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Target‐Activated DNA Polymerase Activity for Sensitive RNase H Activity Assay
Author(s) -
Jung Yujin,
Lee Chang Y.,
Park Ki S.,
Park Hyun G.
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201800645
Subject(s) - rnase h , rnase p , dna polymerase , microbiology and biotechnology , polymerase , primer (cosmetics) , dna , ribonuclease , biology , chemistry , biochemistry , rna , gene , organic chemistry
Herein, the ribonuclease H (RNase H) activity assay based on the target‐activated DNA polymerase activity is described. In this method, a detection probe composed of two functional sequences, a binding site for DNA polymerase and a catalytic substrate for RNase H, serves as a key component. The detection probe, at its initial state, suppresses the DNA polymerase activity, but it becomes destabilized by RNase H, which specifically hydrolyzes RNA in RNA/DNA hybrid duplexes. As a result, DNA polymerase recovers its activity and initiates multiple primer extension reactions in a separate TaqMan probe‐based signal transduction module, leading to a significantly enhanced fluorescence “turn‐on” signal. This assay can detect RNase H activity as low as 0.016 U mL −1 under optimized conditions. Furthermore, its potential use for evaluating RNase H inhibitors, which have been considered potential therapeutic agents against acquired immune deficiency syndrome (AIDS), is successfully explored. In summary, this approach is quite promising for the sensitive and accurate determination of enzyme activity and inhibitor screening.