Single-stranded binding proteins and helicase enhance the activity of prokaryotic argonautes in vitro

PloS One
Eric A HuntNathan A Tanner

Abstract

Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides, some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however, they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome, specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors, including thermostable single-stranded DNA binding proteins and UvrD-like helicase, in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates, including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restrict...Continue Reading

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Citations

Dec 6, 2018·Nature Communications·Lidiya LisitskayaAndrey Kulbachinskiy
May 18, 2021·Frontiers in Molecular Biosciences·Eric A HuntNathan A Tanner
Sep 4, 2021·Nucleic Acids Research·Kok Zhi LeeKevin V Solomon

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Methods Mentioned

BETA
electrophoresis
PCR

Software Mentioned

eAgos
pAgos
Excel
PeakScanner
pAgo
GraphPad Prism

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