卟啉锰修饰的TFO结合并切割HBV-DNA片段体外的实验研究

Sequece-dependent cleavage of HBV DNA by combining with triple helix-forming oligodeoxyribonucleotides modified with manganese porphyrin in vitro

目的观察卟啉锰修饰的螺旋寡核苷酸(TFO)在体外结合并切割HBV-DNA片段的能力。方法以卟啉锰、吖啶修饰TFO末端;在37℃pH7.4体外环境中,使卟啉锰、吖啶修饰的TFO与32P标记的HBV-DNA片段结合,以凝胶滞留试验、酶足迹和切割试验观察其结合的亲和性、特异性以及切割HBV-DNA片段的能力。结果卟啉锰、吖啶修饰的TFO可与HBV靶序列结合成三链DNA,解离常数(Kd)为3.5×107mol/L,相对亲和力为0.008,并具有序列特异性。在KHSO5存在的情况下,卟啉锰、吖啶修饰的TFO可切割靶DNA,切割部位为三链DNA形成区。结论在有KHSO5存在的情况下,卟啉锰TFO吖啶化合物可定点切割靶HBV-DNA。

Objective To observe the ability of triple helix-forming oligonueleotides （TFO） modified with manganese porphyrin to combine with and cleave HBV DNA fractions. Methods The ends of TFO were modified with manganese porphyrin and acridine; At 37℃ and pH7.4 condition in vitro, TFO modified with manganese porphyrin and acridine were bound with ^32 P labeled HBV DNA fragments, the affinity and specificity binding to target sequenee were tested by electrophoretic mobility shift and DNase 1 footprinting assays, the ability to cleave HBV DNA fractions was observed with cleavage experiments. Results TFO modified with manganese porphyrin and acridine could bind to target sequence in a sequeneedependent manner with Kd values of 3.5 × 10^7 mol/L and a relative affinity of 0. 008. In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target sequence in the region forming triplex DNA. Conclusion In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could eleave target HBV-DNA in sequence-dependent manner.