Non-enzymatic primer extension with strand displacement

Abstract

Non-enzymatic RNA self-replication is integral to the emergence of the ‘RNA World’. Despite considerable progress in non-enzymatic template copying, demonstrating a full replication cycle remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible approach to strand displacement synthesis in which short ‘invader’ oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic primer extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued primer extension with strand displacement by employing activated 3′-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been facilitated by short RNA oligonucleotides.

Deep sequencing of non-enzymatic RNA primer extension

“…As we begin to probe higher-order primer extension experiments, the set of reactants will only multiply. Experiments with all four nucleotides are rare, partly because the reaction is inefficient and partly because the results are challenging to interpret without sequencing data ( 56 , 57 ). NERPE-Seq may help us address the relationship between inefficient primer extension with all four bases and the role of the bridged intermediate.…”

The virtual circular genome model for primordial RNA replication

“…Such events would be fatal if genome replication required continuous end-to-end copying of a template strand. In contrast, in the virtual circular genome model, such events would simply generate a fraction of oligonucleotides that are no longer growing, but which can still play useful roles as templates, invaders for strand displacement synthesis (Zhou et al 2019), and downstream helpers for primer extension (Prywes et al 2016).…”

“…In contrast, the upstream fragment will initially terminate in a 2′-3′ cyclic phosphate, which cannot be extended by either primer extension or ligation reactions. However such a fragment could act as a template if long enough, or as an invader strand in RNA catalyzed strand displacement synthesis (Zhou et al 2019). Subsequent hydrolysis of the cyclic phosphate will generate either a 2′ or a 3′-phosphate.…”

Early adolescent adversity alters periaqueductal gray/dorsal raphe threat responding in adult female rats

“…Previous work from our laboratory recorded single-unit activity in the ventrolateral periaqueductal gray (vlPAG) while rats discriminated cues predicting unique foot shock probabilities: danger (p = 1.00), uncertainty (p = 0.375), and safety (p = 0.00). Activity patterns of cue-excited vlPAG neurons 20 – 23 reflected the foot shock probability associated with each cue (threat probability), rather than fear output 24 . Activity patterns of cue-inhibited neurons showed signaling of fear output and threat probability 25 .…”

“…Activity patterns of cue-excited vlPAG neurons 20 – 23 reflected the foot shock probability associated with each cue (threat probability), rather than fear output 24 . Activity patterns of cue-inhibited neurons showed signaling of fear output and threat probability 25 . The vlPAG may then serve a more integrative role in fear 26 – 28 , marking disruptions in periaqueductal threat function as a candidate for adversity-induced latent vulnerability.…”