3 years ago

Programmable RNA recognition using a CRISPR-associated Argonaute

Doudna, Lapinaite, Cate, A., J., J. A.
Argonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5'-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) are destabilized upon binding to highly complementary RNA substrates. Quantitative and site-specific covalent crosslinking of the gRNA to MpAgo stabilizes the MpAgo RNP and significantly improves its specificity and affinity for RNA targets. Using crosslinked MpAgo RNPs, we mapped the seed region of the gRNA that contributes to specific RNA substrate binding, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that crosslinked MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that crosslinked MpAgo RNPs can be used as a highly-specific RNA-targeting platform to probe RNA biology.

Publisher URL: http://biorxiv.org/cgi/content/short/208041v1

DOI: 10.1101/208041

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