An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated a
s x) and pyridin-2-one (designated as y) has been developed for specific tr
anscription. The ribonucleoside triphosphates of y and a modified y, 5-meth
ylpyridin-2-one, are selectively incorporated into RNA opposite x in the te
mplates by T7 RNA polymerase, In addition, the sequences of the DNA templat
es containing x can be confirmed by a dideoxynucleotide chain-terminator me
thod supplemented with the deoxynucleoside triphosphate of y, The bulky dim
ethylamino group of x in the templates effectively eliminates noncognate pa
iring with the natural bases. These results enable RNA biosynthesis for the
specific incorporation of unnatural nucleotides at the desired positions.