【作者】 李辉；

【导师】 梁好均； 林汉华；

【作者基本信息】 中国科学技术大学 ， 高分子化学与物理， 2017， 博士

【摘要】 在过去的几十年中,DNA的研究和应用远远超出了"遗传物质"这一概念范畴,尤其是DNA纳米技术的发展吸引了大量研究者的注意力。DNA纳米技术主要有两个分支:静态的和动态的DNA纳米技术。在动态纳米技术中,DNA链替换反应是一项重要的研究领域,并且它已经在DNA逻辑门,分子机器以及复杂的DNA纳米"建筑"的构建等应用中发挥这重要的作用。另一方面球状核酸探针是一种近年来新型的生物纳米材料。通常这种材料由两部分组成:球状的纳米金核心和核酸链外壳。这种新型的探针具有许多优异的物理化学性能,并极大地方便了体外以及细胞内的检测。综上所述,本论文的研究内容主要集中于DNA链替换反应以及球状核酸探针在活细胞实验中的应用。第一部分内容是基于DNA链替换反应的单碱基突变检测。遗传信息的精确表达依赖于核酸序列的特异性互补配对,因此检测核酸序列中的碱基突变对于遗传疾病的诊断和医药学的发展具有重要意义。酶标记法是一种常用的检测DNA碱基突变的手段。但酶标记试剂通常比较昂贵,而且某些酶的反应条件比较苛刻。升温法(High-temperature)和化学变性(chemical denaturation)是也两种常用的传统的检测手段,但是这两种方法并不适用于存在多个杂交反应的复杂体系,因此其应用受到限制。近年来在DNA纳米技术领域,一种基于粘性反应末端(Toehold)的DNA链替换反应成为新的研究热点,并被广泛应用DNA逻辑门,分子器件的设计与构建。更要的是此反应在室温下,且不需要酶的催化即可实现DNA链构型和序列的重组,而且动力学过程可控。因此,成为一个理想的检测DNA链碱基突变的工具。基于以上所述,我们设计了一个包含两步的,并由DNA-AuNP探针驱动的DNA链替换反应,并借助石英微晶天平(QCM)这个测试平台来检测DNA序列片段中的单碱基突变。接下来我们在目标链选择了几个任意位置并检测单碱基突变并进行检测。结果表明,在目标链的任意位置上,不论是单碱基错配,插入还是碱基缺失,都能准确无误地检测出来。最后我们计算了该方法的检测限。在该设计中,DNA-AuNP探针能够将目标链释放回溶液中,从而使得目标链能够循环往复地出发DNA链替换反应,进而降低检测限,提高监测的灵敏度。该方法的检测限被估算为22pM,比直接加入DNA连接链的策略检测限低两个数量级。第二部分内容是球状核酸探针探测细胞对外源DNA降解活动。向细胞内导入外源的DNA分子探针是一种检测胞内目标分子的常用的策略。同时这种方法还经常用来获取与胞内生理活动,疾病诊断,生物药剂有关的信息。分子信标,DNA链替换反应工作网络,DNA四面体纳米结构经常用来构建探针。另一方面,DNA功能化纳米金(球状核酸)是一种近年来兴起的新型的生物材料探针。带有巯基修饰的DNA链可通过Au-S键连接到球状的纳米金上。其中DNA序列可以与目标基因互补,而纳米金是有效的荧光淬灭基团,因此带荧光标记的球状核酸可以用来细胞内的检测。但是由于细胞的防御机制,外源的DNA很容易被细胞内的酶降解,从而导致假阳性信号。因此在本工作中我们利用球状核酸探针来检测细胞对外源DNA的降解位点和降解行为。以前的观点认为外源的DNA在细胞内被无规降解直至碎片化。然而本论文中的发现与之前截然不同。以MCF-7细胞为例,我们发现降解特定地发生在DNA链的5’端而且只有5’端的第一个核苷酸被酶切,而其余部分保持完好。此外,这个发现提供了一个避免降解引起的假阳信号的方法,即研究者可以将荧光团标记到DNA链的安全区域。例如,检测MCF-7细胞内的目标分子,荧光团可以被标记到DNA链的3’或中间位置。同时我们希望这个发下能够提供一些新的视角去理解细胞内的降解活动以增强DNA探针稳定性。MicroRNA是一类非编码的小RNA片段。其通常能够对特定的mRNA起到负调节的作用,尤其是一些miRNA能够抑制在癌细胞生长,分裂过程中起关键作用的mRNA的表达。因此miRNA也成为一种有前途的新型的抗癌试剂。本工作中,我们以microRNA-34a(miRNA-34a)为例,设计并合成了纳米载体将miRNA-34a导入到细胞中,并诱导细胞凋亡。在这个设计中,survivinmRNA作为触发链,通过两步链替换反应将miR-34a释放到细胞中。利用检测荧光信号的方法验证上述设计原理。经荧光光谱仪,共聚焦显微镜以及流式细胞仪的验证,所设计的纳米载体能够将miRNA-34a运输到细胞中并成功地释放出来。在这个反应中,mRNA作为"触发器",最终导致miRNA-34a的释放。这个设计能够实现mRNA抑制和miRNA-34a过表达同时发生。因此,我们利用qRT-PCR来表征此设计原理的效率。结果表明,实验组中的miRNA的表达水平明显提高,而mRNA的表达量则被显著抑制。我们利用CCK-8试剂以及Annexin V-FITC/PI分别检测细胞活性和细胞凋亡。释放出的miRNA-34a(实验组)能明显地抑制细胞活性并诱导细胞凋亡。证明了此设计方法可行性以及潜在的应用价值。更多还原

【Abstract】 Over the past few decades,the research and application of DNA is far beyond the scope of "biological genetic material".The development of DNA nanotechnology has attracted much attention.There mainly are two subfields in DNA nanotechnology:structural and dynamic DNA nanotechnology.The dynamic DNA nanotechnology,in particular the strand displacement reaction,has made great achievements in DNA logic gates,DNA molecular machine,and artificial DNA nanoactuators.On other hand,the DNA-functionalized gold nanoparticles are also emerging in biodetection.Particularly,these novel probes greatly facilitate intercellular assay.Due to the interests in these subjects,I mainly focus on the DNA strand displacement reaction and DNA-gold nanoparticle probes in the biodetection.The first part is on the four different DNA hybridization