Ligation of RNA Oligomers by the Schistosoma mansoni Hammerhead Ribozyme in Frozen Solution

Abstract

The interstitial liquid phase within frozen aqueous solutions is an environment that minimizes RNA degradation and facilitates reactions that may have relevance to the RNA World hypothesis. Previous work has shown that frozen solutions support condensation of activated nucleotides into RNA oligomers, RNA ligation by the hairpin ribozyme, and RNA synthesis by a RNA polymerase ribozyme. In the current study, we examined the activity of a hammerhead ribozyme (HHR) in frozen solution. The Schistosoma mansoni hammerhead ribozyme, which predominantly cleaves RNA, can ligate its cleaved products (P1 and P2) with yields up to ~23 % in single turnover experiments at 25 °C in the presence of Mg(2+). Our studies show that this HHR ligates RNA oligomers in frozen solution in the absence of divalent cations. Citrate and other anions that exhibit strong ion-water affinity enhanced ligation. Yields up to 43 % were observed in one freeze-thaw cycle and a maximum of 60 % was obtained after several freeze-thaw cycles using wild-type P1 and P2. Truncated and mutated P1 substrates were ligated to P2 with yields of 14-24 % in one freeze-thaw cycle. A pool of P2 substrates with mixtures of all four bases at five positions were ligated with P1 in frozen solution. High-throughput sequencing indicated that 70 of the 1024 possible P2 sequences were represented in ligated products at 1000 or more read counts per million reads. The results indicate that the HHR can ligate a range of short RNA oligomers into an ensemble of diverse sequences in ice.

Nucleic Acid Catalysis under Potential Prebiotic Conditions

“…Freezing favors ligation in reversible transesterification reactions, even from highly fragmented ribozymes . Freezing can enable highly thermodynamically disfavored reactions, such as ligation of monomeric 2′, 3′‐cyclic nucleoside monophosphates to a free 5′ end of RNA .…”

“…Both HP and HH ribozyme ligation yields strongly benefit from repeated freeze‐thaw (FT) cycling . This effect can even be used to enable the in trans assembly of long structured RNAs, such as the ≈200 nt RNA polymerase ribozymes, from fragments between 20–30 nt .…”

“…[192] Freezing favors ligation in reversible transesterification reactions, even from highly fragmented ribozymes. [175,193,194] Freezing can enableh ighly thermodynamically disfavored reactions, such as ligationo fm onomeric 2',3 '-cyclicn ucleoside monophosphates to af ree 5' end of RNA. [195] While this reversal of exonucleolytic cleavage has an equilibrium constanto f % 2.2 m À1 under aqueous conditions (at 0 8C [196] ), it can be decreased % 20-foldb yf reezing to À9 8Ci nt he presence of 25 mm NaCl and 10 mm MgCl 2 ,e nablingq uantitative non-canonical 3'-5' nucleotidyltransferofR NA.…”

“…Both HP and HH ribozyme ligationy ields strongly benefit from repeated freeze-thaw (FT) cycling. [175,193,194] This effect can even be used to enablet he in trans assembly of long structured RNAs, such as the % 200 nt RNA polymerase ribozymes, from fragments between 20-30 nt. [193] The beneficial effects of FT cycles are likely the result of reducing the propensity of small ribozymes to form inactive or poorly active ribozymesubstrate complexes that attenuate bulk catalysis.…”

Taming Prebiotic Chemistry: The Role of Heterogeneous and Interfacial Catalysis in the Emergence of a Prebiotic Catalytic/Information Polymer System

“…The eutectic phase in water/ice has also been shown to promote additional processes that are relevant to the RNA world hypothesis, such as the catalytic activity of ribozymes among other ligases [100,101] and polymerases [74,102], or even permits their self-assembly [103]. …”