Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity
Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity
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DOI：
10.1006/mcpr.2002.0423
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发表时间：
2002-08-01
影响因子：
3.3
通讯作者：
Lu, P
中科院分区：
文献类型：
--
作者：
Rizzo, J;Gifford, LK;Lu, P
目前检测和测定核糖核酸酶H（RNase H）活性的方法是间接的和耗时的。本文介绍了一种基于分子信标荧光猝灭机制的直接灵敏的RNA切割检测方法。开发了用于RNase H酶活性的RNA-DNA嵌合信标测定。底物是用5 '-荧光素和3'-DABCYL标记的单链RNA-DNA嵌合寡核苷酸。探针的荧光团（荧光素）通过RNA：DNA茎环结构与猝灭剂（DABCYL）保持紧密接近。当RNA：DNA杂交茎的RNA序列被切割时，荧光团与猝灭剂分离，并且荧光可以作为时间的函数被检测到。已经调查了具有不同茎长度和序列的嵌合信标用于该测定，其中E. coli RNase H.我们发现，信标动力学参数与以前报道的值使用更繁琐的测定定性一致。该方法允许实时检测RNase H活性，并为RNase H动力学和机理研究提供了一种方便的方法。(C)2002爱思唯尔科技有限公司版权所有。
Current methods to detect and assay ribonuclease H (RNase H) activity are indirect and time-consuming. Here we introduce a direct and sensitive method, based on the fluorescence quenching mechanism of molecular beacons, to assay RNA cleavage in RNA:DNA hybrids. An RNA-DNA chimeric beacon assay for RNase H enzymatic activity was developed. The substrate is a single-stranded RNA-DNA chimeric oligonucleotide labeled with a 5'-fluorescein and a 3'-DABCYL. The fluorophore (fluorescein) of the probe is held in close proximity to the quencher (DABCYL) by the RNA:DNA stem-loop structure. When the RNA sequence of the RNA:DNA hybrid stem is cleaved, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Chimeric beacons with different stem lengths and sequences have been surveyed for this assay with E. coli RNase H. We found that the beacon kinetic parameters are in qualitative agreement with previously reported values using more cumbersome assays. This method permits real-time detection of RNase H activity and a convenient approach to RNase H kinetic and mechanistic study. (C) 2002 Elsevier Science Ltd. All rights reserved.