Regulation of Protein Activity and Cellular Functions Mediated by Molecularly Evolved Nucleic Acids

Regulation of protein activity is essential for revealing the molecular mechanisms of biological processes. DNA and RNA achieve many uniquely efficient functions, such as genetic expression and regulation. The chemical capability to synthesize artificial nucleotides can expand the chemical space of nucleic acid libraries and further increase the functional diversity of nucleic acids. Herein, a versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of aptamers able to regulate protein activity. Specifically, an aptamer that targets integrin alpha3 was identified and this aptamer can inhibit cell adhesion and migration. Overall, this chemical‐design‐assisted in vitro selection approach enables the generation of functional nucleic acids for elucidating the molecular basis of biological activities and uncovering a novel basis for the rational design of new protein‐inhibitor pharmaceuticals.     

Aptamers: molecular tools for analytical applications

Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.     

适配体在癌症诊断与靶向治疗中的研究进展

核酸适配体是利用体外筛选技术,即指数富集的配体系统进化技术(SELEX),从核酸分子文库中得到的寡核苷酸片段。其与靶标物有很高的特异性和亲和力,将适配体作为识别单元的生物传感研究以及适配体偶联成像试剂的生物体内外成像研究在临床诊断中有很大的应用前景,此外,适配体靶向癌细胞或组织的治疗方法相比传统化学治疗副作用更小,在临床上也有极大的应用前景。本文综述了适配体目前在癌症诊断和靶向治疗两个方面的研究进展,并分析现阶段存在的问题以及面临的挑战。     

A microfluidic SELEX prototype

Aptamers are nucleic acid binding species capable of recognizing a wide variety of targets ranging from small organic molecules to supramolecular structures, including organisms. They are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process referred to as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Here we describe an automated microfluidic, microline-based assembly that uses LabView-controlled actuatable valves and a PCR machine, and which is capable of the selection and synthesis of an anti-lysozyme aptamer as verified by sequence analysis. The microfluidic prototype described is 1) a simple apparatus that is relatively inexpensive to assemble, making automated aptamer selection accessible to many investigators, and 2) useful for the continued “morphing” of macro→meso→microfabricated structures until a convergence to a few functional systems evolves and emerges, partly or completely achieving simpler, smaller and more rapid SELEX applications.     

ADLOC: An Aptamer‐Displacement Assay Based on Luminescent Oxygen Channeling

Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high‐throughput screening. Here we describe an unprecedented type of a nucleic acid‐based sensor system and show that it is amenable to high‐throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof‐of‐principle study we demonstrate that the format is feasible for efficient identification of small drug‐like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin‐specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA‐aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer‐displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18 000 drug‐like small molecules two compounds as strong singlet‐oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC‐based assay described here could be used to screen at least 100 000 compounds per day.     

Control of Biocatalytic Transformations by Programmed DNA Assemblies

This study demonstrates the self‐assembly of inhibitor/enzyme‐tethered nucleic acid fragments or enzyme I‐, enzyme II‐modified nucleic acids into functional nanostructures that lead to the controlled inhibition of the enzyme or the activation of an enzyme cascade. In one system, the anti‐cocaine aptamer subunits are modified with monocarboxy methylene blue (MB⁺) as the inhibitor and with choline oxidase (ChOx). The cocaine‐induced self‐assembly of the aptamer subunits complex results in the inhibition of ChOx by MB⁺. In a further configuration, two nucleic acids of limited complementarity are functionalized at their 3′ and 5′ ends with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, or with MB⁺ and ChOx. In the presence of a target DNA sequence, synergistic complementary base‐pairing occurs, thus leading to stable supramolecular Y‐shaped nanostructures of the nucleic acid units. A GOx/HRP bienzyme cascade or the programmed inhibition of ChOx by MB⁺ is demonstrated in the resulting nucleic acid nanostructures. A quantitative theoretical model that describes the nucleic acid assemblies and that results in the inhibition of ChOx by MB⁺ or in the activation of the GOx/HRP cascade, respectively, is provided.     

