Learn about prokaryotic argonautes, which are nucleic acid-guided endonucleases that can be used as programmable nucleases. TtAgo is the first commercially available prokaryotic argonaute.
Prokaryotic argonautes are nucleic acid-guided endonucleases involved in prokaryotic cellular defense against foreign genetic elements. When provided with synthetic nucleic acid guides, prokaryotic argonautes can be used as programmable nucleases. Argonautes from different prokaryotic species may utilize either DNA or RNA guides to target DNA or RNA substrates, leading to many potential combinations of guide and substrate preferences. Prokaryotic argonaute proteins are typically comprised of four domains: the MID domain, the PAZ domain, the PIWI domain, and the N domain. The MID domain of prokaryotic argonaute binds a short, single-stranded oligonucleotide guide, which is typically 16 to 18 nucleotides in length. The guide is often 5-prime-phosphorylated, though some argonautes can utilize guides with other 5-prime chemical modifications. The PAZ domain is responsible for holding the 3-prime end of the guide while the argonaute searches for a matching sequence. The prokaryotic argonaute/guide complex searches for a complementary sequence on the target strand. When a match is found, the 3-prime end is released to allow for complete base pairing between the guide and substrate. This activates prokaryotic argonaute endonuclease activity in the PIWI domain, which contains a metal-dependent RNase H-like active site. A break is created in the phosphodiester backbone of the complementary substrate nucleic acid. In the case of double-stranded substrates, a break is only created in the strand which is complementary to the guide nucleic acid. The N domain is then thought to act as a “wedge” that helps release the target DNA. Thermus thermophilus Argonaute, also known as TtAgo, is the first commercially-available prokaryotic argonaute from NEB. TtAgo is a thermostable prokaryotic argonaute which utilizes short, 5-prime-phosphorylated single-stranded DNA guides to target and cut DNA substrates at temperatures between 65 and 85 degrees celsius. For more information on TtAgo, please visit neb.com/M0665.