Seio Kohji, Sasami Takeshi, Tawarada Ryuya, Sekine Mitsuo
Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Japan.
Nucleic Acids Res. 2006;34(16):4324-34. doi: 10.1093/nar/gkl088. Epub 2006 Aug 26.
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 T(m) experiments were performed to clarify the effects of replacement of the fifth G with c(3)G 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 T(m).
通过使用N，N - 二苯基甲酰基和N，N - 二甲基氨基亚甲基作为碱基保护基团，合成了掺入3 - 脱氮鸟嘌呤（c3G）的2'-O - 甲基RNA，以抑制需要N3原子的剪切型5'-GA-3'/5'-GA-3'串联错配碱基配对。与未修饰的RNA相比，这些修饰的RNA与互补的和含有单个错配的RNA的杂交更弱。进行了熔解温度（T(m)）实验，以阐明用c(3)G取代第五个G对2'-O - 甲基 - (5'-CGGCGAGGAG-3')/5'-CUCCGAGCCG-3'和2'-O - 甲基 - (5'-CGGGGACGAG-3')/5'-CUCGGACCCG-3'双链体稳定性的影响，这两种双链体分别形成剪切型和面对面型5'-GA-3'/5'-GA-3'串联错配碱基对。因此，这种取代导致前一种双链体（其剪切型碱基对需要N3原子）比后一种双链体（其面对面型碱基对不需要N3原子）更明显地不稳定。在2'-O - 甲基RNA/DNA双链体中也观察到类似的趋势。这些结果表明，G的N3原子在稳定标准G/C碱基对以及碱基识别中起重要作用，并且其缺失抑制了不期望的剪切型错配碱基对的形成。基于从头算计算的计算研究表明，c3G较弱的氢键结合能力和较大的偶极矩可能是较低T(m)的原因。
