发光核酸适配体的筛选及应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-1183

摘要/Abstract

摘要：

发光核酸适配体（light-up nucleic acid aptamers,LNAs）是一类能够特异性结合目标分子并增强目标分子发光性能的功能核酸（functional nucleic acids,FNAs）。LNAs具有组成简单、结构稳定、表达时间短等优点,因此在细胞成像、物质检测等传感领域显示出巨大潜力。随着对细胞内基因原位分析的研究深入,现存胞内成像技术的不足逐渐显露,LNAs成像系统应运而生。本文梳理并概括了不同类型LNAs的筛选方法,并从胞内、体外两个方面对LNAs的前沿应用进行了分析,指出了当前LNAs研究以及传感系统存在的不足,展望了LNAs在无细胞传感中的巨大发展前景,以期能够促进LNAs系统成为胞内外传感研究的一件利器。

关键词: 核酸发光适配体, 荧光团, RNA成像, 功能核酸

Abstract:

Light-up nucleic acid aptamers（LNAs）are a class of functional nucleic acids that can specifically bind to target molecules and enhance the luminescence performance of target molecules. LNAs have the advantages of simple composition,stable structure,and short cell expression time,thus they show great potential in the field of cell imaging,substance detection and other sensing fields. With the in-depth research on the in-situ analysis of intracellular genes,the deficiencies of the existing intracellular imaging technology have gradually appeared,and the LNAs imaging system has emerged. This review sorts out and summarizes the selecting methods of different types of LNAs,analyzes the frontier applications of LNAs from both intracellular and in vitro aspects,points out the deficiencies of the current LNAs research and the sensing system,and looks forward to the huge development prospects of LNAs in cell-free sensing,aiming to promote the LNAs system becoming a powerful tool for the research of intracellular and extracellular sensing.

Key words: light-up nucleic acid aptamers, fluorophore, RNA imaging, functional nucleic acids

周子琦, 张洋子, 兰欣悦, 刘洋儿, 朱龙佼, 许文涛. 发光核酸适配体的筛选及应用[J]. 生物技术通报, 2022, 38(5): 240-247.

ZHOU Zi-qi, ZHANG Yang-zi, LAN Xin-yue, LIU Yang-er, ZHU Long-jiao, XU Wen-tao. Selection and Application of Light-up Nucleic Acid Aptamers[J]. Biotechnology Bulletin, 2022, 38(5): 240-247.

图/表 3

图1 采用SELEX等技术对LNAs进行体外筛选

Fig. 1 In vitro selection of LNAs using SELEX and other techniques

图2 LNAs系统在活细胞内的应用 A：瞬时转染COS-7细胞中(CGG)60-Spinach 2病灶形成的监测;B：m6A修饰Broccoli作为荧光底物在FTO检测中的应用;C：在活细胞中使用RNA适配体和Spinach发光核酸适配体检测SAM;D：劈裂Spinach发光核酸适配体的组装与传感

Fig. 2 Application of LNAs system in living cells A: Monitoring the formation of (CGG)60-Spinach 2 foci in transiently transfected COS-7 cell. B: Application of m6A-modified Broccoli as a fluorometric substrate in the FTO assay. C: Imaging SAM in living cells with RNA aptamer and Spinach. D: Assembly and sensing of aptamer in split Spinach

图3 LNAs系统在体外的应用 A：基于核糖开关的荧光二价金属离子传感器示意图,czcD-2的二级结构和厌氧滴定实验;B：蔬菜适配体标记的RNA及其裂解产物的凝胶检测示意图;C：ROSALIND系统的传感设备与原理

Fig. 3 Application of LNAs system in vitro A: Schematic diagram of fluorescent bivalent metal ion sensor based on riboswitch, secondary structure of czcD-2 and anaerobic titration experiment. B: Schematic representation of the in-gel detection of vegetable aptamer-tagged RNAs and their cleavage products. C: Sensing equipment and principle of ROSALIND system

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