Ali M Monsur, Aguirre Sergio D, Mok Wendy W K, Li Yingfu
Department of Biochemistry, McMaster University, Hamilton, ON, Canada.
Methods Mol Biol. 2012;848:395-418. doi: 10.1007/978-1-61779-545-9_25.
Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules that have the ability to catalyze a chemical reaction. Currently, DNAzymes have to be isolated from random-sequence DNA libraries by a process known as in vitro selection (IVS) because no naturally occurring DNAzyme has been discovered. Several IVS studies have led to the isolation of many RNA-cleaving DNAzymes (RNase DNAzymes), which catalyze the transesterification of a phosphodiester linkage in an RNA substrate, resulting in its cleavage. An RNase DNAzyme and its substrate can be modified with a pair of donor and acceptor fluorophores (or a fluorophore and quencher pair) to create a fluorescence-signaling system (a signaling DNAzyme) where the RNA-cleaving activity of the DNAzyme is reported through the generation of a fluorescent signal. A signaling DNAzyme can be further coupled with an aptamer (a target-binding nucleic acid sequence) to generate a fluorogenic aptazyme in which the aptamer-target interaction confers an allosteric control of the coupled RNA-cleaving and fluorescence-signaling activity of the DNAzyme. Fluorogenic aptazymes can be exploited as valuable molecular tools for biosensing applications. In this chapter, we provide both a detailed description of methods for isolation of signaling DNAzymes by IVS and general approaches for rational engineering of fluorogenic aptazymes for target detection.
脱氧核酶（或DNA酶）是具有催化化学反应能力的单链DNA分子。目前，由于尚未发现天然存在的DNA酶，因此必须通过一种称为体外筛选（IVS）的过程从随机序列DNA文库中分离DNA酶。多项IVS研究已导致分离出许多RNA切割DNA酶（核糖核酸酶DNA酶），这些酶催化RNA底物中磷酸二酯键的酯交换反应，从而导致其切割。核糖核酸酶DNA酶及其底物可以用一对供体和受体荧光团（或荧光团和猝灭剂对）进行修饰，以创建一个荧光信号系统（信号DNA酶），其中DNA酶的RNA切割活性通过荧光信号的产生来报告。信号DNA酶可以进一步与适体（一种靶标结合核酸序列）偶联，以产生荧光适体酶，其中适体与靶标的相互作用赋予对偶联的DNA酶的RNA切割和荧光信号活性的变构控制。荧光适体酶可作为生物传感应用中有价值的分子工具。在本章中，我们详细描述了通过IVS分离信号DNA酶的方法，以及合理设计用于靶标检测的荧光适体酶的一般方法。
