Systematic Evolution Of Ligands Research Articles

Development and classification of RNA aptamers for therapeutic purposes: an updated review with emphasis on cancer.

Today, RNA aptamers are being considered promising theranostic tools against a wide variety of disorders. RNA aptamers can fold into complex shapes and bind to diverse nanostructures, macromolecules, cells, and viruses. It is possible to isolate RNA aptamers from a vast pool of nucleic acids via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. As therapeutics, aptamers have great potential because of their ability to bind to proteins and selectively limit their activities with negligible side effects. Several RNA aptamers with potential implications in cancer diagnosis are known to confer a great affinity for single-stranded DNA molecules, long non-coding RNAs, circulating tumor cells, vascular endothelial growth factors, and tissue and sera-derived exosomes in patients with different malignancies. Furthermore, clinical investigations have revealed the efficacy of RNA aptamer-based agents in imaging modalities. This review seeks to deliver new insights into the development, classification, nanomerization, and modification of RNA aptamers, as well as their applications in cancer theranostics. The aptamers' mechanism of action and their interest to clinical trials as theranostic agents are also discussed.

Advances in Aptamers-Based Applications in Breast Cancer: Drug Delivery, Therapeutics, and Diagnostics.

Aptamers are synthetic single-stranded oligonucleotides (such as RNA and DNA) evolved in vitro using Systematic Evolution of Ligands through Exponential enrichment (SELEX) techniques. Aptamers are evolved to have high affinity and specificity to targets; hence, they have a great potential for use in therapeutics as delivery agents and/or in treatment strategies. Aptamers can be chemically synthesized and modified in a cost-effective manner and are easy to hybridize to a variety of nano-particles and other agents which has paved a way for targeted therapy and diagnostics applications such as in breast tumors. In this review, we systematically explain different aptamer adoption approaches to therapeutic or diagnostic uses when addressing breast tumors. We summarize the current therapeutic techniques to address breast tumors including aptamer-base approaches. We discuss the next aptamer-based therapeutic and diagnostic approaches targeting breast tumors. Finally, we provide a perspective on the future of aptamer-based sensors for breast therapeutics and diagnostics. In this section, the therapeutic applications of aptamers will be discussed for the targeting therapy of breast cancer.

Foot-and-mouth disease virus: DNA aptamer selection for the 3ABC protein

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family. The 3ABC is a non-structural protein of FMDV, produced during viral replication and absent from inactivated FMD vaccines. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain DNA aptamers specific for 3ABC protein with a view of their application in the FMD diagnosis.Aptamers are usually obtained through SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. In this study, an aptamer (termed FMDV1) was selected by a variation of this technique called Capillary Electrophoresis SELEX (CE-SELEX). The FMDV1 aptamer showed high binding affinity to the 3ABC protein with Kd value in the nano molar range: 22.69 ± 1.79 nM. The FMDV1 aptamer binding to 3ABC was significantly higher when compared with the BSA protein, used as control, demonstrating its specificity.

Antithrombin-Specific RNA Aptamers As a Potential Therapy to Restore Hemostatic Balance in Hemophilia Patients

Hemophilia A and B are inherited blood bleeding disorders due to insufficient blood coagulation factors. Individuals with hemophilia A have a deficiency in factor VIII, while individuals with hemophilia B have a deficiency in factor IXa. Both of these factors are important in the intrinsic pathway of blood coagulation. In the absence of these factors, hemophilia patients are prone to bleeding. Thrombin, the primary factor in the blood coagulation pathway, promotes blood clotting and prevents bleeding. Hemophilia patients develop insufficient thrombin, resulting in an imbalance between the procoagulant and anticoagulant systems. Thus, generating more thrombin activity in these patients is an ideal therapeutic approach. We propose to increase thrombin's activity by inhibiting the primary inhibitor of thrombin and the natural anticoagulant protein, antithrombin (AT), using RNA molecules (aptamers) that bind to their target protein with high affinity and specificity. Several aptamers are evaluated in clinical trials as therapies for various blood diseases. We hypothesized that AT-specific aptamers could inhibit AT activity by preventing AT from forming a high molecular weight complex with thrombin, resulting in an overall increase in thrombin activity and restoring hemostatic equilibrium. The main goal is to design novel therapeutic molecules that promote thrombus formation in hemophilia patients. Using Systematic evolution of ligands by exponential enrichment (SELEX), we developed a library of RNA aptamers molecules that bind to AT with high to moderate affinity. We assessed the individual aptamers' ability to inhibit AT and increase thrombin activity using various in vitro methods. Three individual aptamers inhibited AT activity by preventing AT from forming a complex with thrombin in the presence and absence of heparin, resulting in a significant increase in thrombin activity. We also showed a decrease in thrombin generation using an ex vivo thrombin generation assay in the presence of aptamers. Our data illustrate that AT-specific RNA aptamers can inhibit AT activity and increase thrombin activity. Thus, these molecules could potentially be used to treat patients with both hemophilia A and B. Additional studies are being conducted to assess their ability to increase thrombin activity in a pre-clinical model.

DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX.

Staphylococcus aureus is the most common bacteria found in skin, soft tissues, bone, and bone prostheses infections. The aim of this study was to select DNA aptamers for S. aureus to be applied inthe diagnosis of bacteria. We used SELEX (Systematic Evolution of Ligands by EXponencial Enrichment) for peptidoglycan followed by cell-SELEX with S. aureus cells as target. Four sequences showed significantly higher binding to S. aureus distinguishing it from the control cells of other significant microbial species:Escherichiacoli,Candida albicans,Streptococcus pyogenes and Streptococcus pneumoniae. In particular, ApSA1 (Kd = 62.7 ± 5.6 nM) and ApSA3 (Kd = 43.3 ± 3.0 nM) sequences combined high affinity and specificity for S. aureus, considering all microorganisms tested. Our results demonstrated that these aptamers were able to identify peptidoglycan in the S. aureus surface and have great potential for use in the development of radiopharmaceuticals capable to identify S. aureus infectious foci, as well as in other aptamer-based methodologies for bacteria diagnosis.

Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. NanodropTM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process.

Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system

Accurate genome editing based on various molecular tools has always been the focus of gene-editing research and the primary goal for therapeutic application. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a well-established gene-editing method that is preferred due to its simplicity and high efficiency. In this study, a group of single-stranded DNA aptamers with high affinity and high specificity for the Cas9 protein were obtained by the systematic evolution of ligands through the exponential enrichment method. Their binding affinity and possible binding domains to theCas9 protein were analyzed. In addition, we demonstrated the effectiveness of aptamers in regulating dCas9-modulated gene transcription, in terms of both transcriptional activationand repression. Additionally, the aptamers successfully reduced the off-target effect and improved the efficiency of gene homologous recombination repair mediated by CRISPR-Cas9. The findings suggest a potential method to better control precise gene editing and enrich the diversity of modulating tools for the CRISPR-Cas9 system.

An antifouling polydopamine-based fluorescent aptasensor for determination of arginine kinase

Simple yet efficient detection methods for food allergens are in urgent need to help people avoid the risks imposed by allergenic food. In this work, a polydopamine (PDA)-based fluorescent aptasensor was developed to detect arginine kinase (AK), one of the major allergens in shellfish. The aptamer towards AK was firstly selected via systematic evolution of ligands by exponential enrichment method and labeled with fluorescein amidite (FAM) to build a fluorescence resonance energy transfer (FRET) system with PDA particles. Polyethylene glycol (PEG) was employed to construct an antifouling surface for the aptasensor to eliminate food matrix interferences. With the presence of AK, the PDA-based aptasensor exhibited elevated fluorescent signals as the FAM-labeled aptamer bound to AK and detached from the PDA particles. The aptasensor showed great stability and resistance to nonspecific interference of background proteins and had a limit of detection (LOD) of 0.298 μg/mL. The proposed aptasensor was further proved to be feasible for quantitative analysis of AK in nine species of shrimps and five commercial processed products, which indicated its high potential in tracing the presence of AK in complex aquatic products.

On the Move-Sensitive Fluorescent Aptassay on Board Catalytic Micromotors for the Determination of Interleukin-6 in Ultra-Low Serum Volumes for Neonatal Sepsis Diagnostics.

