Highly sensitive detection of RNase H via a novel DNA/RNA heteroduplex combining isothermal exponential amplification strategy
Zhang X. Jia Q. Shi Y. Gao Z. Amin A. Xie Y. Li Y.
December 2024Elsevier Inc.
Microchemical Journal
2024#207
Ribonuclease H (RNase H) plays a crucial role in a variety of cellular processes and is emerging as an essential therapeutic target for many diseases. Various methods have been constructed to assay RNase H activity, but these methods are either time-consuming or have poor sensitivity. In this study, we developed a novel strategy that combined a specially designed DNA/RNA chimeric substrate with exponential amplification reaction (EXPAR) for rapid and sensitive detection of RNase H activity. In the presence of RNase H, the RNA strand of the DNA/RNA heteroduplex was specifically degraded, forming a 3′-hydroxyl group in the locking primer for recognition and extension by DNA polymerase. This ultimately triggered EXPAR and produced a large number of single-stranded DNA, which was monitored in real-time with fluorescent dye. Under optimized conditions, the proposed strategy can detect as low as 6 × 10−10 U/μL of RNase H, which was at least 1000 times more sensitive than several reported methods. Furthermore, we demonstrated the usage of this method for RNase H inhibitor analysis and practical application in complex biological samples, including serum and tumor cell extracts. Therefore, these results suggested that the developed method is a promising tool for highly sensitive detection of RNase H and RNase H-associated disease diagnosis.
DNA/RNA duplex , Exponential amplification reaction , Inhibitor , Locking primer , RNase H
Text of the article Перейти на текст статьи
Sino-UAE International Cooperative Joint Laboratory of Pathogenic Microorganism Rapid Detection, Qingdao Nucleic Acid Rapid Detection Engineering Research Center, Qingdao Key Laboratory of Nucleic Acid Rapid Detection, College of Biological Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China
Arcadia High School, Arcadia, 91007, CA, United States
Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah, 27272, United Arab Emirates
Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, 010000, Kazakhstan
Sino-UAE International Cooperative Joint Laboratory of Pathogenic Microorganism Rapid Detection
Arcadia High School
Basic Medical Sciences
Department of Biology
10 лет помогаем публиковать статьи Международный издатель
Книга Публикация научной статьи Волощук 2026 Book Publication of a scientific article 2026