The capping of the 3′-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not affected by the capping. The capping could be simply executed via a one step enzymatic process using 2′,4′-bridged nucleoside 5′-triphosphate and terminal deoxynucleotidyl transferase.The capping of the 3′-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not affected by the capping. The capping could be simply executed via a one step enzymatic process using 2′,4′-bridged nucleoside 5′-triphosphate and terminal deoxynucleotidyl transferase.Figure optionsDownload full-size imageDownload as PowerPoint slide

Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been replaced with an amide-type linker [–CH2CONH–] or an amine-type linker [–CH2CH2NH–] were synthesized to investigate the effect of these backbone modifications on polymerase reactions. In addition, a triphosphate analogue of thymidine dinucleotide with the amide-type linker was synthesized and enzymatic insertion of the amide linkage into ODN was attempted using this analogue for the polymerase reaction. Primer extension reactions using three types of thermostable DNA polymerases, KOD(exo-), Vent(exo-) and Taq were performed for the assays. Analysis of these data indicate that (i) the polymerase reaction tends to be affected much more by insertion of the cationic flexible amine-type linker than by insertion of the neutral rigid amide-type linker; (ii) the backbone modification has a greater effect on the polymerase reaction when it is adjacent to the 3′-end of a primer as the elongation terminus than when it is on the template, as well as in base or sugar modification; (iii) although the modified linker in the modified DNA template is passed beyond by the polymerase, it still affects the extension reaction several bases downstream from its location; (iv) the modified linker in the template, in some cases, also affects the extension reaction upstream from its location; (v) further improvement of the chemical structure is required for dinucleotide-mimic incorporation.Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been replaced with an amide-type linker or an amine-type linker were synthesized to investigate the effect of these backbone modifications on polymerase reactions. A triphosphate analogue of thymidine dinucleotide with the amide-type linker was synthesized and enzymatic insertion into ODN was attempted.

Incorporation of 2′,4′-bridged nucleotides into the 3′-end of oligodeoxyribonucleotide (ODN) was examined using terminal deoxynucleotidyl transferase (TdT). The three types of 2′,4′-bridged nucleoside-5′-triphospates with different bridging structures used were incorporated efficiently into the 3′-end of DNA by TdT, although only single nucleotide incorporation was observed. Nuclease resistance was conferred on DNA, depending on the types of bridging nucleotides added.The enzymatic addition of BNA nucleotides to the 3′-end of oligodeoxyribonucleotide (ODN) was examined using terminal deoxynucleotidyl transferase (TdT); nuclease resistance was conferred on DNA, depending on the types of BNA nucleotides added.