Biology subjects

Chandradoss, S. D.

Publications and source records attributed to Chandradoss, S. D..

2 recordsLinked to original sources

bioRxiv2019

Argonaute bypasses cellular obstacles without hindrance during target search

Argonaute (Ago) proteins are key players in gene regulation in eukaryotes and host defense in prokaryotes. For specific interference, Ago relies on base pairing between small nucleic acid guides and complementary target sequences. To efficiently scan nucleic acid chains for potential targets, Ago must bypass both secondary structures in mRNA and single stranded DNA as well as protein barriers. Through single-molecule FRET, we reveal that lateral diffusion is mediated mainly through protein-nucleic acid interactions, rather than interactions between the guide and targeted strand. This allows Ago to scan for targets with high efficiency but without maintaining tight contact with the DNA backbone. Real-time observations show that Ago "glides" short distances over secondary structures while using intersegmental jumps to reduce scanning redundancy and bypass protein barriers. Our single-molecule method in combination with kinetic analysis may serve as a novel platform to study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

biophysics↗

bioRxiv2019

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures ([≥]65{degrees}C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5-end and thymidines in the sub-seed segment (siDNA nucleotides 2-4). Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37{degrees}C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

molecular biology↗