核酸识体的研究及应用

核酸识体(aptamer)是近年来发展起来的一类经体外人工进化程序筛选出的寡聚核苷酸。它能高效、特异地结合各种配体，在蛋白质的分析检测、医学诊断治疗、生物传感器和分子开关的开发等方面有很大的应用前景，文章对核酸识体的研究和应用进展进行了综述。     

Validation of an empirical RNA-ligand scoring function for fast flexible docking using RiboDock®

We report the design and validation of a fast empirical function for scoring RNA-ligand interactions, and describe its implementation within RiboDock, a virtual screening system for automated flexible docking. Building on well-known protein-ligand scoring function foundations, features were added to describe the interactions of common RNA-binding functional groups that were not handled adequately by conventional terms, to disfavour non-complementary polar contacts, and to control non-specific charged interactions. The results of validation experiments against known structures of RNA-ligand complexes compare favourably with previously reported methods. Binding modes were well predicted in most cases and good discrimination was achieved between native and non-native ligands for each binding site, and between native and non-native binding sites for each ligand. Further evidence of the ability of the method to identify true RNA binders is provided by compound selection ('enrichment factor') experiments based around a series of HIV-1 TAR RNA-binding ligands. Significant enrichment in true binders was achieved amongst high scoring docking hits, even when selection was from a library of structurally related, positively charged molecules. Coupled with a semi-automated cavity detection algorithm for identification of putative ligand binding sites, also described here, the method is suitable for the screening of very large databases of molecules against RNA and RNA-protein interfaces, such as those presented by the bacterial ribosome.     

Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time. Selection rules based on dissociation equilibria and kinetics were devised to obtain families of analogous sequences. Selected sequences of the same family were shown to bind thrombin with high affinity. Furthermore, data acquired from SR-CE selection was mined by creating sub-libraries that were categorized by the functionality of the aptamers (e. g., pre-organized aptamers versus structure-induced aptamers). Using this approach, a novel fluorescent molecular recognition sensor for thrombin with nanomolar detection limits was discovered. Thus, in this proof-of-concept report, we have demonstrated the potential of a “DNA Aptaomics” approach to systematically design functional aptamers as well as to obtain high affinity aptamers.     

Molecule-binding dependent assembly of split aptamer and γ-cyclodextrin: A sensitive excimer signaling approach for aptamer biosensors

A highly sensitive and selective fluorescence aptamer biosensors for the determination of adenosine triphosphate (ATP) was developed. Binding of a target with splitting aptamers labeled with pyrene molecules form stable pyrene dimer in the γ-cyclodextrin (γ-CD) cavity, yielding a strong excimer emission. We have found that inclusion of pyrene dimer in γ-cyclodextrin cavity not only exhibits additive increases in quantum yield and emission lifetime of the excimer, but also facilitates target-induced fusion of the splitting aptamers to form the aptamer/target complex. As proof-of-principle, the approach was applied to fluorescence detection of adenosine triphosphate. With an anti-ATP aptamer, the approach exhibits excimer fluorescence response toward ATP with a maximum signal-to-background ratio of 32.1 and remarkably low detection limit of 80 nM ATP in buffer solution. Moreover, due to the additive fluorescence lifetime of excimer induced by γ-cyclodextrin, time-resolved measurements could be conveniently used to detect as low as 0.5 μM ATP in blood serum quantitatively.     

光学适配体传感器检测赭曲霉毒素A的研究进展

赭曲霉毒素A(Ochratoxin A,OTA)是真菌产生的次级代谢产物,性质稳定,不易去除,人体摄入后将产生严重的健康危害。数十年来,核酸适配体不断发展,成为生物传感器的重要识别元件之一,适体传感器被广泛用于生物、医药、疾病等分析检测。本文总结了用于检测OTA的经典方法和基于核酸适配体的生物传感器方法,并主要从光学适配体传感器方面阐述了近年用于检测赭曲霉毒素A的适配体传感器,并对其进行了总结和展望。     

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed K_d values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with K_d values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.     

Ultrafast Fluorescence Spectroscopy via Upconversion and Its Applications in Biophysics

In this review, the experimental set-up and functional characteristics of single-wavelength and broad-band femtosecond upconversion spectrophotofluorometers developed in our laboratory are described. We discuss applications of this technique to biophysical problems, such as ultrafast fluorescence quenching and solvation dynamics of tryptophan, peptides, proteins, reduced nicotinamide adenine dinucleotide (NADH), and nucleic acids. In the tryptophan dynamics field, especially for proteins, two types of solvation dynamics on different time scales have been well explored: ~1 ps for bulk water, and tens of picoseconds for “biological water”, a term that combines effects of water and macromolecule dynamics. In addition, some proteins also show quasi-static self-quenching (QSSQ) phenomena. Interestingly, in our more recent work, we also find that similar mixtures of quenching and solvation dynamics occur for the metabolic cofactor NADH. In this review, we add a brief overview of the emerging development of fluorescent RNA aptamers and their potential application to live cell imaging, while noting how ultrafast measurement may speed their optimization.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

Aptamer Laden Liquid Crystals Biosensing Platform for the Detection of HIV-1 Glycoprotein-120

We report a label-free and simple approach for the detection of glycoprotein-120 (gp-120) using an aptamer-based liquid crystals (LCs) biosensing platform. The LCs are supported on the surface of a modified glass slide with a suitable amount of B40t77 aptamer, allowing the LCs to be homeotropically aligned. A pronounced topological change was observed on the surface due to a specific interaction between B40t77 and gp-120, which led to the disruption of the homeotropic alignment of LCs. This results in a dark-to-bright transition observed under a polarized optical microscope. With the developed biosensing platform, it was possible to not only identify gp-120, but obtained results were analyzed quantitatively through image analysis. The detection limit of the proposed biosensing platform was investigated to be 0.2 µg/mL of gp-120. Regarding selectivity of the developed platform, no response could be detected when gp-120 was replaced by other proteins, such as bovine serum albumin (BSA), hepatitis A virus capsid protein 1 (Hep A VP1) and immunoglobulin G protein (IgG). Due to attributes such as label-free, high specificity and no need for instrumental read-out, the presented biosensing platform provides the potential to develop a working device for the quick detection of HIV-1 gp-120.     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

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(K_D)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。