综述与专论: 核酸适配体筛选与亲和力评价技术主要研究方向、进展与挑战

核酸适配体是一类具有特异性分子识别能力的单链DNA或者RNA分子，通过指数富集的配体系统进化技术（SELEX）筛选得到。核酸适配体相比抗体具有热稳定性高、便于化学合成与修饰、免疫原性低等优点，在生物分析、生物医学、生物技术等众多领域引起广泛关注。高质量的核酸适配体是应用的基础，然而目前能够满足实际应用的核酸适配体数量还非常有限。如何获得高亲和力、高特异性、高体内稳定性的核酸适配体是核酸适配体领域的技术瓶颈。本文首先简单介绍了SELEX技术的基本原理和核酸库的设计、筛选过程监控、次级文库制备、测序和候选适配体筛选等关键步骤。接着归纳总结了30多年来核酸适配体筛选技术的6个主要研究方向、研究进展和局限性。这6个主要研究方向分别是提高适配体特异性的筛选方法、提高适配体稳定性（抗核酸酶降解能力）的筛选方法、快速筛选方法、复杂靶标适配体筛选方法、小分子靶标适配体筛选方法、提高适配体亲和力的筛选方法。其中快速筛选技术是长期以来持续关注的研究方向，几乎所有物理分离手段都已用于提高SELEX的筛选效率。最近，高效化学反应与SELEX技术的结合为核酸适配体的快速筛选提供了新的策略。本文随后对适合小分子靶标核酸适配体筛选的3类方法进展和存在的问题进行了重点评述。这3类方法分别是基于靶标固定的筛选技术、基于文库固定的筛选技术（捕获-SELEX，Capture-SELEX）和均相筛选技术（氧化石墨烯-SELEX，GO-SELEX）。基于靶标固定的筛选技术尽管存在空间位阻等众多问题，由于其操作的简单性，目前依然应用广泛。近年来Capture-SELEX应用广泛。结合36种靶标适配体的筛选实验条件（文库设计、正筛靶标浓度、负筛靶标的选择和浓度）和所获得的适配体的亲和力（K_D，解离常数，dissociation constant）和特异性，对Capture-SELEX的实验条件与适配体性能的关系进行了讨论。统计数据表明，降低正筛靶标浓度有利于提高适配体的亲和力，但不是必要条件。负筛选是目前提高适配体特异性的主要技术手段，但适配体的特异性还不能满足实际需求。负筛选靶标及其浓度的选择差异很大，而且36种靶标中有20种靶标的适配体筛选没有进行负筛选。如何提高核酸适配体的特异性是目前小分子靶标核酸适配体所面临的难题，急需寻找新的策略。本文还列表归纳了近三年利用GO-SELEX进行的13种小分子靶标的实验条件和所获得的适配体的K_D和特异性。统计数据表明，GO-SELEX比Capture-SELEX所需要的筛选轮数少，两种方法所获得的适配体的亲和力多在纳摩尔每升水平。Capture-SELEX相对较低的筛选效率应该主要由于文库的自解离问题。核酸适配体的亲和力评价是候选核酸适配体结构与性能评价的重要组成部分。常用的核酸适配体亲和力评价技术包括基于分离、基于固定、均相体系三大类十多种方法。假阳性和假阴性是各种评价技术都有可能存在的问题。本文以纳米金比色法和等温热滴定技术为例评述技术进展，讨论导致不同亲和力评价技术结果不一致性问题的根本原因。本文最后对核酸适配体筛选技术、亲和力评价技术和技术的标准化的未来发展趋势进行了展望。

Review: Main Research Directions, Advances and Challenges in Nucleic Acid Aptamer Isolation and Affinity Evaluation Technologies

Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules with specific molecular recognition capability, obtained by a process called systematic evolution of ligands by exponential enrichment (SELEX). They have the advantages of high thermal stability, ease of chemical synthesis and modification, and low immunogenicity compared to antibodies, and have attracted widespread interest in many fields such as bioanalysis, biomedicine, and biotechnology. High-quality aptamers are the basis of applications, however, the number of them that meet requirements of practical applications is very limited. How to obtain aptamers with high affinity, high specificity, and high in vivo stability is the technical bottleneck in the field of aptamers. Firstly, this review briefly introduces the basic theory of SELEX and its critical experimental steps including design of nucleic acid library, monitoring selection process, preparation of secondary library, sequencing and screening of candidate aptamers. The six main research directions of SELEX during the past thirty years are then concluded. They are respectively (1) how to improve the specificity of aptamers, (2) how to improve the stability of aptamers against nuclease degradation, (3) rapid SELEX, (4) how to isolate aptamers for complex targets, (5) how to isolate small molecule-binding aptamers, and (6) how to isolate high affinity aptamers. The development of rapid SELEX technologies has attracted tremendous attention and almost all physical separation methods have been applied to improve the SELEX efficiency. Very recently, several methods involving the highly efficient chemical reactions have been reported, providing novel strategies for the rapid isolation of aptamers. The key research progresses of SELEX technologies suitable for the isolation of small molecule-binding aptamers are subsequently reviewed and the challenges of each method are critically commented. There are three types of SELEX methods including the target-immobilized SELEX, library-immobilized SELEX (Capture-SELEX), and homogeneous SELEX (GO-SELEX). Even though the target-immobilized SELEX suffers from many issues such as steric hindrance, it is still a popularly used method due to its simplicity. In recent years, Capture-SELEX has been widely applied. The experimental conditions of Capture-SELEX (concentration of positive-SELEX target, choice of negative-SELEX targets and their concentrations) and the affinity (K_D, dissociation constant) and the specificity of the isolated aptamers for the 36 targets are listed in a table. Based on the information from the table, the effect of the experimental conditions on the affinity and the specificity is discussed. The statistical data indicates that the lower concentration of the positive-SELEX targets favors the isolation of the higher affinity aptamers, while it is not a necessary condition. Negative-SELEX is currently the dominant strategy to improve the specificity of aptamers. However, the specificity of many aptamers cannot meet the requirement for practical applications. The choice of negative-SELEX targets and their concentrations in each case are quite different. In 20 out of the 36 targets, no negative-SELEX was performed for the aptamer isolation. How to obtain the aptamers with high specificity is the most difficult challenge for small molecule targets. It is in urgent need to establish novel strategies beyond negative-SELEX to improve the specificity of aptamers. The experimental conditions of GO-SELEX and the K_D and the specificity of the isolated aptamers for the 13 small molecule targets are also list for comparison. The comparison data shows the less numbers of the enrichment cycles required for GO-SELEX than Capture-SELEX, while the obtained aptamers all commonly have K_D in the nanomolar range. The lower enrichment efficiency of Capture-SELEX should be due to the self-dissociation of the immobilized library. The affinity evaluation is the important part of the characterization of aptamer structure and performance. More than ten affinity assays are frequently used for aptamer characterization, which are roughly divided into three categories: separation-based, immobilization-based, and homogeneous methods. All techniques could generate false-positive and false-negative results. Taking gold nanoparticle-based colorimetric assay and isothermal thermal titration as examples, we review the technical progresses and comment on the fundamental reasons resulting in the inconsistent results when the different affinity assays are conducted. The final part of this review provides an outlook on the future trends of aptamer isolation technologies, affinity characterization techniques, and the technical standardization.