核酸适体偶联药物的生物偶联构建技术与应用

核酸适体被称为“化学抗体”, 具有与抗体类似或更加优异的特异性和亲和力, 可以精准地靶向靶蛋白, 与靶蛋白特异性结合. 此外, 核酸适体还具有获取简单、 合成简便、 易于进行化学修饰、 不易变性、 靶标范围广、 免疫原性低及细胞内化快等优点, 已被广泛应用于众多研究领域. 在癌症治疗领域, 核酸适体作为一种优异的靶向识别工具和药物递送载体, 可实现抗肿瘤药物的精准递送. 将核酸适体与药物分子偶联, 可通过核酸适体的靶向作用使药物分子随核酸适体共同进入靶细胞, 实现药物分子在靶细胞内的富集, 进而促进靶细胞的死亡. 近年来, 核酸适体偶联药物已成为癌症靶向治疗的前沿新兴领域, 希望通过该领域的深入研究为癌症靶向治疗领域提供新思路. 本文综合评述了以生物偶联技术构建的核酸适体偶联药物及其应用研究.     

Label-free and reagentless aptamer-based sensors for small molecules

A label free, reagentless aptasensor for adenosine is developed on an ISFET device. The separation of an aptamer/nucleic acid duplex by adenosine leads to the aptamer/adenosine complex that alters the gate potential of the ISFET. The sensitivity limit of the device is 5 x 10-5 M. Also, the immobilization of the aptamer/nucleic acid duplex on an Au-electrode and the separation of the duplex by adenosine mono-phosphate (AMP) enable the electrochemical detection of adenosine by faradaic impedance spectroscopy. The separation of the aptamer/nucleic acid duplex by adenosine and the formation of the aptamer/adenosine complex results in a decrease in the interfacial electron-transfer resistance in the presence of [Fe(CN)6]3-/4- as redox active substrate.     

Binary malachite green aptamer for fluorescent detection of nucleic acids

A new probe that can fluorescently report the presence of specific nucleic acids in solution with extremely high selectivity was developed. The probe consists of malachite green-a triphenylmethane dye-and two short RNA strands, each of which comprises a fragment complementary to an analyte molecule and a fragment of a malachite green aptamer (MGA). The two RNA strands form MGA upon hybridization to the adjacent positions of the nucleic acid analyte. MGA is able to bind malachite green and enhance the fluorescence of the dye, thus monitoring the presence of the nucleic acid in solution. The probe reliably discriminates against 41 out of 42 possible single nucleotide substitutions in 14-mer DNA analyte at room temperature in physiological buffer. Consisting of unmodified RNA strands, which can be expressed in living cells, binary MGA probe represents a promising instrument for real-time nucleic acid monitoring in vivo.     

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。     

小分子靶标的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化(SELEX)技术筛选得到的核糖核酸(RNA)或单链脱氧核糖核酸(ssDNA)。核酸适配体通过高亲和力特异性地识别小分子、蛋白质、细胞、微生物等多种靶标,在生物、医药、食品和环境检测等领域的应用日渐增多。但目前实际可用的核酸适配体有限,其筛选过程复杂,筛选难度大,制约了其应用。与生物大分子、细胞和微生物等靶标不同,小分子靶标与核酸分子的结合位点少、亲和力弱,且靶标通常需要固定在载体上。此外,小分子靶标结合核酸形成的复合物与核酸自身的大小、质量、电荷性质等方面差异较小,二者的分离难度大。故小分子靶标的核酸适配体筛选过程与大分子和细胞等复合靶标相比有明显差异,筛选难度更大。因此需要根据其自身结构特点和核酸适配体的应用目的选定靶标或核酸库的固定方法,优化靶标核酸复合物的分离方法。本文介绍了不同类型小分子(具有基团差异的单分子、含相同基团分子和手性分子等)靶标的选择及其核酸适配体的筛选方法,并对核酸库的设计、与靶标结合的核酸的分离方法和亲和作用表征方法进行了介绍,列出了自2008年以来报道的40余种小分子靶标的核酸适配体序列和复合物的平衡解离常数(K_d)。     

Secondary Structure Libraries for Artificial Evolution Experiments

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.     

Development of a Dual‐Functional Hydrogel Using RGD and Anti‐VEGF Aptamer

Synthetic molecular libraries hold great potential to advance the biomaterial development. However, little effort is made to integrate molecules with molecular recognition abilities selected from different libraries into a single biomolecular material. The purpose of this work is to incorporate peptides and nucleic acid aptamers into a porous hydrogel to develop a dual‐functional biomaterial. The data show that an anti‐integrin peptide can promote the attachment and growth of endothelial cells in a 3D porous poly(ethylene glycol) hydrogel and an antivascular endothelial growth factor aptamer can sequester and release VEGF of high bioactivity. Importantly, the dual‐functional porous hydrogel enhances the growth and survival of endothelial cells. This work demonstrates that molecules selected from different synthetic libraries can be integrated into one system for the development of novel biomaterials.     

