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Biosens. Bioelectron., 2 March 2024, 253 S0956-5663(24)00179-9 | https://doi.org/10.1016/j.bios.2024.116174 A novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension reaction

Junhyeok Yoon, Jinhwan Lee, Jaemin Kim, Sang Mo Lee, Soohyun Kim, Hyun Gyu Park

Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

Corresponding author : Hyun Gyu Park

Abstract

We herein present a novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction. The detection probe employed as a key component in this technique serves as a substrate for RNase H and triggers the PS-THSP reaction upon the RNase H-mediated degradation of the probe. As a consequence, a large number of long concatemeric amplification products could be produced and used to identify the RNase H activity through the fluorescence signals produced by the nucleic acid-specific fluorescent dye, SYTO 9. Importantly, the use of the gp32 protein allowed the PS-THSP reaction to be performed at 37 °C, ultimately enabling an isothermal one-step RNase H assay. Based on this sophisticated design principle, the RNase H activity was very sensitively detected, down to 0.000237 U mL ^-1 with high specificity. We further verified its practical applicability through its successful application to the screening of RNase H inhibitors. With its operational convenience and excellent analytical performance, this technique could serve as a new platform for RNase H assay in a wide range of biological applications.

논문정보

형식Research article
게재일2024년 03월 (BRIC 등록일 2024-03-27)
연구진국내 연구진
분야 생명과학 > 생물공학

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