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 restriction enzyme to generate long, unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.
Highlights
Argonautes are nucleic acid-guided endonucleases generally divided into two main groups: eukaryotic argonautes and prokaryotic argonautes
Thermostable helicase TthUvrD improves Thermus thermophilus argonaute (TtAgo) activity on plasmid substrates As many UvrDs are well characterized and are functional on a variety of different substrates, including blunt double-stranded DNA (dsDNA) substrates and nicked/relaxed plasmid DNA substrates [55], we investigated the effects of UvrD-like helicases on TtAgo activity
We observed that the addition of thermostable TthUvrD to the reaction enhanced the ability of TtAgo to cut a plasmid substrate in 4 hr using two guides targeting complementary strands to generate a double-strand break (Fig 1)
Summary
Argonautes are nucleic acid-guided endonucleases generally divided into two main groups: eukaryotic argonautes (eAgos) and prokaryotic argonautes (pAgos). The eAgos are well known for their role in RNA interference (RNAi) pathways where they are a principle component of the RNA-induced silencing complex (RISC). Within the RISC, eAgos are involved in binding small RNA guides which are used as a directive template for various mechanisms of downstream post-transcriptional regulation and translational disruption of mRNA, depending on the level of sequence complementarity [1,2,3]. While the scope of eAgo activity with relation to RNAi comprises a broad, well-established field of study, pAgos, which are putatively involved in cellular defense against foreign and mobile genetic elements [3,4,5,6], exhibit a much broader range of enzymatic activity in relation to substrate and guide preferences.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
