2′-O-Methyl-RNAs incorporating 3-deazaguanine (c3G) were synthesized by use of N,N-diphenylcarbamoyl and N,N-dimethylaminomethylene as its base protecting groups to suppress sheared-type 5′-GA-3′/5′-GA-3′ tandem mismatched base pairing which requires the N3 atom. These modified RNAs hybridized more weakly with the complementary and single mismatch-containing RNAs than the unmodified RNAs. The Tm experiments were performed to clarify the effects of replacement of the fifth G with c3G on stabilization of 2′-O-methyl-(5′-CGGCGAGGAG-3′)/5′-CUCCGAGCCG-3′ and 2′-O-methyl-(5′-CGGGGACGAG-3′)/5′-CUCGGACCCG-3′duplexes, which form sheared-type and face-to-face type 5′-GA-3′/5′-GA-3′ tandem mismatched base pairs, respectively. Consequently, this replacement led to more pronounced destabilization of the former duplex that needs the N3 atom for the sheared-type base pair than the latter that does not need it for the face-to-face type base pair. A similar tendency was observed for 2′-O-methyl-RNA/DNA duplexes. These results suggest that the N3 atom of G plays an important role in stabilization of the canonical G/C base pair as well as the base discrimination and its loss suppressed formation of the undesired sheared-type mismatched base pair. Computational studies based on ab initio calculations suggest that the weaker hydrogen bonding ability and larger dipole moment of c3G can be the origin of the lower Tm.