Random Oligonucleotide Library Research Articles

In vitro selection of a single-strand DNA aptamer targeting the receptor-binding domain of SARS-CoV-2 spike protein

Nucleic acid aptamers are developed from a pool of random oligonucleotide libraries with an in vitro selection through systematic evolution of ligands via exponential enrichment (SELEX) process, which are capable of specific and high-affinity molecular binding against targets. The receptor-binding domain (RBD) of spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is involved in the early stages of viral infection, is a promising target for aptamer selection. Currently, there are no effective approaches to prevent virus from spreading. In this study, a new ssDNA aptamer RBD/S-A1 binding to the RBD of spike protein from SARS-CoV-2 with high affinity (Kd=1.74 ± 0.2 nM) and low cross-binding activity was selected and evaluated. Although aptamers targeting the RBD of spike protein from SARS-CoV-2 have been described in a handful of previous studies, the RBD/S-A1 aptamer identified in this work may be considered as a potential supplementation for the current diagnosis and research of coronavirus SARS-CoV-2.

Efficient screening for 8-oxoguanine DNA glycosylase binding aptamers via capillary electrophoresis

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(KD)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。

Aptamer Applications in Emerging Viral Diseases.

Aptamers are single-stranded DNA or RNA molecules which are submitted to a process denominated SELEX. SELEX uses reiterative screening of a random oligonucleotide library to identify high-affinity binders to a chosen target, which may be a peptide, protein, or entire cells or viral particles. Aptamers can rival antibodies in target recognition, and benefit from their non-proteic nature, ease of modification, increased stability, and pharmacokinetic properties. This turns them into ideal candidates for diagnostic as well as therapeutic applications. Here, we review the recent accomplishments in the development of aptamers targeting emerging viral diseases, with emphasis on recent findings of aptamers binding to coronaviruses. We focus on aptamer development for diagnosis, including biosensors, in addition to aptamer modifications for stabilization in body fluids and tissue penetration. Such aptamers are aimed at in vivo diagnosis and treatment, such as quantification of viral load and blocking host cell invasion, virus assembly, or replication, respectively. Although there are currently no in vivo applications of aptamers in combating viral diseases, such strategies are promising for therapy development in the future.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.

The Role of RNA and DNA Aptamers in Glioblastoma Diagnosis and Therapy: A Systematic Review of the Literature

Glioblastoma (GBM) is the most lethal primary brain tumor of the central nervous system in adults. Despite advances in surgical and medical neuro-oncology, the median survival is about 15 months. For this reason, initial diagnosis, prognosis, and targeted therapy of GBM represent very attractive areas of study. Aptamers are short three-dimensional structures of single-stranded nucleic acids (RNA or DNA), identified by an in vitro process, named systematic evolution of ligands by exponential enrichment (SELEX), starting from a partially random oligonucleotide library. They bind to a molecular target with high affinity and specificity and can be easily modified to optimize binding affinity and selectivity. Thanks to their properties (low immunogenicity and toxicity, long stability, and low production variability), a large number of aptamers have been selected against GBM biomarkers and provide specific imaging agents and therapeutics to improve the diagnosis and treatment of GBM. However, the use of aptamers in GBM diagnosis and treatment still represents an underdeveloped topic, mainly due to limited literature in the research world. On these bases, we performed a systematic review aimed at summarizing current knowledge on the new promising DNA and RNA aptamer-based molecules for GBM diagnosis and treatment. Thirty-eight studies from 2000 were included and investigated. Seventeen involved the use of aptamers for GBM diagnosis and 21 for GBM therapy. Our findings showed that a number of DNA and RNA aptamers are promising diagnostic and therapeutic tools for GBM management.

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases.

One of the pivotal steps in aptamer selection is the amplification of target‐specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in‐depth study of using 5‐indolyl‐AA‐dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo‐) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water‐in‐oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high‐throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.

Application and development of aptamer in cancer: from clinical diagnosis to cancer therapy.

Traditional anticancer therapies can cause serious side effects in clinical treatment due to their nonspecific of tumor cells. Aptamers, also termed as 'chemical antibodies', are short DNA or RNA oligonucleotides selected from the synthetic large random single-strand oligonucleotide library by systematic evolution of ligands by exponential enrichment (SELEX) to bind to lots of different targets, such as proteins or nucleic acid structures. Aptamers have good affinities and high specificity with target molecules, thus may be able to act as drugs themselves to directly inhibit the proliferation of tumor cells, or own great potentialities in the targeted drug delivery systems which can be used in tumor diagnosis and target specific tumor cells, thereby minimizing the toxicity to normal cells. Here we review the unique properties of aptamer represents a great opportunity when applied to the rapidly developing fields of biotechnology and discuss the recent developments in the use of aptamers as powerful tools for analytic, diagnostic and therapeutic applications for cancer.

Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences.

Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an iterative method for in vitro selection of aptamers from a random synthetic oligonucleotide library. Successful retrieving of aptamers by SELEX relies on optimization of various steps including target immobilization, aptamer partitioning, amplification, and ssDNA generation, which all require spending considerable effort and cost. Furthermore, due to the random nature of the initial library, SELEX may redirect toward the selection of low-affinity aptamers that are over-represented in the ssDNA population due to PCR bias. Thus, precise monitoring of the SELEX process is crucial to ensure the selection of target-specific aptamers. In the present study, we investigated the reliability and simplicity of different direct and indirect monitoring methods including UV-Vis spectroscopy, real-time PCR quantification and melt-curve analysis, electrophoretic mobility shift assay (EMSA) and enzyme-linked oligonucleotide assay (ELONA) for selection of DNA aptamers for a protein target. All the examined methods were capable of illustrating the gradual evolution of specific aptamers by the progression of SELEX and showed almost similar results regarding the identification of the enriched round of selection. Moreover, we describe the use of melt-curve analysis in the colony real-time PCR method as a simple, robust, and repeatable tool for pre-sequencing separation of distinct aptamer clones.

Screening of Oligonucleotide Aptamers and Application in Detection of Pesticide and Veterinary Drug Residues

Aptamers are a type of single stranded short deoxyribonucleic acid or ribonucleic acid sequences screened from a random oligonucleotide library, which have high affinity and specific recognition for specific target molecules. Since the advent of screening process called the systematic evolution of ligands by exponential enrichment (SELEX), there have been various SELEX-based improved screening processes and capillary electrophoresis-based SELEX or non-SELEX selection processes. As a molecular recognition element similar to antibody, aptamers have been applied to the detection of residues of pesticide and veterinary drugs associated with food and environmental safety. In these applications, aptamers are usually formed composite probes with other materials to produce signals, such as gold nanoparticles, quantum dots, or constitute sensors with electrochemical electrode. These screening methods and the applications of aptamers in the detection of pesticide and veterinary drug residues are summarized to provide a helpful reference for these issues.

The application of aptamers in receptor- and molecule-based biosensors

Aptamer is an oligonucleotide sequence that can bind to a specific target molecule, which is screened from a random oligonucleotide library. It has shown promising prospects in biomedical applications. Due to its decisive advantages such as simple preparation, easy modification, good stability and wide range of targets compared with traditional antibodies, aptamer has been widely applied in the development of biosensors, which can not only be used to realize the identification and detection of target molecules, but also be utilized as the bridge between sensitive elements and transducers to improve the coupling efficiency. In addition, in recent years, the introduction of various nanomaterials such as metal nanoparticles, carbon materials and functional nanospheres to aptamer-based biosensors has contributed to their improvement in analytical performances and commercial applications. First, this review briefly introduces the methods for aptamer screening based on the SELEX technique and summarizes basic properties of aptamers. Second, the basic principle and classification of aptamer-based biosensors are introduced in detail. Third, based on the most recent progress in the field of aptamer-based biosensors, we focus on the applications of aptamers in biosensor development, including the applications in the field of biomimetic olfactory and taste receptor biosensors as well as applications in the detection of small molecules and proteins. Finally, we analyze and discuss the problems and development trends of applications of aptamers in biosensors.

Multiplex Immunoassays Utilizing Differential Affinity Using Aptamers Generated by MARAS

Disease diagnosis typically requires to determine concentration of multiple biomarkers in patient serums. Here, a novel method for multiplex immunoassays is proposed and the feasibility is demonstrated. The method utilizes the differential affinity between aptamers and multiple analytes for multiplex immunoassays. During the selection, aptamers capable of binding to multiple analytes with different affinities are screened from a random oligonucleotide library using the MARAS procedure with different magnetic field conditions for different target analytes. During the detection, the same magnetic field conditions are applied to differentiate different target analytes in blind serums. The results show that the recovery rates of the spiked targets in BD buffer and blind serums are similar. Moreover, there is a minimal interference resulting from non-specific binding of molecules in serums other than the target molecules. Therefore, the use of differential affinities between aptamers and different analytes for multiplex immunoassays is proved to be feasible.

Base‐Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology

The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5 kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1 kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.

Improved human plasma identification coverage based on random oligonucleotide library immobilized magnetic particles

Improved human plasma identification coverage based on random oligonucleotide library immobilized magnetic particles

Subtractive SELEX using agar beads for screening DNA aptamers with specific affinity to HIV gp41 antigen

To obtain DNA aptamers with a highly specific affinity to HIV gp41 antigen using SELEX screening for detection of HIV. The specific DNA aptamers of HIV gp41 antigen were screened from the double-stranded DNA derived from the single-stranded DNA (ssDNA) library with agarose beads as the supportive medium and HIV gp41 antigen as the target molecule using SELEX technique and real-time quantitative PCR. The secondary ssDNA library obtained after 6 rounds of screening was amplified by PCR to obtain dsDNA. The dsDNA was linked with pMDTM 18-T vector, cloned and sequenced to obtain 4 aptamers of HIV gp41 antigen. The affinities of the 4 aptamers (Kd) all reached the nanomolar level. Among the 4 aptamers, the No.15 aptamer showed the strongest affinity. Specificity analysis of the aptamers revealed that all these 4 aptamers had specific affinity to HIV gp41 antigen with no affinity to other non-specific proteins. We successfully obtained DNA aptamers with highly specific affinity to the HIV gp41 antigen from random single-stranded oligonucleotide library, and the obtained aptamers have the ability to antagonize HIV gp41 antigen.

