1,3-Dideazaguanosine in Atomic Mutagenesis Provides Unprecedented Insight Into Hydrogen Bonding and Stacking Interactions in Folded RNA

TL;DR

Researchers studied a modified RNA base to understand how RNA structures form and function, revealing insights into hydrogen bonds and RNA stability.

Contribution

The first synthesis and application of 1,3-deazaguanosine in RNA mutagenesis to probe hydrogen bonding and stacking interactions.

Findings

c1c3G destabilizes RNA helices but integrates without disrupting neighboring base pairs.

c1c3G–C base pairing is weaker than A–U due to reduced stacking capability.

c1c3G reveals a critical double contact in the twister ribozyme's active site affecting catalytic activity.

Abstract

The central goal
of RNA atomic mutagenesis is to evaluate the presumed
contacts between individual atoms and their interaction partners with
regard to function. This is made possible, for instance, by deaza-modified
nucleobases, which are introduced site-specifically into RNA. Mostly,
nucleotides with a single nitrogen-to-carbon exchange have been used
so far while double exchanges are largely missing although such modification
patterns would be highly useful. Here, a systematic study on 1,3-deazaguanosine
(c1c3G) is reported. We present the first synthesis
of this nucleoside and an appropriately protected c1c3G phosphoramidite for RNA solid-phase synthesis. Comprehensive
experimentation on c1c3G modified RNAs, using
UV melting profile analysis together with NMR spectroscopy, shed light
on the thermodynamics and base pairing properties. We found that c1c3G destabilizes RNA double helices, but it can
integrate well therein without impairing neighboring base pairs. Our
data also show that, although two hydrogen bonds are possible in a
c1c3G – C Watson–Crick base pair
geometry, the pairing strength is significantly weaker than that of
an A–U pair. This can be explained by a loss of stacking capability
when the guanine heterocyclic core is replaced by the shape-complementary
benzimidazole analog. This observation has implications for the etiology
nucleic acids and may explain why purines have evolved as a dominating
heterocyclic component of these fundamental biomacromolecules. Furthermore,
our findings help to properly apply c1c3G in
atomic mutagenesis experiments, particularly for probing the transition
state of self-cleaving nucleolytic RNA. We demonstrate this for the
twister ribozyme by identifying a double contact of a guanine in its
active site that impacts catalytic activity by 5 orders of magnitude.

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