The role of sugar-backbone heterogeneity and chimeras in the simultaneous emergence of RNA and DNA

Abstract

Hypotheses of the origins of RNA and DNA are generally centred on the prebiotic synthesis of a pristine system (pre-RNA or RNA), which gives rise to its descendent. However, a lack of specificity in the synthesis of genetic polymers would probably result in chimeric sequences; the roles and fate of such sequences are unknown. Here, we show that chimeras, exemplified by mixed threose nucleic acid (TNA)-RNA and RNA-DNA oligonucleotides, preferentially bind to, and act as templates for, homogeneous TNA, RNA and DNA ligands. The chimeric templates can act as a catalyst that mediates the ligation of oligomers to give homogeneous backbone sequences, and the regeneration of the chimeric templates potentiates a scenario for a possible cross-catalytic cycle with amplification. This process provides a proof-of-principle demonstration of a heterogeneity-to-homogeneity scenario and also gives credence to the idea that DNA could appear concurrently with RNA, instead of being its later descendent.

Figure 1.. The prebiotic clutter generated heterogeneity-to-homogeneity scenario versus the biology inspired paradigm of replacing one homogeneous genetic system with its homogeneous genetic successor.

(a) Constitutional formula representation of the three oligonucleotide building blocks investigated in this study. (b) Three possible scenarios for the emergence of RNA and DNA from prebiotic chemistry. Middle: the classical RNA world concept where the formation of a pristine and homogeneous RNA (or pre-RNA) leads to its homogeneous-backbone successor DNA (or RNA). Top: a heterogeneous mixture of TNA (pre-RNA) and RNA forming chimeric TRNA sequences that transition to homogenous RNA, which then gives rise to DNA. Bottom: a heterogeneous RNA-DNA mixture progressing/co-evolving via chimeric RDNA sequences directly to homogeneous RNA and DNA simultaneously.

Figure 2.. The preferential association with, and ligation of homogeneous ligands by, chimeric TRNA template over chimeric ligands.

(a) Thermal stability of TRNA chimeric duplexes in 1 M NaCl, 10 mM Na₂HPO₄, 100 μM EDTA, pH 7.2; a = [5μM], b = [2μM] duplex concentration; c = Entry 7, no clear sigmoidal transition in UV-thermal melt was observed. (b) Comparison of the rate of ligation reaction at 4°C of homogeneous-RNA (R_L1+R_L2) and heterogeneous TNA-RNA ligands (C_L1+C_L2) on heterogeneous TNA-RNA template, C_T1. (c) EDC-mediated ligation reaction at 4 °C of mixture of homogeneous-RNA (R_L1, R_L2) and chimeric TNA-RNA ligands (C_L1, C_L2) using TRNA chimeric sequence (C_T1) as template. (d) comparison of the amounts of products R_P1 and C_P1 produced in the reaction mixture in 2c; see supplementary Fig. 24, for conditions. A, T = RNA; T = DNA; a, t = TNA. Line in graph (2b) is drawn as guide to indicate the trend and is not a mathematical curve fitting. % yields are calculated with respect to the template C_T1. Experiments were run in triplicate and the error range is less than ± 5%; error bars represent standard deviation.

Figure 3.. Chimeric RDNA templates preferentially associate and ligate homogeneous RNA and DNA ligands over chimeric ligands.

(a) The list of homogeneous and chimeric sequences used in this study. (b) Comparison of ligation efficiency by hexadecameric (AU)-RDNA template C_T2, with RNA (R_L3, R_L4), DNA (D_L1, D_L2) and chimeric RDNA (C_L3, C_L4) ligands, showing the consistent preferential formation of homogeneous ligation products, R_P2 and D_P1 over chimeric ligation products C_P2. (c and d) Comparison of ligation efficiency by octameric (A, U/T, G, C)-RDNA template C_T4, with RNA (R_L7, R_L8), and chimeric RDNA (C_L5, C_L6) ligands, showing the influence of temperature on the preferential formation of homogeneous ligation products, R_P4 over chimeric ligation products C_P3. See supplementary figs. 50–52 for EDC-ligation reaction conditions. A, U, G, C = RNA; A, T, G, C = DNA. Lines in graph (3a) are drawn as guide indicating the trend and are not mathematical curve fittings. % yields were calculated with respect to the template C_T2 or R_T2 or C_T4 respectively. Experiments were run in triplicate and the error range is less than ± 5%; error bars represent standard deviation.

