能与HBV-DNAC启动子结合成三链结构的寡核苷酸的设计与筛选

Design and screening of-oligo-nucleotides forming triplex through binding with homopurine in HBV-DNA core-promoter

目的 :筛选出能与HBV DNAC启动子形成三链结构的高亲和力寡核苷酸。方法 :以体外合成的3 2 P标记的HBV DNAC启动子区序列 ( 173 4～ 175 4)为靶序列 ,合成 5种有可能与该靶序列形成三链结构的寡核苷酸链 (TFO) ,同时设计对照靶序列和对照TFO ,用凝胶滞留试验、酶足迹试验等方法对比分析这些TFO与靶序列是否形成三链结构及其结合的亲和性、特异性。结果 :在 3 7°C、pH7 4条件下 ,CT TFO和GT TFOp与3 2 P 靶序列DNA结合的亲和力十分微弱 ,其Kd均 >10 -6mol/L ;GT TFOap和AG TFOs与靶序列DNA结合的亲和力较高 ,Kd分别为 5× 10 -7mol/L和 2 5× 10 -8mol/L ;AG TFO1与靶序列结合的亲和力最高 ,Kd为 3× 10 -9mol/L ,而且两者的结合具有序列特异性。结论 :含AG或GT的TFO可与HBVC启动子区类同聚嘌呤链逆平行方向结合成三链DNA ,其中AG TFO1与靶序列结合的亲和力最高 ,反应完全 ,可使靶双链DNA均转变成三链DNA ,经一定修饰后 ,可试用于HBV基因转录抑制实验。

Objective: To find oligodeoxyribonucleotides that can bind with HBV core promoter target site with high affinity and specificity Methods: Similar homopurine domain (1 734～1 754) in HBV core promoter was chosen as the target sequence Several corresponding oligodeoxyribonucleotides were synthesized The binding affinity and specificity of the nucleotides were tested by electrophoretic mobility shift and DNase 1 footprinting assay Results: At 37℃and pH7 4, CT TFO and GT TFOp could not bind with the target sequence with Kd values>10 -6 mol/L GT TFOap and AG TFOs could bind with the target sequence with Kd values of 5×10 -7 mol/L and 2 5×10 -8 mol/L respectively AG TFO1 had the highest affinity among the 5 kinds of oligodexyribonucleotides with a Kd value of 3×10 -9 mol/L in a sequence dependent manner Conclusion: TFO containing AG or GT can bind with antiparallel homopurine in HBV core promoter to form triple helix AG TFO1 might be the best candidate among all the oligodeoxyribonucleotides designed in this study to inhibit HBV gene transcription