duplexes Formed During SELEX Procedure

Background: Aptamers, oligos-based (ssDNA or RNA) and peptide-based, are now emerging as the first-choice biomolecules in various applications in medicine and biology. DNA aptamers selection process includes repeatable steps of DNA amplification and digestion. The amplification process of the enriched DNA aptamers includes using PCR. Objective of study: In this article, we sought to describe an efficient and robust method that corrects or permanently removes the PCR by–products during the cycles of DNA aptamers selection. Materials and Methods: Our method is simple, efficient, and requires no enzymatic treatment. In this method, we applied repetitive cycles of thermal treatment to the enriched aptamers final products during the enrichment cycles. The procedure starts with loading the PCR-induced DNA on 1% agarose gel to evaluate the presence of ladder-like DNA or smearing band-like DNA. Next, we cut and purified these by–products and applied thermal treatment. The thermal treatment was by heating the DNA to 65ºC for 10 minutes and then cool them down to 20ºC for 1 minute. Results: PCR artifacts evaluation has been revealed two types of unwanted DNA sequences; the ladder-like and the smearing bands. The next–Generation illume sequencing specified the presence of long and short sequences. Upon the thermal treatment of the PCR products, the next–generation illume sequencing method results showed that the longer sequences and the shorter sequences of the DNA have been reduced considerably and effectively in the DNA pool over the cycles of the systemic evolution of ligands by exponential enrichment (SELEX) procedure. Furthermore, we could reduce the formation of the hetero–duplexes in the next cycles of (SELEX) procedure. Conclusion: This method suggests practical and routine application to prevent or reduce DNA aptamer by–products during the selection process. This treatment could reduce the mis-shaped or irrelevant non-functional aptamers in SELEX procedure

Advances in the study of DNA-metal complexes in biomedical applications

DNA-metal composites are emerging as emerging materials for biomedical applications due to their unique DNA and metal nanoparticle properties. DNA is a multifunctional macromolecule that enables targeted drug delivery through aptamer selection and gene silencing through nuclease activity. These properties make it highly selective and targeted in therapy. While conventional chemotherapy faces significant drawbacks, including poor targeting and severe side effects, DNA-metal composites with their excellent targeting and therapeutic efficiencies can significantly enhance therapeutic efficacy. This paper first describes the structural properties, synthesis methods of DNA-metal composites and their therapeutic applications. The combination of DNA and metal nanoparticles provides a novel approach to improve the effectiveness of anticancer and antimicrobial therapies, addressing key limitations in current therapeutic strategies. Innovative applications of DNA-metal composites in the anticancer and antimicrobial fields are then explored, highlighting their multiple synthesis and application possibilities through interactions with metal ions or particles. DNA-metal composites address several of the limitations of conventional chemotherapeutic and antimicrobial drugs, providing better targeting and resolving the issue of drug resistance. DNA-metal composites in biomedicine show DNA-metal composites show great promise in the biomedical field, and future research will continue to promote their application and development in clinical therapy.

Selection of novel ssDNA aptamer for ultra-sensitive colorimetric detection of acetamiprid using ultra-small dot assay

An innovative ssDNA aptamer-mediated colorimetric biosensor utilizing an ultra-small dot assay has been developed for the ultra-sensitive detection of acetamiprid, a hazardous insecticide widely used in agriculture. This “Dot assay” leverages ultra-low volumes of less than 20 µL, allowing for rapid visual detection within seconds and addressing the urgent need for accessible and efficient detection methods. Through a non-immobilized graphene oxide-based systematic evolution of ligands by exponential enrichment (SELEX) approach, a specific 79-mer aptamer (Apt-SS5) was identified after 15 enrichment cycles. The Apt-SS5 aptamer demonstrated a remarkable binding affinity for acetamiprid, with a melting temperature (Tm) of 53 °C and a Gibbs free energy (ΔG) of −7.24 kcal/mol at 37 °C. Notably, the binding pocket is formed by nucleotides C-16 to T-32, with critical interactions occurring at C-10, C-16, G-25, A-26, G-27, G-28, and A-29. The optimized colorimetric assay achieved an impressive limit of detection (LOD) of 0.039 ppb and resulted in a significant reduction in assay costs. The target specificity and cross-reactivity of Apt-SS5 were tested against other pesticides, including imidacloprid, thiachloprid, thiamethoxam, chlorpyrifos, pyriproxyfen, and chlorantraniliprole, with no significant color change observed for these non-targets. In contrast, a clear visual color change was noted in the presence of acetamiprid, confirming the aptamer’s high specificity. Additionally, the aptasensor proved effective for testing river and water samples, yielding recovery rates of 84.40 % to 101.66 %. This innovative approach enhances sensitivity and simplifies the detection process, paving the way for rapid screening of pesticide residues in various samples.

Exploring the Lower Limit of Target Concentration in Capture-SELEX Using Guanine as a Model Target.

During an aptamer selection, using a lower target concentration may result in aptamers with a higher binding affinity. Consequently, this begs the question of whether there is a lower limit for target concentration. In this work, we conducted three aptamer selections using 5 μM, 500 nM and 50 nM guanine as the targets, respectively. Successful enrichment of the same guanine aptamers was achieved at both 5 μM and 500 nM guanine, but not with 50 nM. Using 5 μM guanine, the aptamer was enriched in eight rounds of selection, compared to that for 500 nM, which was accomplished in 17 rounds. We discuss the relation of optimal target concentration to the observed Kd value of the resulting aptamers, of which the highest affinity aptamer had a measured Kd of 200 nM. Additionally, we investigated the binding of the aptamers through mutation studies, revealing a critical cytosine. Mutating this cytosine to a thymine switched the selectivity from guanine to adenine, which is reminiscent of the guanine riboswitch. This study revealed a limit in using low target concentration, and the insights described in this article will be useful for guiding the choice of target concentration during capture-SELEX.

Pre-Defined Stem-Loop Structure Library for the Discovery of L-RNA Aptamers that Target RNA G-Quadruplexes.

L-RNA aptamers have been developed to target G-quadruplexes (G4s) and regulate G4-mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subject to high failure rates. By analyzing the previously reported G4-binding L-RNA aptamers, we found that the stem-loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4-SLSELEX-Seq platform specifically for G4 targets by introducing a pre-defined stem-loop structure library during the SELEX process. Using G4-SLSELEX-Seq, we identified an L-RNA aptamer, L-Apt1-12, for the Epstein-Barr nuclear antigen 1 (EBNA1) RNA G4 (rG4) in just three selection rounds. L-Apt1-12 maintained the stem-loop structure initially introduced, and possessed a unique G-triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. L-Apt1-12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards Epstein-Barr virus (EBV)-positive cancer cells, highlighting its potential for targeted therapy against EBV-associated cancers. Furthermore, we demonstrated the robustness and generality of G4-SLSELEX-Seq by selecting L-RNA aptamers for the amyloid precursor protein (APP) rG4 and the hepatitis C virus subtype 1a (HCV-1a) rG4, obtaining high-affinity aptamers in three selection rounds. These findings demonstrated G4-SLSELEX-Seq as a robust and efficient platform for the selection of rG4-targeting L-RNA aptamers.