2′/3′ Regioselectivity of Enzyme‐Free Copying of RNA Detected by NMR

Abstract

The RNA‐templated extension of oligoribonucleotides by nucleotides produces either a 3′,5′ or a 2′,5′‐phosphodiester. Nature controls the regioselectivity during RNA chain growth with polymerases, but enzyme‐free versions of genetic copying have modest specificity. Thus far, enzymatic degradation of products, combined with chromatography or electrophoresis, has been the preferred mode of detecting 2′,5′‐diesters produced in enzyme‐free reactions. This approach hinges on the substrate specificity of nucleases, and is not suitable for in situ monitoring. Here we report how 1 H NMR spectroscopy can be used to detect the extension of self‐templating RNA hairpins and that this reveals the regioisomeric nature of the newly formed phosphodiesters. We studied several modes of activating nucleotides, including imidazolides, a pyridinium phosphate, an active ester, and in situ activation with carbodiimide and organocatalyst. Conversion into the desired extension product ranged from 20 to 90 %, depending on the leaving group. Integration of the resonances of H1′ protons of riboses and H5 protons of pyrimidines gave regioselectivities ranging from 40:60 to 85:15 (3′,5′ to 2′,5′ diester), but no simple correlation between 3′,5′ selectivity and yield. Our results show how monitoring with a high‐resolution technique sheds a new light on a process that may have played an important role during the emergence of life.

RNA auto-polymerisation from 2’,3’-cyclic nucleotides at air-water interfaces

“…The linkage type in polymerisation was also confirmed by 31 P NMR (Figure 1d) and the peaks were assigned based on the literature values. 24,25 Copolymerisation of cNMP We tested the polymerisation tendencies of cAMP, cUMP and cCMP under the same heat-drying conditions and found that these monomers did not polymerise to the same lengths and concentrations as cGMP. Figure 2a shows the polymerisation trend decreases in the order cGMP> cUMP> cAMP > cCMP.…”

Carbodiimide‐Driven Dimerization and Self‐Assembly of Artificial, Ribose‐Based Amphiphiles

“…in a 0.5 m m 3‐(N‐morpholino)propanesulfonic acid (MOPS) buffer, containing 1‐ethylimidazole (0.15 m m ) and MgCl 2 (0.08 m m ) (pH 7.5). These reaction conditions were derived from Richert and co‐workers and are referred to as “condensation buffer.” [46] As shown in Figure 3B, the 31 P NMR signal (∼3.8 ppm) of X 15 MP gradually decreased and vanished after 3 h. Meanwhile, a new signal around ‐11.0 ppm appeared and accumulated, indicating the formation of pyrophosphate X 15 ppX 15 , based on the characteristic chemical shift [47,67] . While non‐substituted adenosine monophosphates also form some phosphodiesters, [47,67] we did not observe this for X 15 MP .…”

RNA polymerisation without catalyst from 2’,3’-cyclic nucleotides by drying at air-water interfaces

“…The linkage type in the polymerisation was also confirmed by 31 P NMR (Figure 1d) and the peaks were assigned based on the literature values. [32,33] Figure 1a and 1b compare the effect of pH on the lengths and concentrations of the n-mers formed by drying with K + (Cl -) and imidazole respectively. We determined the optimal reaction temperature to be 40 °C (S5, S8b).…”