reactions:direct-linker-addition(DLA),hybridized-linker-addition(HLA),direct-complex-addition(DCA),and hybridized-complex-addition(HCA).In fact,the first two methods are traditional way to tune DNA hybridization.The other methods are based on the strand displacement reaction.The experiments were performed on the quartz crystal microbalance(QCM)using DNA-AuNP nanoconjugates as signal amplifiers.In the two first methods,the HLA is faster and have stronger signal intensity than DLA,because the pre-hybridization can enhance the rigidity strand and decrease conformational entropy.The two factors are beneficial to the DNA hybridization.In the strand-displacement-based strategies,two components are employed:catalyst strand and complex.The complex is pre-hybridized by linker strand and protector.The catalyst is a single strand which can displace the protector.Then,the DNA-AuNP can release the catalyst back into solution and the catalyst triggers another strand displacement again.Hence,the last two methods show strong signal intensity of QCM sensor and faster reaction rate.In the second part,we first designed a two-step DNA strand displacement reaction with random sequences for the unambiguous discrimination of a single-base change on the sensing platform of quartz crystal microbalance(QCM).Given that no study has been performed to investigate the design principle of the strand displacement fueled by DNA-gold nanoparticle conjugate(DNA-AuNP)on gold-electrode surface,we use DNA-AuNP as a probe to achieve the discrimination;moreover the probe can release the target sequence back into the solution for the target sequence to circularly initiate the DNA strand displacement,and this reaction circuit can improve the sensitivity.After optimizing and testing the detection strategy,it has been successfully applied to detect mutation of a realistic sequence(p53 gene fragment)associated with human cancer,thereby indicating that this method has potential applicability in general.In addition,we also show some differences in the design between strand displacement in solution and that on gold-electrode surface.The third section focuses on the intracellular assay by using DNA-AuNP.Spherical nucleic acid(SNA)nanoconjugates are powerful tools for intracellular assay.Especially,by using highly effective fluorescence quenching capability of gold particle core,SNA labeled with fluorophore can visualize,track,and quantify targets in living cells.However,sometimes false positive signal may emerge,such as the instability of thermodynamics or signaling properties which can be eliminated by careful design of oligonucleotide shell.The major challenge is the measurable fluorescence leakage caused by intracellular degradation.To avoid this fundamentally,the site and behavior of intracellular degradation need to be investigated.Herein,we mainly describe degradation events happening to SNAs in MCF-7 cell.We find that degradation is specific to the 5’ region of oligonucleotide.More interestingly,only the first nucleotide of 5’ terminal is excised from DNA strand and residual