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 ...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.展开更多
PDGF (platelet derived growth factor) has been shown to play animportant role in tumorigenesis, tumor growth, atherosclerosis and inflammation and other various pathologic settings. PDGF-B chain gene is 92% homologous...PDGF (platelet derived growth factor) has been shown to play animportant role in tumorigenesis, tumor growth, atherosclerosis and inflammation and other various pathologic settings. PDGF-B chain gene is 92% homologous to v-sis oncogene of the simian sarcoma virus. Thus PDGF-B gene is also called c-sis proto-oncogene. This report provides 3 TFOs (triplex-forming oligonucleotides) to inhibit the expression of c-sis/PDGF-B gene. The results from gel mobility shift analysis, in vitro transcription, DNase I footprinting and protein binding assays demonstrate that the TFOs we designed can form sequence-specific stable triplex with the target, and can effectively suppress the downstream gene transcription and inhibit transcription factors binding. They can be used for preparation of drugs to inhibit tumor growth and for the therapy of atherosclerosis, inflammation, etc.展开更多
文摘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.
基金the State Key Programs Basic Research of China (Grant No. G1998051103) and National High Technology Programs of China (Grant No. 863-102-08-8).
文摘PDGF (platelet derived growth factor) has been shown to play animportant role in tumorigenesis, tumor growth, atherosclerosis and inflammation and other various pathologic settings. PDGF-B chain gene is 92% homologous to v-sis oncogene of the simian sarcoma virus. Thus PDGF-B gene is also called c-sis proto-oncogene. This report provides 3 TFOs (triplex-forming oligonucleotides) to inhibit the expression of c-sis/PDGF-B gene. The results from gel mobility shift analysis, in vitro transcription, DNase I footprinting and protein binding assays demonstrate that the TFOs we designed can form sequence-specific stable triplex with the target, and can effectively suppress the downstream gene transcription and inhibit transcription factors binding. They can be used for preparation of drugs to inhibit tumor growth and for the therapy of atherosclerosis, inflammation, etc.