Selection and Identification of Chloramphenicol-Specific DNA Aptamers by Mag-SELEX.

Chloramphenicol (CAP) has been widely used to treat bacterial infections in livestock and aquatic animals. To reduce the risk of CAP residues, an efficient technology to rapidly detect CAP residues in animal-sourced food is expressly needed. In this study, magnetic bead-based systematic evolution of ligands by exponential enrichment (Mag-SELEX) strategy was performed to select and identify CAP-specific single-stranded DNA (ssDNA) aptamers from a random oligonucleotide library. After nine rounds of selection, five potential ssDNA aptamers were selected. Low homology indicated that they might belong to different families. To identify an aptamer with the highest affinity for CAP, the dissociation constant (K d) values of these selected aptamers were determined. The lowest K d values of two potential aptamers (i.e., No. 4 and No. 5) were, respectively, 0.10162±0.0111 and 0.03224±0.00819μM, which were much lower than previously reported lowest K d value (i.e., 0.766μM) of CAP aptamer. Moreover, compared with No. 4, aptamer No. 5 had higher binding rate, which is quite different among those with CAP and with CAP's structural analogs (i.e., thiamphenicol (TAP) and florfenicol (FF)). These results indicated that the potential aptamer No. 5 with highest specificity and affinity for CAP would be an ideal aptamer for future detection of residual CAP in animal-sourced food.

Selection and Application of DNA Aptamer Against Oncogene Amplified in Breast Cancer 1.

Amplified in breast cancer 1 (AIB1), also known as steroid receptor coactivator 3 (SRC-3), is a transcriptional coactivator that interacts with nuclear receptors and other transcription factors to enhance their effects on target gene transcription. AIB1, which acts as a major oncogene, is highly expressed in many human cancers, and has been demonstrated to be a key regulator for tumor initiation, progression, metastasis, invasion, and survival. Recruitment of the transcriptional factor CBP/p300 by CBP/p300-interaction domain (CID) of AIB1 is essential for its transcriptional activation function. In this research, we isolated a DNA aptamer AY-3 that binds to AIB1-CID from a random oligonucleotide library using in vitro screening technology-Systematic Evolution of Ligands by EXponential enrichment (SELEX). The binding affinity of the aptamer to AIB1-CID fusion protein is in the nanomolar range. More importantly, the aptamer was found to disrupt in the interaction between p300 and AIB1. This aptamer has great potential to serve as a therapeutic agent for cancer by inhibiting the coactivation of AIB1.

Aptamer for imaging and therapeutic targeting of brain tumor glioblastoma.

Aptamers are short single-stranded nucleic acids (RNA or ssDNA), identified by an in vitro selection process, denominated SELEX, from a partially random oligonucleotide library. They bind to a molecular target, a protein or other complex macromolecular structures of interest with high affinity and specificity, comparable to those of antibodies. Recently, aptamer selection protocols were developed for targeting living cells, including tumors. Chemical modifications of the aptamers and modalities of their detection and delivery systems are already available with high selectivity and targeting ability for the desired cancer cell type, making them promising for diagnosis and therapy. Glioblastoma multiformae represents the most malignant and fatal stage of glioma, and is also the most frequent brain tumor. Glioblastoma-specific aptamers were developed by either targeting the whole cell surface or known glioma biomarkers. These aptamers may gain importance for imaging, tumor cell isolation from biopsies and drug delivery. In biomedical imaging techniques, aptamers coupled with radionuclide or fluorescent labels, bioconjugates and nanoparticles offer an advanced, noninvasive manner for defining the glioblastoma tissue border. Though single modality aptamer imaging probes have some limitations, these are overcome by the use of multimodal probes. Due to selectivity and chemical characteristics, aptamers can be coupled to functionalized nanoparticles and loaded with a drug, appeared promising for in vivo targeting of glioblastoma. Finally, aptamers are effective mediators for gene silencing when coupled to small interfering RNA and a viral vector, thus providing a novel tool with enhanced targeting capability in drug delivery, designed for tailored treatment of glioblastoma patients.

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application

Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wide variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer–AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer–AuNPs-based assay was found to be 5ng/ml.

SELEX aptamer used as a probe to detect circulating tumor cells in peripheral blood of pancreatic cancer patients.

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15+/CD45- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.

Targeting Th17 Cells with Small Molecules and Small Interference RNA.

T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn's disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4+ T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage.