Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8‐oxo‐7,8‐dihydropurine, and 8‐bromopurine analogues within A‐form duplexes of RNA

Abstract

Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8-oxo-7,8-dihydropurine (common products of oxidation at the C8-position of purines), and 8-bromopurine (as probes to explore conformational changes), derivatives, namely 8-oxoG, 8-oxoI, 8-oxoA, 8-BrG, and 8-BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid-phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (T m) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H-bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti-I:syn-A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8-oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble-pair is only stabilized by G; and (d) 8-oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.

The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis

“…We have shown that the 8-oxo-purines (8-oxo-adenosine, 8-oxo-guanosine, and 8-oxo-inosine) all perform poorly as substrates for nonenzymatic RNA copying chemistry, regardless of whether they are present as activated mononucleotides, in the template, or at the end of a primer . Activated 8-oxo-purine monomers are incorporated very poorly in primer extension reactions, at rates comparable to those observed with mismatched canonical nucleotides, consistent with the strongly destabilizing effects of these modified nucleotides on duplex thermodynamic stability. − …”

Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1

“…The former two are of interest given that 1) alkylative damage was recently explored alongside oxidative stress and m 1 G was one of the studied species; ( Yan et al, 2019 ) 2) both m 1 G and m 6,6 A are naturally occurring modifications with essential biological functions, particularly in the case of the former; ( McCown et al, 2020 ) and 3) the H-bonding pattern of these purine derivatives can serve to probe the hypothesis that, changes in H-bonding may play a role in the enzymatic stalling. Furthermore, 8-BrG is a chemically modified nucleoside that can be used to potentially provide information about conformational changes ( anti-syn ) around the glycosidic bond, ( Skinner et al, 2020 ) given that substitution at the C8-position of purine rings has been shown to favor the equilibrium towards the syn-isomer. Thus, we proceeded to incorporate each of these chemical probes/modifications into the same sequence context and obtained ONs 6–8 , which were converted to radiolabeled-phosphorylated ONs 6 ″– 8 ″, and treated with Xrn-1 as discussed previously ( Figure 2B ).…”

Conformational preferences of inosine and its methyl derivatives: Comparison of the AMBER derived force field parameters and reparameterization of the glycosidic torsion parameters

“…An NMR study reports a large predominance of SYN population and preference for the C2′-endo (SOUTH) sugar pucker conformation of inosine in solution [48]. Several theoretical and experimental studies have been conducted to investigate the structural and thermodynamic consequences due to the presence of inosine in RNA duplexes [6, [49][50][51][52][53]. Notably, the nearest neighbor thermodynamic parameters for I•U and I•C base pairs have been developed by Znosko and colleagues [50,51].…”