吡柔比星与c-myc G4-DNA相互作用的研究

The interaction of c-myc G4-DNA with pirarubicin

目的研究吡柔比星与c-mycG4-DNA的相互作用．并探讨其可能的抗肿瘤作用机制。方法采用紫外可见吸收光谱、圆二色光谱（CD）、等温滴定微量量热试验（ITC）、核磁NMR滴定以及荧光能量共振转移（FRET）熔点试验等方法，研究吡柔比星与c-mycG4-DNA的相互作用；采用PCR-Stop方法研究吡柔比星对c-mycG4-DNA复制的抑制。结果在电子吸收光谱中，吡柔比星的特征吸收峰随c-mycG4-DNA浓度的增加出现明显的减色效应和红移，减色率AH=9．2％（△λ=4nm）；根据热力学计算的结合常数K11为1．0×10^4μmol／L；随着吡柔比星的加入pmycG4-DNA的圆二色光谱在265nrn处的信号变化均匀缓慢，在300nm处出现1个负的诱导信号：在吡柔比星作用下，c-mycG4-DNA5＇-末端表面碱基G21-A25发生明显变化，并且熔点升高20．2℃；PCR-Stop试验证实，随着吡柔比星浓度的增加，c-mycG4一DNA的复制能力不断减弱。结论吡柔比星能够以沟槽方式与c-mycG4-DNA相互作用，稳定c-mycG4-DNA四链体结构，并抑制其复制功能．吡柔比星抗肿瘤活性可能与它能够结合并稳定c-mycG4-DNA密切相关。

Objective To investigate the interaction of pirarubicin with c-myc G4-DNA. Methods The interaction of pirarubicin with c-myc G4-DNA was investigated by UV-Vis spectroscopy, circular dichroism （CD） spectroscopy, ITC experiments, NMR and FRET melting profiles, as well as PCR-Stop experiment. Results It＇s found that there was hypochromise of AH= 9.2% （△λ=4 urn） at the characterized absorption in the electronic spectra of pirarubicin in the presence of c-myc G4-DNA,and the binding constant Kb was 1.0×10^4 μmoL/L. In addition,the strengths of CD signal of c-myc G4-DNA at the maximum of 265 nm decreased in the presence of emodin,and an induced CD signal was observed at 300 nm. Besides,the I H-NMR spectroscopy experiments indicated that pirarubicin could bind closely to the 5＇-terminal face of the G-quadruplex DNA by the groove constitute G21 - A25 in c-myc G4-DNA molecules. Furthermore, the melting point of c-myc C,4-DNA was increased about AT = 20,2 ~ in the presence of pirarubicin. As a result,the replieation of c-mye C,4-DNA was blocked.Conclusion The results show that pirarubicin ean bind to c-mye G4-DNA in groove binding mode with high affinity, indicating that the inhibitory activity of this compound is in relate to c-rnyc.