Abstract
Prokaryotic Argonautes (pAgo) are an increasingly well-studied class of guided endonucleases, and the underlying mechanisms by which pAgo generate nucleic acid guides in vivo remains an important topic of investigation. Recent insights into these mechanisms for the Argonaute protein from Thermus thermophilus has drawn attention to global sequence and structural feature preferences involved in oligonucleotide guide selection. In this work, we approach the study of guide sequence preferences in T. thermophilus Argonaute from a functional perspective. Screening a library of 1,968 guides against randomized single- and double-stranded DNA substrates, endonuclease activity associated with each guide was quantified using high-throughput capillary electrophoresis, and localized sequence preferences were identified which can be used to improve guide design for molecular applications. The most notable preferences include: a strong cleavage enhancement from a first position dT independent of target sequence; a significant decrease in activity with dA at position 12; and an impact of GC dinucleotides at positions 10 and 11. While this method has been useful in characterizing unique preferences of T. thermophilus Argonaute and criteria for creating efficient guides, it could be expanded further to rapidly characterize more recent mesophilic variants reported in the literature and drive their utility toward molecular tools in biology and genome editing applications.
Highlights
Prokaryotic Argonautes are programmable endonucleases involved in cellular defense against foreign genetic elements (Olovnikov et al, 2013; Swarts et al, 2014b, 2015; Koonin, 2017; Kuzmenko et al, 2020)
Reactions were carried out in 96-well PCR plate format where one individual guide was mixed with Thermus thermophilus pAgo (TtAgo) and the corresponding single- or double-stranded DNA substrate, and specific cleavage activity was measured by Capillary electrophoresis (CE) analysis of the resulting fluorescently-labeled product fragment size (Greenough et al, 2016)
The most prominent guide sequence motif affecting overall guided-endonuclease activity of TtAgo was the identity of the nucleotide at g1
Summary
Prokaryotic Argonautes (pAgo) are programmable endonucleases involved in cellular defense against foreign genetic elements (Olovnikov et al, 2013; Swarts et al, 2014b, 2015; Koonin, 2017; Kuzmenko et al, 2020). While no specific sequence motifs such as the PAM sequence requirements of Cas proteins have been identified for pAgo, variability among targetable sequences has been observed (Swarts et al, 2014a; Enghiad and Zhao, 2017) Much of this variation has been attributed to global sequence attributes such as GC content or secondary structure (Swarts et al, 2017), but the bulk of knowledge concerning guide characteristics has been limited to sequencing pools of guides which are co-purified with pAgo (Olovnikov et al, 2013; Swarts et al, 2014a; Hegge et al, 2019; Kuzmenko et al, 2020). Some studies have analyzed a small number of guide sequences in vitro to determine base content and positional effects, with evidence for TtAgo having a preference of a 5 dC for guides created in vivo (Swarts et al, 2014a, 2017), but the scale is still much smaller than the sequencing of co-purified, associated guides
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