Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by T_m measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).