期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 304, 期 -, 页码 -出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2019.127380
关键词
RNase H activity; DNA walker; DNAzymes; Signal amplification; RNase H inhibitor
资金
- National Natural Science Foundation of China [21804046, 21778020]
- Fundamental Research Funds for the Central Universities [2662018QD012]
- Natural Science Foundation of Hubei Province, China [2018CFB368]
- Sci-tech Innovation Foundation of Huazhong Agriculture University [2662017PY042, 2662018PY024]
The evaluation of Ribonuclease H (RNase H) activity is very important for anti-HIV drug development and the study of cellular processes, including DNA replication, DNA repair and transcription. However, the highly sensitive detection of RNase H activity with a simple strategy remains challenging. Here, a DNAzyme-powered on-particle DNA walker was proposed as a signal amplifier to evaluate the RNase H activity at levels as low as 0.00847 U mL(-1). Pre-caged enzyme strands and substrate strands of Mn2+-specific DNAzyme were modified at the interface of gold nanoparticles, forming the DNA walker probes. In the absence of RNase H, a minimum fluorescent signal was observed due to excellent ability of gold nanoparticles to quench the fluorophore that labeled the substrate strand. In the presence of RNase H, the DNA/RNA hybrid complex was hydrolyzed to release single stranded DNA, which was able to hybridize to the locker strand that caged the enzyme strand. Following the addition of Mn2+, the active forms of DNAzymes were cleaved at the RNA site of substrate strands, releasing enhanced fluorescent molecules. Finally, the walker strands traveled over the AuNPs like a machine to amplify the signal, thus indirectly measuring the activity of RNase H. This approach proved to be an ultrasensitive and simple method for the evaluation of RNase H activity in cell extracts and has also been successfully used to evaluate the effects of inhibitors.
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