portion,3’ and internal nucleotide,remains stably attached to gold particle surface.This unusual degradation action totally differs from previous studies that foreign oligonucleotides would be randomly digested into tiny fragments or single nucleotide in intracellular environment.Importantly and helpfully,these findings offer a route to avoid degradation-caused false positive signal that one can choose a secure region for labeling fluorophore along DNA strand.For instance,in this work,fluorophore can be tagged at 3’ terminal or internal nucleotide of oligonucleotide,except 5’ terminus which is specially targeted by nuclease degradation in MCF-7 cell.The final section aims at microRNA(miRNA)delivery by using SNA nanocarriers.MiRNAs are a class of endogenous,noncoding small RNAs.Recently,vast studies have shown that miRNAs are very promising anticancer reagents,because they play an important role in downregulating the gene expression by mRNA degradation or translational repression.In particular,microRNA-34a(miR-34a)is one of the best-studied cancer suppressors.It has been exhibited that overexpression of miR-34a can inhibit proliferation,migration,and invasion in various cancer cells,including breast cancer which remains a major cause of cancer death for women.However,some biopharmaceutical problems,such as rapid degradation by nuclease,poor cellular uptake,limit the development and application of miRNA biotherapy.Chemical modification and transfection reagents can increase miRNA lifetime and cellular uptake,but also may affect the function of miRNA,induce unwanted toxicity and immune response.Fortunately,SNA nanoconjugates,due to their unique properties,offer a route to extremely weaken these challenges.Typically,a SNA nanostructure consists of an AuNP core and an oligonucleotide shell.The AuNP core is used as scaffold and the oligonucleotides are anchored on the surface of AuNP core by AuNP-S bond.SNA nanoconjugates are capable of autonomous cell entry without assistance of transfection reagents,have stronger nuclease resistance than linear counterparts and do not elicit observable cytotoxicity or immune response.Hence,we believe SNA can be an ideal nanocarrier for miRNA delivery and releasing in living cell.In this work,we deliver and release miR-34a in MCF-7 cell line by means of two sets of SNA nanocarriers:R-F SNA and miR-34a SNA.This strategy contains a two-step strand displacement reaction:the survivin mRNA transcript firstly swaps out an intermediate strand(R-F strand)from the R-F SNA,and then the free R-F strand invades the miR-34a SNA to release the miR-34a mimics.Therefore,this design rationale allows concurrence of mRNA depletion and miRNA overexpression.Validated by quantitative RT-PCR,the expression of miR-34a is significantly elevated and survivin mRNA level is reduced by-77%.Meanwhile,the released miR-34a largely reduces cell viability and induces apoptosis of MCF-7 cancer cell.These measurements indicate the high-efficiency and availability of this SNA-based delivery system.更多还原