A novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension reaction

• Published in: Biosensors & bioelectronics Vol. 253; p. 116174
• Main Authors: Yoon, Junhyeok, Lee, Jinhwan, Kim, Jaemin, Lee, Sang Mo, Kim, Soohyun, Park, Hyun Gyu
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01.06.2024
• Subjects: Enzyme assay; Isothermal amplification; Phosphorothioate (PS) DNA; RNase H; Phosphorothioate (PS) DNA; RNase H; Enzyme assay; Isothermal amplification
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2024.116174

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.