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

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.