Figure 4.. The beneficial role for chimeric RDNA template in overcoming the template-product inhibition based on thermodynamic stability of duplexes.

(a) The expected difference between chimeric RDNA-RNA duplex and the homogeneous RNA-RNA duplex in being able to overcome the template-product inhibition. (b) Schematic representation of the proposal that hexadecameric (AU)-RDNA template C_T2 with RNA ligands R_L3+R_L4 produces R_P2, which in the presence of R_L5, R_L6 is expected to lead to R_P3, based on the greater thermodynamic stability of the R_P2:R_P3 duplex over the R_P2:C_T2 duplex, and release the C_T2 for another round of ligation reaction. (c) Time course of the EDC-mediated-ligation experiments documenting the effect of change in ratio of ligands, and the sequential-addition of ligands R_L5+R_L6 (0 h) followed by R_L5+R_L6 (at 20 h) versus all-in-one-pot reaction on the production of R_P2 and R_P3. (d) Comparison of the amount of R_P3 formed by the homogeneous RNA template R_T2 versus chimeric RDNA template C_T2 (at 48 h) demonstrating the higher efficiency of C_T2 in mediating the formation of R_P3 by overcoming the template-product inhibition. See supplementary figs. 63–78 for EDC-ligation conditions. A, U = RNA; A, T = DNA. Lines in graph (4c) are drawn as guide indicating the trend and are not mathematical curve fittings. % yields were calculated with respect to the template C_T2 or R_T2 respectively. Experiments were run in triplicate and the error range is less than ± 5%; error bars represent standard deviation.

Figure 5.. Comparison of the efficiency between chimeric RDNA (C_T2) and RNA (R_T2) templates in producing the final ligation product R_P3 under step-wise dilution conditions, demonstrating the superior ability of C_T2 to act as a template for ligation with turn over.

(a) Production of the ligation products R_P2 and R_P3 in the stepwise-dilution (in 24 h intervals) experiment with templates C_T2 and R_T2, over a period of 96 h, containing all four ligands R_L3, R_L4, R_L5 and R_L6; the drops at 24, 48, and 72 h indicate the dilution step. (b) Time course contrast between the templates C_T2 and R_T2 for the production of the first ligation product R_P2 formed from R_L3 and R_L4. (c) Comparison of the efficiency of production of the second ligation product R_P3 (from R_L5 and R_L6) between the templates C_T2 and R_T2. (d) Chromatogram traces at 96 h after three stepwise-dilution juxtaposing the three parallel experiments in the presence of C_T2 (top trace), R_T2 (middle trace) and containing no template (bottom trace). See supplementary figs. 91–93 for EDC-ligation conditions (at 4 °C). For C_T2, R_T2, R_P2, R_P3, R_L3, R_L4, R_L5 and R_L6 see Fig. 4a. Lines in graphs (5a-c) are drawn as guide indicating the trend and are not mathematical curve fittings. % yields were calculated with respect to the template C_T2. Experiments were run in triplicate and the error range is less than ± 5%; error bars represent standard deviation.

Figure 6.. Experiment for testing the possibility of cross-catalytic amplification in oligonucleotide replication via regeneration of the chimeric RDNA (C_T2) template.

(a) The sequences of oligonucleotides used in this investigation; C_T2^NH is the same as chimeric template C_T2 but with a single phosphoramidate (NP) link at the ligation junction. (b) Schematic representation of the hypothesis that the presence of chimeric ligands C_L7 and C_L8 (complementary to R_P2) could induce the regeneration of the chimeric template C_T2^NH leading to further production of R_P2. The concomitant release of C_T2 also creates the potential for another round of ligation reaction. (c) Comparison of the amount of R_P2 produced from the combination of C_T2+R_L3+R_L4 (1:5:5) versus the combination of C_T2+R_L3+R_L4+C_L7+C_L8 (1:5:5:2:2), demonstrating the regeneration of chimeric template C_T2^NH along with higher and increasing production of R_P2 in the latter combination. See supplementary Fig. 94 for experimental conditions. % yields were calculated with respect to the template C_T2. Experiments were run in duplicate and the error range is less than ± 5%; error bars represent standard deviation.