A graphene oxide/nickel/platinum nanoparticle micromotor (MM)-based fluorescent aptassay is proposed to determine interleukin-6 (IL-6) in serum samples from low-birth-weight infants (gestational age of less than 32 weeks and birthweight below 1000 g) with sepsis suspicion. In this kind of patients, IL-6 has demonstrated good sensitivity and specificity for the diagnosis of sepsis, both for early and late onset sepsis. The approach was based on the adsorption of the aptamer for IL-6 tagged with 6-FAM as a fluorescent label (AptIL-6, λem = 520 nm) on the graphene oxide external layer (MMGO-AptIL-6) inducing fluorescence quenching (OFF state) and a subsequent on-the-move affinity recognition of IL-6 from AptIL-6 (IL-6-AptIL-6 complex) recovering the fluorescence (ON state). An aptamer against IL-6 was selected and developed by the systematic evolution of ligands by exponential enrichment technology. This approach displayed a suitable linear range of 0.07-1000 pg mL-1 (r = 0.995) covering the cut-off and clinical practice levels, allowing direct determination without any dilution and simplifying the analysis as well as exhibiting an excellent sensitivity (LOD = 0.02 pg mL-1) in ultralow volumes of diagnostic clinical samples (2 μL). A high agreement between IL-6 levels obtained from our MM-based approach and the method used by the Hospital was obtained (relative error < 3%). The MM-based aptassay is competitive in comparison with that of the Hospital, in terms of a significant reduction of the sample volume (15 times less) and enhanced sensitivity, employing similar analysis times. These results position MM technology with enough potential to achieve high sensitivities in low sample volumes, opening new avenues in diagnosis based on low sample volumes.

In vitro selection and optimization of high-affinity aptamer for milk allergen α-lactalbumin and its application in dual-mode detection.

Milk is one of the most common sources of protein in people’s daily lives, and it is also recognized by the World Health Organization (WHO) as one of the eight categories of food allergies to human beings. α-lactalbumin (α-La) is the main cause of milk allergy. In this study, a single-stranded DNA aptamer with high binding affinity to α-La were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Compared with the full-length sequence, the binding affinity of the truncated aptamer LA-1t for α-La was increased six times using fluorescence analysis. Circular dichroism (CD) indicated that the secondary structure of LA-1t contained a typical hairpin structure. Through the docking simulation of LA-1t and α-La, these experimental results were further explained theoretically, and the recognition mechanism was explained. Finally, the colorimetric and fluorescence signal of boron nitride quantum dots anchored to porous CeO2 nanorods (BNQDs/CeO2) were modulated by FAM-labeled LA-1t to achieve highly selective and sensitive determination of α-La. This dual-mode sensing strategy displayed sensitive recognition for α-La in a linear range of 5–4,000 ng/ml with the LOD was 3.32 ng/ml (colorimetry) and 0.71 ng/ml (fluorescence), respectively. Simultaneously, the colorimetry/fluorescence dual-mode sensing strategy was applied for detecting α-La in spiked real samples and demonstrated good stability and reliability.

Automated ssDNA SELEX Using Histidine-Tagged Target Proteins.

The systematic evolution of ligands by exponential enrichment (SELEX) enables the identification of ssDNA or RNA sequences binding to different target molecules, highly specific and with high affinity. In this chapter, we describe a selection strategy with ssDNA for a histidine-tagged protein that could be either performed hands-on manually or fully automated by an appropriate robotic selection platform.

