A Korea Advanced Institute of Science and Technology (KAIST) research team has developed a technology that can detect RNA-degrading enzymes using the incidental cleavage activity of the CRISPR-Cas12a system.
Ribonuclease H, a kind of RNA degrading enzyme, is an essential region in reverse transcriptase viruses, including human immunodeficiency virus (HIV)-1 that cause AIDS and hepatitis B viruses and relates to the proliferation of reverse transcribing viruses.
Therefore, ribonuclease H is an important target for developing antiviral agents. The university stressed that researchers use electrophoresis or high-performance liquid chromatography to detect the activity of ribonucleic acid hydrolase H. However, these techniques have disadvantages such as low specificity and sensitivity, a complicated detection process, and a long detection time.
To overcome this limitation of the current technology, the research team, led by Professor Park Hyun-kyu of the Department of Biochemical Engineering, used the CRISPR-Cas12a system to improve the detection sensitivity significantly and succeeded in detecting ribonuclease H within an hour of the test, showing the highest sensitivity among technologies reported so far.
The research team used a short DNA/RNA chimera complex as a substrate for ribonuclease H and designed to release activator DNA under the activity of ribonuclease H.
The team stressed that when the Cas12a/crRNA complex recognizes the released activator DNA, it activates the incidental cleavage activity of Cas12a and cuts the surrounding reporter DNA to generate a fluorescent signal.
The research team could detect ribonuclease H activity in cancer cells through this technology. Researchers explained that when considering that ribonuclease H is involved in the proliferation of HIV, the results of this study may contribute to the development of an AIDS treatment.
“We expect to utilize our research for target discovery of antiviral agents by highly sensitive detection of ribonuclease H using the incidental cleavage activity of the CRISPR-Cas12a system,” Professor Park said.
The results of the research were published in Chemical Communications.