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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Cleavage sequence specificity of Nsp15
John V McGuire1, Scott Horowitz1
1Department of Chemistry & Biochemistry, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, USA.
None:
Nsp15 is an EndoU nuclease that is partially responsible for SARS-CoV-2's ability to evade the immune system response. Despite its importance, the sequence specificity of Nsp15 remains difficult to fully determine. In this work, we use a systematic approach to measure Nsp15's sequence specificity by testing all 16 dinucleotides for cleavage activity. The results show a preference for uridine in the first dinucleotide position, but with varying specificity in the second position. Using Alphafold3 predictions to examine the structural basis of this specificity suggests important contacts 3' of the dinucleotide sequence as well as contacts to the dinucleotides that agree with the cleavage specificity.
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