Application of nucleic acid aptamer-based lateral flow assay in microbe detection

<p indent="0mm">Microbial contamination and infection are global issues in the food and environmental fields that seriously threaten human health. Bacteria and fungi can easily cause food spoilage, resulting in diarrhea and vomiting; viruses can infect humans through different transmission routes, causing severe or even fatal harm. Hence, rapid analysis and identification of pathogenic microorganisms and simultaneous detection of multiple types of microbes have become hot research topics in biochemical analysis and molecular diagnosis. The lateral flow assay (LFA) is a simple, rapid, economical, and efficient detection technology with high sensitivity, simple operation, and environmental friendliness. It can provide instant test results under non-laboratory circumstances, hence becoming an ideal choice for point-of-care testing, which has been applied to rapidly detect various targets. The current conventional principle of the LFA is still based on the specific recognition of the antigen by the antibody. However, as a commonly used target recognition molecule in conventional biochemical and medical detection, the application of antibodies also has certain limitations for rapid and accurate identification of certain targets due to strict control of the production and purification process, as well as susceptibility to the interference of the operating environment, pH, temperature, and other conditions, such as long production cycle, high cost, poor stability, and cross-reactivity. Aptamers are a class of single-stranded DNA (ssDNA) or RNA obtained through the systematic evolution of ligands by exponential enrichment (SELEX), which can usually form a stable secondary structure. Aptamers can be folded into a three-dimensional structure through conformational change and interact with the target through conformation complementarity, π-π stacking between aromatic rings, base stacking, electrostatic interaction, and hydrogen bonding. So far, nearly 300 kinds of aptamers have been discovered. As alternatives, aptamers are easy and facile to modify and label with high sensitivity and specificity. Accordingly, the innovative rapid detection technique can be developed by combining the LFA with an aptamer. This aptamer-based LFA technology can be widely used in qualitative, semi-quantitative, and quantitative detection in food safety, environment, clinical, and other fields. Nowadays, most microbe detection methods are constructed based on this approach. The common strategies of aptamer-based LFAs include the sandwich method, competitive method, and adsorption–desorption method. Diverse ingenious materials such as gold nanoparticles and quantum dots have also been proposed for signal read-out. Different signal capture models, such as colorimetric and fluorescence methods, have been applied for sensitive and accurate detection of a single or multiple target microbe. Furthermore, in view of the unique properties of nucleic acid aptamers, several signal amplification methods can be further involved in the LFA to enhance the sensitivity for target detection. This review introduces the use of aptamers with different structural patterns and labeling types in recent years, as well as a variety of methods to detect microbes, especially for the rapid detection of pathogenic bacteria. Based on the excellent characteristics, the aptamer-based LFA presents more flexibility and selectivity for microbe detection with good applicability, specificity, and sensitivity and can better achieve low-cost, rapid detection. This study is expected to provide a reference for developing nucleic acid aptamer-based LFA technologies, especially for efficient and accurate diagnosis of corona virus disease 2019 (COVID-19), exploiting the novel application scope of LFA technology.

Selection of RNA aptamers targeting hypoxia in cancer.

Hypoxia plays a crucial role in tumorigenesis and drug resistance, and it is recognised as a major factor affecting patient clinical outcome. Therefore, the detection of hypoxic areas within the tumour micro-environment represents a useful way to monitor tumour growth and patients’ responses to treatments, properly guiding the choice of the most suitable therapy. To date, non-invasive hypoxia imaging probes have been identified, but their applicability in vivo is strongly limited due to an inadequate resistance to the low oxygen concentration and the acidic pH of the tumour micro-environment. In this regard, nucleic acid aptamers represent very powerful tools thanks to their peculiar features, including high stability to harsh conditions and a small size, resulting in easy and efficient tumour penetration. Here, we describe a modified cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach that allows the isolation of specific RNA aptamers for the detection of the hypoxic phenotype in breast cancer (BC) cells. We demonstrated the effectiveness of the proposed method in isolating highly stable aptamers with an improved and specific binding to hypoxic cells. To our knowledge, this is the first example of a cell-SELEX approach properly designed and modified to select RNA aptamers against hypoxia-related epitopes expressed on tumour cell surfaces. The selected aptamers may provide new effective tools for targeting hypoxic areas within the tumour with great clinical potential.

A Flow Cytometry-based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer.

