摘要
Objective: To screen a triple helix-forming oligodeoxyribonucleotide (TFO) that can bind HBV core promoter at target site with high affinity and specificity, and to observe the ability of manganese porphyrin modified TFO to combine and cleave HBV DNA. Methods: Similar homopurine domain (1 734 - 1 754) in HBV core promoter was selected as target sequence. Several corresponding TFOs were synthesized. The affinities and specificities of TFOs binding target sequence were tested with electrophoretic mobility shift and DNase I footprinting assays. The selected best TFO was modified with manganese porphyrin and acridine. The ability of the TFO derivative to cleave HBV DNA was observed with cleavage experiment. Results: Under the condition of 371 and pH 7. 4, the TFO consisting of cytidylate and thymidylate (CT-TFO) and the parallel TFO consisting of guanylate and thymidylate (GT-TFOp) bound the target sequence weakly with Kd values much more than 10 -6 mol/L. The affinities of anti-parallel GT-TFO ( GT-TFOap) and short TFO consisting of adenine nucleotide and guanylate (AG-TFOsh) binding the target sequence were higher than those of the formers, with Kd values of 5 μ 10-7 mol/L and 2. 5 μ 10-8 mol/L respectively. Long AG-TFO (AG-TF01) had the highest binding affinity with a Kd value of 3 μ 10 -9 mol/L among all the TFOs studied for sequence specificity. In the presence of potassium monopersulfate, KHSO5, TFO modified with manganese porphyrin and acridine cleaved the target sequence where the triplex DNA formed. Conclusion: TFO containing AG or GT binds homopurine in HBV core promoter in adverse parallel direction to form triple helix. AG-TFO1 has the highest binding affinity among all the TFOs studied. After modified with manganese porphyrin, AG-TFO1 completely binds and cleaves the target HBV DNA sequence where triplex DNA is formed.
Objective: To screen a triple helix-forming oligodeoxyribonucleotide (TFO) that can bind HBV core promoter at target site with high affinity and specificity, and to observe the ability of manganese porphyrin modified TFO to combine and cleave HBV DNA.Methods: Similar homopurine domain (1 734-1 754) in HBV core promoter was selected as target sequence.Several corresponding TFOs were synthesized.The affinities and specificities of TFOs binding target sequence were tested with electrophoretic mobility shift and DNase Ⅰ footprinting assays.The selected best TFO was modified with manganese porphyrin and acridine.The ability of the TFO derivative to cleave HBV DNA was observed with cleavage experiment.Results: Under the condition of 37℃ and pH 7.4, the TFO consisting of cytidylate and thymidylate (CT-TFO) and the parallel TFO consisting of guanylate and thymidylate (GT-TFOp) bound the target sequence weakly with Kd values much more than 10-6 mol/L.The affinities of anti-parallel GT-TFO (GT-TFOap) and short TFO consisting of adenine nucleotide and guanylate (AG-TFOsh) binding the target sequence were higher than those of the formers, with Kd values of 5×10-7 mol/L and 2.5×10-8 mol/L respectively.Long AG-TFO (AG-TFOl) had the highest binding affinity with a Kd value of 3×10-9 mol/L among all the TFOs studied for sequence specificity.In the presence of potassium monopersulfate, KHSO5, TFO modified with manganese porphyrin and acridine cleaved the target sequence where the triplex DNA formed.Conclusion: TFO containing AG or GT binds homopurine in HBV core promoter in adverse parallel direction to form triple helix.AG-TFOl has the highest binding affinity among all the TFOs studied.After modified with manganese porphyrin, AG-TFOl completely binds and cleaves the target HBV DNA sequence where triplex DNA is formed.
