Structural Superimposition

Abstact

Synthetic RNAs bearing deazapurine nucleobases are powerful probes for dissecting RNA-catalyzed reactions by atomic mutagenesis. Here we systematically characterize RNA containing 1-deazainosine (c(1)I) and compare it with inosine (I). We first report the synthesis of a suitably protected c(1)I phosphoramidite and its incorporation into RNA by solid-phase synthesis. We then provide a comprehensive thermodynamic analysis of base-pair stability from UV-melting experiments, showing that c(1)I-C pairs are less stable than the corresponding I-C pairs. Although a two-hydrogen-bond Hoogsteen interaction between c(1)I and protonated C is conceivable, NMR spectroscopy indicates that c(1)I-C predominantly adopts a Watson-Crick-like geometry with a single hydrogen bond. These pairs are accommodated within RNA duplexes without disrupting neighboring base pairing. We also use c(1)I to probe poly(I:C) motifs that mimic viral double-stranded RNA, assessing how strand length governs duplex versus hairpin formation. Finally, atomic mutagenesis of the twister ribozyme with c(1)I supports the hypothesis that an active-site guanine participates directly in phosphodiester-bond cleavage. Together, these results clarify how deazapurines modulate nucleic-acid properties and provide guidance for their use in atomic mutagenesis to interrogate RNA catalysis.