In this paper, we report a new modified base, 2-N-acetyl-3-deazaguanine (a2c3G), and the synthesis of the 2‘-O-methyl-RNA incorporating a2c3G. It should be noted that a2c3G is the first example of guanine analogues that recognize cytosine more selectively than guanine, keeping the stability of the Watson−Crick base pair essentially unchanged. The higher selectivity might be attributed to the relatively weak hydrogen bonding ability of a2c3G to U in comparison to that of a2c3G−C pair as suggested by the ab initio calculations. Moreover, incorporation of a2c3G destabilized a tandem GA mismatch in a certain sequence probably because, due to the lack of the nitrogen atom, the sheared-type GA pairing was avoided. In combination with the previously reported highly selective artificial nucleobases, such as 2-thiouracil, the a2c3G must become a useful material to develop new hybridization probes or antisense molecules having higher sequence selectivity.