【作者】 张黎明；

【导师】 朱莉娜；

【作者基本信息】 天津大学 ， 化学， 2019， 硕士

【摘要】 G-四链体是一种核酸二级结构,广泛存在于人类基因组的许多重要区域,如端粒与致癌基因的启动子区域。越来越多的研究表明,这些区域中G-四链体的形成与一些关键的生物学功能密切相关,G-四链体有望成为新型抗癌药物研究的靶点。目前已报道的G-四链体探针以荧光探针和比色探针为主,迫切需要新型探针丰富G-四链体探测家族。基于此我们创新性地提出了一种新型G-四链体探针——基于G-四链体比率识别的共振光散射(RLS)探针。我们课题组之前的工作已经表明,长侧臂水溶性阳离子卟啉可以作为G-四链体识别的比色探针和荧光探针。在本论文工作中,我们尝试将阳离子卟啉作为RLS探针进行G-四链体识别。首先,对比比色和荧光识别效果,说明RLS探针识别G-四链体的可行性。由于阳离子卟啉侧臂取代基种类的不同,会在G-四链体识别方面表现出p H依赖性行为。通过梳理不同侧臂的阳离子卟啉衍生物识别G-四链体的p H条件,我们选择四种阳离子卟啉作为RLS探针,包括一种新合成的八电荷水溶性阳离子卟啉(OMHEPz EOPP),在重要的生理p H范围内(6.0-7.4),实现对G-四链体的RLS比率探测。由于RLS比率探测具有自参考能力,可以降低光源强度、仪器灵敏度以及探针浓度等因素造成的影响,具有较高的G-四链体探测精准度。RLS作为G-四链体探测家族的新成员,为研究与G-四链体相关聚合物的形成、破坏以及聚合物大小、形状和聚集变化等信息,提供了一个有用的工具。其次,由于阳离子卟啉衍生物可以作为G-四链体比色探针、荧光探针以及RLS探针,我们运用了一种统计方法——线性判别分析(LDA),对三种探针识别结果进行统计分析,实现了G-四链体的量化区分。再次,对阳离子卟啉与G-四链体作用的RLS响应机制进行研究,两者之间末端堆积的结合方式,在一定程度上促进卟啉的N质子化,从而引起RLS信号变化。最后,研究RLS探针的实用性,我们将卟啉衍生物/G-四链体RLS信号应用于Micro RNA检测,实现了Micro RNA无标记和序列特异性的检测。本论文考察了RLS探针的应用性和实用性,RLS探针实现了对G-四链体的高特异性识别,并指示了G-四链体类聚合物聚集变化的信息。同时本论文报道的OMHEPz EOPP,为高正电荷阳离子卟啉的制备,提供了一种简单实用的方法。更多还原

【Abstract】 G-quadruplexes are noncanonical nucleic acid secondary structure formed by guanine(G)-rich DNAs or RNAs.Since genomic sequences with G-quadruplexforming potential are widely found in many important regions of human genome and increasing evidences reveal that G-quadruplex formation in these regions is closely related with some crucial biological functions,G-quadruplexes represent promising targets for development of new anticancer drugs.Specific G-quadruplex-probing is also crucial for both biological sciences and biosensing applications.Most reported probes are focused on fluorescent or colorimetric recognition of G-quadruplexes.Herein,for the first time,we reported a new specific G-quadruplex-probing technique——resonance light scattering(RLS)-based ratiometric recognition.Four water soluble cationic porphyrin derivatives,including an unreported octacationic porphyrin,with large side arm substituents were synthesized and developed as RLS probes.By testing these four water soluble cationic porphyrins,we demonstrated that these four RLS probes showed their individual best performance at different pH values,highlighting the important roles of side arm substituents.Combing these four RLS probes makes specific G-quadruplex recognition in the important physiological pH range of 7.4-6.0 become possible.These RLS probes were demonstrated to work well for ratiometric recognition of G-quadruplexes with high specificity against singleand double-stranded DNAs,including long double-stranded ones.RLS probe as an important member in G-quadruplex probe family,thus providing a useful tool for studies on G-quadruplex-related events concerning G-quadruplex formation,destruction and changes in size,shape and aggregation.Since the tested porphyrin derivatives can simultaneously provide three ways(UV-vis absorption,fluorescent and RLS)to specifically recoginize G-quadurplexes,we can apply a statistical method——linear discriminant analysis(LDA)to achieve a quantifiable discrimination of Gquadruplexes from ss DNAs and ds DNAs.The working mechanism was speculated to be based on the RLS signal changes caused by porphyrin protonation that was promoted by the end-stacking of porphyrins on G-quadruplexes.As a proof-of-concept example of applications,the RLS probes were demonstrated to work well for label-free and sequence-specific sensing of Micro RNA.This work adds an important member in G-quadruplex probe family,thus providing a useful tool for studies on G-quadruplex-related events.A new porphyrin derivative with eight positive charges was synthesized and characterized.Thus,this work also provides a simple and useful way for the preparation of cationic porphyrins with high charges.更多还原