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

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 utiliz...

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Bibliographic Details
Published in:PloS one 2018-08, Vol.13 (8), p.e0203073-e0203073
Main Authors: Hunt, Eric A, Evans, Jr, Thomas C, Tanner, Nathan A
Format: Article
Language:English
Subjects:
Argonaute Proteins - metabolism
Bacterial Proteins - metabolism
Binding
Biology and Life Sciences
Cellular apoptosis susceptibility protein
Complications
CRISPR
Deoxyribonucleic acid
DNA
DNA Breaks, Double-Stranded
DNA helicase
DNA Helicases - metabolism
DNA, Single-Stranded - metabolism
DNA-Binding Proteins - metabolism
Endonuclease
Enzymatic activity
Enzyme Stability
Enzymes
Escherichia coli - genetics
Eukaryotes
Gene editing
Genetic modification
Genome editing
Genomes
Homology
Nanoarchaeota
Palate
Physical Sciences
Preferences
Prokaryotes
Protein binding
Proteins
RNA polymerase
Single-stranded DNA
Substrates
Temperature
Thermus thermophilus - enzymology
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Summary: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 restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0203073