Abstract
A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson–Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2′-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5–4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3–4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.
Highlights
A-form RNA (A-RNA) and B-form DNA (B-DNA) double helices have several important differences (Figure 1A)
Our results indicate that the A-form geometry destabilizes purine-pyrimidine and purine-purine HG bps due to the energetic cost associated with changing the sugar-backbone conformation to accommodate the syn purine base
We predict that removal of the 2 -hydroxyl at a purine nucleotide in RNA should not result in a resurgence of stably formed HG bps on N1-methylation, as long as the local conformation remains A-form
Summary
A-form RNA (A-RNA) and B-form DNA (B-DNA) double helices have several important differences (Figure 1A). The ribose primarily adopts the C3 ́-endo conformation in ARNA This brings into proximity adjacent nucleotides shortening the double helix and moving bps away from the helical axis [6] (Figure 1A). The resulting A-RNA double helix is more rigid than its B-DNA counterpart [7,8,9] These differences between A-RNA and B-DNA have important biological implications for their recognition by proteins [10,11] and ligands [12], the templated processes of replication, transcription and translation, the consequences of ribonucleotide and deoxyribonucleotide misincorporation [13,14], and the impact of damage [15] and chemical modifications
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