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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 screen high-affinity oligonucleotides that form triple-stranded structure with HBV DNAC promoter. METHODS: Five pairs of oligonucleotide chains (TFO) which could form a triple-stranded structure with the target sequence were synthesized using in vitro synthesized 3 2 P-labeled HBV DNAC promoter sequence (173 4 ~ 175 4) At the same time, the control target sequence and control TFO were designed. The affinity and specificity of TFO and target sequence to form triple-stranded structure and their binding were compared and analyzed by gel retention test and enzyme footprint test. Results: The binding affinity of CT TFO and GT TFOp to 3 2 P target DNA was very weak at 37 ℃ and pH 7 4 with Kd> 10 -6 mol / L, respectively. GT TFOap, AG TFOs and target DNA Kd was 5 × 10 -7 mol / L and 25 × 10 -8 mol / L, respectively. The binding affinity of AG TFO1 with the target sequence was the highest, Kd was 3 × 10 -9 mol / L, and the binding affinity of the two Binding is sequence specific. CONCLUSION: TFO containing AG or GT can bind to triple-stranded DNA in the anti-parallel orientation of the same homopurine in the promoter region of HBVC. Among them, AG TFO1 binds to the target sequence with the highest affinity and complete reaction, Into the three-stranded DNA, after a certain modification, can be used for HBV gene transcriptional inhibition test.