Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying - PubMed (original) (raw)

Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying

Stephanie J Zhang et al. Proc Natl Acad Sci U S A. 2022.

Abstract

Nonenzymatic template-directed RNA copying using chemically activated nucleotides is thought to have played a key role in the emergence of genetic information on the early Earth. A longstanding question concerns the number and nature of different environments that might have been necessary to enable all of the steps from nucleotide synthesis to RNA copying. Here we explore three sequential steps from this overall pathway: nucleotide activation, synthesis of imidazolium-bridged dinucleotides, and template-directed RNA copying. We find that all three steps can take place in one reaction mixture undergoing multiple freeze-thaw cycles. Recent experiments have demonstrated a potentially prebiotic methyl isocyanide-based nucleotide activation chemistry. However, the original version of this approach is incompatible with nonenzymatic RNA copying because the high required concentration of the imidazole activating group prevents the accumulation of the essential imidazolium-bridged dinucleotide. Here we report that ice eutectic phase conditions facilitate not only the methyl isocyanide-based activation of ribonucleotide 5′-monophosphates with stoichiometric 2-aminoimidazole, but also the subsequent conversion of these activated mononucleotides into imidazolium-bridged dinucleotides. Furthermore, this one-pot approach is compatible with template-directed RNA copying in the same reaction mixture. Our results suggest that the simple and common environmental fluctuation of freeze-thaw cycles could have played an important role in prebiotic nucleotide activation and nonenzymatic RNA copying.

Keywords: RNA copying; RNA world hypothesis; chemical nucleotide activation; origin of life; prebiotic chemistry.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Activation in the ice eutectic phase enhances the yield of activated nucleotides (*pN) and promotes formation of bridged dinucleotides (Np*pN). (A) Scheme showing role of ice eutectic concentration. Left: At room temperature, the excess 2AI needed to drive nucleotide activation inhibits bridged dinucleotide accumulation. Right: activation in ice eutectic phase requires only equimolar 2AI and ribonucleotides, allowing bridged dinucleotide accumulation and template copying. (B) Solutes are concentrated in the ice eutectic phase. (C) Nucleotide activation in the ice eutectic phase (left) and at room temperature (Right) with one round of MeNC addition and equimolar initial nucleotides and 2AI (

SI Appendix, Fig. S3_A_

). (D) Cycles of MeNC addition and eutectic freezing drive continued nucleotide activation. Curvy arrows indicate addition of MeNC after each thawing of the reaction mixture. Note that the reaction mixture is frozen as eutectic ice for the entire reaction course except during the brief thawing steps (∼30 min at room temperature for the mixture to thaw completely) (

SI Appendix, Fig. S3_B_

). (E) Activation is inefficient at room temperature in the absence of ice eutectic freezing. Straight arrows indicate the addition of MeNC at room temperature (

SI Appendix, Fig. S3_C_

Fig. 2.

The frequency of MeNC delivery at long time intervals does not affect the overall yield of activated products. The yields of nucleotide activation were measured after each of four freeze-thaw cycles with periodic addition of MeNC. (A) Yields of activated species are low with time intervals on the order of hours in between MeNC additions. (B_–_D) However, lengthening of the time interval beyond 24 h does not significantly affect the final yield (

SI Appendix, Fig. S5

). Reaction conditions: 10 mM pA, 10 mM 2AI, 100 mM 2MBA, 50 mM MeNC, 30 mM MgCl2, 50 mM Na+-Hepes pH 8, and subsequent periodic addition of MeNC in three aliquots of 50 mM. After the last addition of MeNC, the solution was left under eutectic ice phase conditions for the indicated time, then the solution was thawed for room temperature NMR measurement.

Fig. 3.

Efficient eutectic ice phase activation of all four canonical ribonucleotides. (A) Each of the four canonical ribonucleotides can be activated using the same approach as optimized for pA (Fig. 1_D_). However, pG exhibits lower activation compared to the other three. (B) All four nucleotides can be activated efficiently when incubated simultaneously. Indicated concentrations are sum totals across all the nucleotides. (C) GMP activation yields decrease as GMP concentration is increased, suggesting that the low activation of GMP is partly due to its low solubility in aqueous phase (compare with A). Total [pN] = 10 mM, 10 mM 2AI, 100 mM 2MBA, 50 mM MeNC, 30 mM MgCl2, 50 mM Na+-Hepes pH 8, and periodic addition of MeNC in three aliquots of 50 mM each (

SI Appendix, Fig. S6

Fig. 4.

Ice eutectic phase activation enables a complete pathway from unactivated mononucleotides through activation to primer extension. (A) Schematic of primer-template complex. (B) Time course of primer extension using pure activated mononucleotides (5 mM *pG and 5 mM *pC, room temperature). (C) Time course of primer extension reactions using pNs (n = C and G) generated with MeNC activation chemistry under ice eutectic phase. (D) Primer-template was added to already-activated mononucleotides generated by MeNC-based nucleotide activation under ice eutectic phase conditions, yielding extended primer (as in C). (E) Primer-template duplex was directly treated with pNs (n = C and G) and MeNC activation chemistry under ice eutectic phase. The products of the reaction were then incubated for an additional 24 h at room temperature (F) without and (G) with bridge-forming activation. Each set of three lanes in (D_–_G) represents independent experimental replicates under the indicated condition. (H) Quantification of primer extension products. Positions of primer and +1 to +3-nucleotide extension products are indicated. Gel image of replicates for (B, C) is included in

SI Appendix, Fig. S7

Fig. 5.

Ice eutectic phase activation enables efficient primer extension with all four nucleotides. (A) Schematic of primer-template complex. (B) Time course of primer extension using pure activated mononucleotides and trinucleotides (5 mM each *pN and 0.5 mM each *pN(pN)2, room temperature). (C) Primer-template duplex was directly treated with pNs (n = A, U, C, and G) and (pN)3 and isocyanide activation chemistry under ice eutectic phase. The products of the reaction were then incubated for 24 h at room temperature, and finally treated with additional two rounds of bridge-forming activation for another 48 h (see details in Materials and Methods). (D and E) Quantification of primer extension products under conditions (B and C).

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Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying - PubMed (original) (raw)