【作者】 郑克威；

【导师】 谭铮；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 核酸可以形成一种叫做G-quadruplex的四聚体结构,在不同物种的基因组中,具有形成G-quadruplex能力的DNA序列数量非常庞大。越来越多的研究显示这些G-quadruplex结构参与基因转录渊控,因而成为癌症和其他疾病的重要治疗靶点。除了端粒3’悬突以外,基因组G-quadruplex序列大部分都分布在双链DNA中间,附近不仅有互补的DNA与之配对,其两侧还有双链DNA限制。目前为止,有关双链DNA中G-quadruplex/duplex竞争的研究很少。在本研究中,我们制备了来自人基因组的含有不同G-quadruplex形成序列的双链DNA,分别在稀释溶液和PEG制造的分子拥挤环境下将双链进行加热变性退火和体外转录,结果发现,在分子拥挤条件下,加热变性退火和发生RNA转录的双链DNA中有G-quadruplex形成。我们使用DMS footprinting以及非变性聚丙烯酰胺凝胶电泳实验对G-quadruplex的形成进行了证明。我们的实验结果还发现,加热变性退火后非变性聚丙烯酰胺凝胶电泳可以提供一种衡量G-quadruplex在双链DNA中形成能力的简单方法。利用此方法我们研究了K离子和PEG200的浓度对G-quadruplex形成能力的影响,结果发现,PEG制造的分子拥挤环境是双链DNA中G-quadruplex稳定存在所必须的。大多数G-quadruplex在双链DNA中形成需要的PEG浓度在30-40%,和体内细胞中牛物分子浓度接近。这个现象指示分子拥挤环境可能是体内基因组中G-quadruplex形成的生理基础。基因组中G-quadruplex序列数量巨大且分布广泛,它们在基因表达调控中的作用使得G-quadruplex结构成为人们设计药物分子治疗疾病的靶点。研究生理条件下基凶组双链DNA由G-quadruplex的结构特征对寻找靶向G-quadruplex的药物具有重要的指导意义。利用上述加热变性退火后非变性聚丙烯酰胺凝胶电泳的方法,我们筛选到一种特异性识别并结合G-quadruplex的酞菁分子Zn-TTAPc,这种小分子具有两个特殊的性质,1)可以特异性结合于G-quadruplex的G-quatert平面；2)在紫外光照射下可以在结合位点处切割DNA。在本研究中,我们发展了一种可以解析G-quadruplex折叠方式的光切割footprint方法。通过比较酞菁分子对G-quadruplex两个末端平面切割程度的差异,我们可以解析出形成G-quadruplex的阴条G-tract的5’-3’走向,从而了解整个G-quadruplex的结构类型。这种方法简单明了,而且不需要非常专业的知识。并且,该酞菁分子对G-quadruplex的选择性远高于双链以及单链DNA,这种方法还可以应用于鉴定长单链和双链DNA中G-quadruplex的形成。更多还原

【Abstract】 Nucleic acid can adopt a four-stranded DNA structures known as G-quadruplex. Large numbers of guanine-rich sequences with potential to form G-quadruplexes have been identified in genomes of various organisms. It is now believed that G-quadruplexes play important role in regulating gene expression and thus constitute valuable therapeutic targets against cancer and other diseases. In addition to Telomere 3’overhang, G-quadruplex-forming sequences are constrained at both ends by long DNA duplex with a complementary strand in close proximity to compete for duplex formation. G-quadruplex/duplex competition in long double-stranded DNA has rarely been studied. In this work, we prepared long dsDNA from human genome carrying G-quadruplex-forming sequences with flanking duplex at both sides and studied G-quadruplex formation under both dilute and molecular crowding conditions during the process of in vitro transcription and heat denaturation/renaturation. Our data revealed that molecular crowding creates an essential environment for stable G-quadruplex to form in dsDNA. Using DMS footprinting and gel electrophoresis, we prove the formation of G-quadruplex. Our results also showed that the heat denaturation/renaturation treatment followed by gel electrophoresis could provide a simple method to quantitatively access the ability of G-quadruplex formation in long double-stranded DNA. The effect of K+ and PEG concentration was investigated and we found that stable G-quadruplexes could only form under the crowding condition with PEG at concentrations near the physiological concentration of biomass in living cells. This observation reveals a physicalbasis for the formation of stable G-quadruplexes in genome and supports its presence under the in vivo molecular crowding condition.The wide spread of G-quadruplex-forming sequences in genomic DNA and their role in regulating gene expression has made G-quadruplex structures attractive therapeutic targets against a variety of diseases. Information on the structure of G-quadruplexes is crucial for understanding their physiological roles and designing effective drugs against them. Resolving the structures of G-quadruplexes in double-stranded DNA is critic for searching and designing drugs.Using the quantitatively method to measure the ability of G-quadruplex formation in long double-stranded DNA as previous, we find a ligand tetrakis(2-trimethylaminoethylethanol) phthalocyaninato zinc tetraiodine (Zn-TTAPc). This ligand have especially good shape complementarity with the quartet plane and interact with G-quadruplex via stacking externally to the terminal G-quartets. Furthermore, under light irradiation, phthalocyanines cleave DNA via generation of singlet oxygen. Because of these properties, we developed a photocleavage footprinting technique to determine the folding orientation of each individual G-tract in intramolecular G-quadruplex formed in both single- and double-stranded nucleic acids. Based on the differential photocleavage induced by a ligand tetrakis(2-trimethylaminoethylethanol) phthalocyaninato zinc tetraiodine (Zn-TTAPc) to the guanines between the two terminal G-quartets in a G-quadruplex, this method identifies the guanines hosted in each terminal G-quartets to reveal G-tract orientation. The method is extremely intuitive, straightforward, and requires little expertise. Besides, it also detects G-quadruplex formation in long single- and double-stranded nucleic acids.更多还原