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

A scalable framework for the discovery of functional helicase substrates and helicase-driven regulatory switches

Helicases are ubiquitous motor enzymes that remodel nucleic acids (NA) and NA-protein complexes in key cellular processes. To explore the functional repertoire and specificity landscape of helicases, we devised a screening scheme-Helicase-SELEX (Systematic Evolution of Ligands by EXponential enrichment)-that enzymatically probes substrate and cofactor requirements at global scale. Using the transcription termination Rho helicase of Escherichia coli as a prototype for Helicase-SELEX, we generated a genome-wide map of Rho utilization (Rut) sites. The map reveals many features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and cofactor-dependent Rut sites with inverted G > C skewed compositions. We also implemented an H-SELEX variant where we used a model ligand, serotonin, to evolve synthetic Rut sites operating invitro and invivo in a ligand-dependent manner. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.

Selection of DNA aptamer and its application as an electrical biosensor for Zika virus detection in human serum

Zika virus is a highly infectious virus that is part of the flavivirus group. Precise diagnosis of the Zika virus is significant issue for controlling a global pandemic after the COVID-19 era. For the first time, we describe a zika virus aptamer-based electrical biosensor for detecting Zika virus in human serum. The electrical biosensor composed of a Zika virus aptamer/MXene nanoparticle heterolayer on Au micro-gap electrode (AuMGE)/print circuit board (PCB) system. The Zika virus aptamer was designed to bind the envelope protein of the Zika virus by systematic evolution of ligands by exponential enrichment (SELEX) technique. The binding affinity of the aptamer was determined by fluorescence. For improving the sensor signal sensitivity, Ti3C2Tx MXene was introduced to surface of Au micro-gap electrode (AuMGE). The immobilization process was confirmed by atomic force microscopy (AFM). The prepared aptamer/MXene immobilized on AuMGE can detect the Zika virus through capacitance change according to the target concentration. The capacitance signal from the biosensor increased linearly according to increment of envelope proteins in the human serum. The limit of detection was determined to 38.14 pM, and target proteins could be detected from 100 pM to 10 μM. Thus, the developed electrical aptabiosensor can be a useful tool for Zika virus detection.

On-site detection of fish furunculosis by combining DNAzyme and carboxyl-functionalized graphene.

Furunculosis, which is caused by Aeromonas salmonicida, can induce septicemia, leading to the rapid death of fishes belonging to Salmonidae, Cyprinidae, and Fuscheridae, and lamprey. Targeting A. salmonicida, five DNAzyme sequences with the highest enrichment rates were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The enrichment rates were 34.78, 23.60, 8.91, 2.89, and 2.34%, respectively. The DNAzyme with the highest activity, named D-AS-2, showed specificity and sensitivity. D-AS-2 was combined with carboxyl-functionalized graphene to construct a biosensor, which showed good fluorescence response to scabies lesion samples. The diagnostic procedure was completed in <2min and can be used for the on-site diagnosis of fish diseases. A low-cost, rapid, simple, and highly specific biosensor for the diagnosis of furunculosis was established based on DNAzyme and carboxyl-functionalized graphene.

In vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses.

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

Optimization of Gonyautoxin1/4-Binding G-Quadruplex Aptamers by Label-Free Surface-Enhanced Raman Spectroscopy

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures.

Evolution of Interferon-Gamma Aptamer with Good Affinity and Analytical Utility by a Rational In Silico Base Mutagenesis Post-SELEX Strategy.

The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is conventionally an effective method to identify aptamers, which are oligonucleotide sequences with desired properties to recognize targets specifically and sensitively. However, there are some inherent limitations, e.g., the loss of potential high-affinity sequences during biased iterative PCR enrichment processes and the limited structural diversity of the initial library, which seriously restrict their real-world applications. To overcome these limitations, the in silico base mutagenesis post-SELEX strategy based on the low Gibbs free energy (ΔG) and genetic algorithm was developed for the optimization of the interferon-gamma aptamer (B1-4). In the process of evolution, new sequences were created and the aptamer candidates with low ΔG values and advanced structures were produced. After five rounds of selection, systematic studies revealed that the affinity of the newly developed evolutionary aptamer (M5-5) was roughly 10-fold higher than that of the parent aptamer (B1-4), and an aptasensor detection system with a limit-of-detection (LOD) value of 3.17 nM was established based on the evolutionary aptamer. The proposed approach provided an efficient strategy to improve the aptamer with low energy and a high binding ability, and the good analytical utility thereof.