L. Ortolevadonnelly et al., IDENTIFYING RNA MINOR-GROOVE TERTIARY CONTACTS BY NUCLEOTIDE ANALOG INTERFERENCE MAPPING WITH N-2-METHYLGUANOSINE, Biochemistry (Easton), 37(37), 1998, pp. 12933-12942
Nucleotide analogue interference mapping (NAIM) is a general biochemic
al method that rapidly identifies the chemical groups important for RN
A function. In principle, NAIM can be extended to any nucleotide that
can be incorporated into an in vitro transcript by an RNA polymerase.
Here we report the synthesis of 5'-O-(1-thio)-N-2-methylguanosine trip
hosphate (m(2)G alpha S) and its incorporation into two reverse splici
ng forms of the Tetrahymena group I intron using a mutant form of T7 R
NA polymerase. This analogue replaces one proton of the N2 exocyclic a
mine with a methyl group, but is as stable as guanosine (G) for second
ary structure formation. We have identified three sites of m(2)G alpha
S interference within the Tetrahymena intron: G22, G212, and G303. Al
l three of these guanosine residues are known to utilize their exocycl
ic amino groups to participate in tertiary hydrogen bonds within the r
ibozyme structure. Unlike the interference pattern with the phosphorot
hioate of inosine (I alpha S, an analogue that deletes the N2 amine of
G), m(2)G alpha S substitution did not cause interference at position
s attributable to secondary structural stability effects. Given that t
he RNA minor groove is likely to be widely used for helix packing, m(2
)G alpha S provides an especially valuable reagent to identify RNA min
or groove tertiary contacts in less well-characterized RNAs.