Energy Landscape of Aptamer/Protein Complexes Studied by Single‐Molecule Force Spectroscopy

Aptamers are single‐stranded nucleic acid molecules selected in vitro to bind to a variety of target molecules. Aptamers bound to proteins are emerging as a new class of molecules that rival commonly used antibodies in both therapeutic and diagnostic applications. With the increasing application of aptamers as molecular probes for protein recognition, it is important to understand the molecular mechanism of aptamer–protein interaction. Recently, we developed a method of using atomic force microscopy (AFM) to study the single‐molecule rupture force of aptamer/protein complexes. In this work, we investigate further the unbinding dynamics of aptamer/protein complexes and their dissociation‐energy landscape by AFM. The dependence of single‐molecule force on the AFM loading rate was plotted for three aptamer/protein complexes and their dissociation rate constants, and other parameters characterizing their dissociation pathways were obtained. Furthermore, the single‐molecule force spectra of three aptamer/protein complexes were compared to those of the corresponding antibody/protein complexes in the same loading‐rate range. The results revealed two activation barriers and one intermediate state in the unbinding process of aptamer/protein complexes, which is different from the energy landscape of antibody/protein complexes. The results provide new information for the study of aptamer–protein interaction at the molecular level.     

Functional Xeno Nucleic Acids for Biomedical Application

Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.     

Fluorescence detection of DNA by the catalytic activation of an aptamer/thrombin complex

A conjugate consisting of a thrombin aptamer tethered to the thrombin, Th, with a sensing nucleic acid (1) is used for the optical detection of DNA. The thrombin/aptamer complex blocks the biocatalytic functions of Th. Hybridization of the analyte DNA (2) to the sensing nucleic acid 1 yields a rigid duplex that detaches the aptamer from Th, a process that activates the protein toward the hydrolysis of bis(p-tosyl-Gly-Pro-Arg)-R110 (3) to the rhodamine 110 fluorophore (4). The system allows the DNA sensing with a sensitivity limit of 1 x 10-8 M. The aptamer/Th conjugate is also immobilized on glass slides for the optical detection of DNA. The dissociation of the aptamer/Th complex upon hybridization and the subsequent dehybridization of the duplex and the regeneration of the catalytically inactive Th/aptamer complex duplicate machinery functions.     

Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution‐phase aptamers into “aptamer particles”, each displaying many copies of a single sequence on its surface. We then use fluorescence‐activated cell sorting (FACS) to individually measure the relative affinities of >10⁸ aptamer particles and sort them in a high‐throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high‐affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high‐quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.     

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G‐quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G‐quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence‐coupled capillary electrophoresis method. A 29‐nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G‐quadruplex from unstructured 29‐nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6‐carboxyfluorescein labeled G‐quadruplex aptamer was measured. Third, the stability of G‐quadruplex aptamer–thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29‐nucleic acid thrombin binding aptamer could compete with G‐quadruplex aptamer and thus disassociated the G‐quadruplex structure into an unstructured aptamer. These data suggest that our in‐house established fluorescence‐coupled capillary electrophoresis assay could be applied to binding studies of the G‐quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.     

Monoclonal Antibodies Application in Lateral Flow Immunochromatographic Assays for Drugs of Abuse Detection

Lateral flow assays (lateral flow immunoassays and nucleic acid lateral flow assays) have experienced a great boom in a wide variety of early diagnostic and screening applications. As opposed to conventional examinations (High Performance Liquid Chromatography, Polymerase Chain Reaction, Gas chromatography-Mass Spectrometry, etc.), they obtain the results of a sample’s analysis within a short period. In resource-limited areas, these tests must be simple, reliable, and inexpensive. In this review, we outline the production process of antibodies against drugs of abuse (such as heroin, amphetamine, benzodiazepines, cannabis, etc.), used in lateral flow immunoassays as revelation or detection molecules, with a focus on the components, the principles, the formats, and the mechanisms of reaction of these assays. Further, we report the monoclonal antibody advantages over the polyclonal ones used against drugs of abuse. The perspective on aptamer use for lateral flow assay development was also discussed as a possible alternative to antibodies in view of improving the limit of detection, sensitivity, and specificity of lateral flow assays.     

Kinetic capillary electrophoresis-based affinity screening of aptamer clones

DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer-target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K_d values for the in-capillary reaction of an aptamer and a target and that the obtained K_d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3-5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.