Biology subjects

Chong, Y.

Publications and source records attributed to Chong, Y..

2 recordsLinked to original sources

bioRxiv2019

The stepwise endonuclease activity of a thermophilic Argonaute protein

Thermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the PfAgo stepwise endonuclease activity followed by target DNA amplification, we establish a rapid and specific platform for the unambiguously multiplex gene detection, termed RADAR (Renewed-gDNA Assisted DNA-cleavage by Argonaute). In the end, RADAR was applied to distinguish of human papillomavirus of serotypes in patient samples in a single reaction, suggesting that our technique would be adopted for diagnosing application.

bioengineering↗

bioRxiv2019

A-Star, an Argonaute-directed System for Rare SNV Enrichment and Detection

The ability to detect rare mutations has revolutionized diagnosis and monitoring of tumors, but is limited by the shortage of sensitive, cost-effective and high coverage methods for identification of extremely low abundant mutations. Here, we establish a single-tube multiplex PCR system by employing thermophilic Argonaute-derived DNA-guided nuclease for highly efficient rare mutation detection, referred to as A-Star (Argonaute-directed specific target enrichment and detection), that combines the selective cleavage of the wild type DNA in the DNA denaturation step and the followed amplification of mutant DNA during PCR. A-Star enables easy detection and quantitation of rare mutations originally as low as 0.01% in allele frequency with a [>=] 5500-fold efficiency. We also demonstrate the feasibility of A-Star for detecting oncogenic mutations in complex biological systems such as solid tumors tissues and blood samples. Remarkably, A-Star could achieve the detection of multiple oncogenic genes simultaneously through a simple single-tube reaction. Taken together, our work illustrates a supersensitive and rapid nucleic acid detection system, thereby extending the utility for both research and therapeutic applications.

bioengineering↗