| Literature DB >> 35822842 |
Marianna Rossetti1, Rosa Merlo2, Neda Bagheri1, Danila Moscone1, Anna Valenti2, Aakash Saha3, Pablo R Arantes3, Rudy Ippodrino4, Francesco Ricci1, Ida Treglia5, Elisabetta Delibato5, John van der Oost6, Giulia Palermo3, Giuseppe Perugino2,7, Alessandro Porchetta1.
Abstract
The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.Entities:
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Year: 2022 PMID： 35822842 PMCID： PMC9371913 DOI： 10.1093/nar/gkac578
Source DB: PubMed Journal: Nucleic Acids Res ISSN： 0305-1048 Impact factor: 19.160