N2-methylguanosine is iso-energetic with guanosine in RNA duplexes and GNRA tetraloops

Modified nucleotides are resource-intensive alternatives to the four nucleotides that constitute the bulk of natural RNAs. Yet, even in cases where modifications are highly conserved, their functions are difficult to identify. One possible function might be to modulate the stability of RNA structures. To investigate this possibility for N-2-methylguanosine (m(2)G), which is present in a wide variety of RNAs, we have determined the thermodynamic consequences of substituting m(2)G for Gi in G-C Watson-Crick pairs and G-U wobble pairs within RNA duplexes. The m2G substitution is iso-energetic with G in all cases, except for an internal m(2)G-U pair, where it has a modest (0.3 kcal/mol) stabilizing effect, We have also examined the consequences of replacing G by m(2)G, and A by N-6,N-6-dimethyladenosine (m(2)(6)A) in the helix 45 tetraloop of 16S rRNA, which would otherwise be a standard GNRA tetraloop, This loop is a conserved, hypermethylated region of the ribosome where methylation appears to modulate activity m(2)(6)A substitution destabilizes the tetraloop, presumably because it prevents the formation of the G A sheared pair it would otherwise contain, m(2)G substitution has no effect on tetraloop stability. Together, these results suggest that m(2)G is equally stable as either the s-cis or s-trans rotamer, The lack of a significant effect on secondary structural stability in these systems suggests that m(2)G is introduced into naturally occurring RNAs for reasons other than modulation of duplex stability.