基于G四联体DNA模拟酶和接合探针的核酸检测新方法研究
发布时间：2019-04-24 19:01
【摘要】：G四联体(G4)是一种特殊的核酸二级结构,其由富含鸟嘌呤碱基(G)的寡核苷酸序列通过Hoogsteen键连接而成。在含有一些单价金属离子的溶液中,G4可与氯化血红素单体(Hemin)结合形成具有过氧化物酶活性的功能性寡核苷酸结构,又被称为 DNA 模拟酶(peroxidase-mimicking DNAzyme)。利用 DNA 模拟酶作为信号检测元件对于核酸检测具有极强的便携性,然而现今基于DNA模拟酶的核酸均相检测方式具有较大的检测背景,从而阻碍了 DNA模拟酶在核酸均相检测中的广泛应用。接合探针(junction probe,JP)源于双探针概念(binary probe,BP),在一个系统中,当且仅当两条探针同时与靶核酸序列杂交时可产生杂交信号,使其具有极强的杂交特异性,并且有潜力被运用于核酸的均相检测。传统的接合探针由于有较大的非特异性杂交概率,而使其需要经过复杂的序列设计及优化,这增加了检测的成本。本研究在前人研究的基础上,通过构建基于链置换技术(strand displacement)的接合探针,并整合G四联体用于核酸的便携检测,并初步评价其检测效能。论文主要分为以下两个章节:在第一章的研究中,我们将分裂的G-四联体形成序列整合于两个探针中。只有在含有特异性可识别剪接位点的靶基因转录本存在时,探针可相互组装形成三向接合构象,从而提供功能性的G-四联体构象,其与氯化血红素单体结合后可大大增强氯化血红素的过氧化酶活性。该系统对通过比色测定对靶基因转录本的检测限为0.063 μM,并且可特异地识别靶序列中的单碱基突变(约需3倍量的突变序列才能使检测体系获得与靶序列等同的信号)。该接合探针识别的靶序列可长达46 bp,并且无需复杂的序列设计步骤,显示出其用于进一步构建简便、高效的核酸检测系统的潜力。第二章中,我们构建了一种用于核酸靶序列识别和信号放大的方法。核酸扩增基于链置换扩增技术(strand displacement amplification,SDA),其中两条 DNA 探针在抑制链(inhibitor)作用下不相互组装触发下游链置换扩增。但在靶序列存在时,探针可通过靶序列诱导的三向接合结构形成(核心原理见第一章节)释放抑制链,随后触发SDA反应扩增富C(胞嘧啶)序列模板,这导致一系列富G(鸟嘌呤)序列生成,其与氯化血红素单体结合表现出强大的过氧化物酶活性。用TMB作为底物并将产物在450nm的波长下测定时时,其具有0.8 pM的检测限。该检测系统具有极低的检测背景以及核酸检测的高选择性。
[Abstract]:G-tetraad (G-4) is a special secondary structure of nucleic acid, which is composed of oligodeoxynucleotide sequences rich in guanine base (G) through Hoogsteen bonds. In solutions containing some monovalent metal ions, G4 binds to the hemin monomer (Hemin) to form a functional oligodeoxynucleotide structure with peroxidase activity, also known as the DNA mimicase (peroxidase-mimicking DNAzyme). Using DNA mimetic enzyme as signal detection element is very portable for nucleic acid detection. However, nowadays, homogenous detection of nucleic acid based on DNA mimetic enzyme has a large detection background. This hinders the wide application of DNA mimetic enzyme in nucleic acid homogenous detection. Conjugation probe (junction probe,JP) is derived from the concept of double probe (binary probe,BP). In a system, if and only if two probes are hybridized with the target nucleic acid sequence at the same time, the hybridization signal can be generated, which makes it very specific for hybridization. And it has the potential to be used for homogenous detection of nucleic acids. Because of the large non-specific hybridization probability, the traditional conjugation probe needs complex sequence design and optimization, which increases the cost of detection. In this study, based on the previous studies, the conjugation probe based on the chain substitution technique (strand displacement) was constructed, and the G-tetraad was integrated into the portable detection of nucleic acid, and its detection efficiency was evaluated preliminarily. The thesis is divided into two chapters: in the first chapter, we integrate the split G-tetraad formation sequence into two probes. Only in the presence of transcripts containing specific splicing sites, probes can be assembled together to form a three-way conjugation conformation, thus providing a functional G-tetraad conformation. After binding with hemin monomer, the peroxidase activity of hemin can be greatly enhanced. The detection limit for target gene transcripts by colorimetric assay is 0.063 渭 M, and the system can specifically identify single base mutations in the target sequence (about 3 times the amount of mutation sequence is required for the detection system to obtain the same signal as the target sequence). The target sequence identified by the conjugation probe can be up to 46 bp, long and does not require complex sequence design steps, showing its potential for further construction of a simple and efficient nucleic acid detection system. In the second chapter, we construct a method for nucleic acid target sequence recognition and signal amplification. Nucleic acid amplification is based on chain substitution amplification (strand displacement amplification,SDA), in which two DNA probes do not assemble each other to trigger downstream strand substitution amplification under the action of inhibitory chain (inhibitor). However, in the presence of the target sequence, the probe can release the inhibitory chain through the three-way conjugation structure induced by the target sequence (see chapter I for the core principle), and then trigger the SDA reaction to amplify the C (cytosine)-rich sequence template. This leads to the formation of a series of G-rich (guanine) sequences, which combine with hemin monomers and exhibit strong peroxidase activity. When TMB was used as substrate and the product was determined at the wavelength of 450nm, it had a detection limit of 0.8 pM. The detection system has very low detection background and high selectivity of nucleic acid detection.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440
，
本文编号：2464700
[Abstract]:G-tetraad (G-4) is a special secondary structure of nucleic acid, which is composed of oligodeoxynucleotide sequences rich in guanine base (G) through Hoogsteen bonds. In solutions containing some monovalent metal ions, G4 binds to the hemin monomer (Hemin) to form a functional oligodeoxynucleotide structure with peroxidase activity, also known as the DNA mimicase (peroxidase-mimicking DNAzyme). Using DNA mimetic enzyme as signal detection element is very portable for nucleic acid detection. However, nowadays, homogenous detection of nucleic acid based on DNA mimetic enzyme has a large detection background. This hinders the wide application of DNA mimetic enzyme in nucleic acid homogenous detection. Conjugation probe (junction probe,JP) is derived from the concept of double probe (binary probe,BP). In a system, if and only if two probes are hybridized with the target nucleic acid sequence at the same time, the hybridization signal can be generated, which makes it very specific for hybridization. And it has the potential to be used for homogenous detection of nucleic acids. Because of the large non-specific hybridization probability, the traditional conjugation probe needs complex sequence design and optimization, which increases the cost of detection. In this study, based on the previous studies, the conjugation probe based on the chain substitution technique (strand displacement) was constructed, and the G-tetraad was integrated into the portable detection of nucleic acid, and its detection efficiency was evaluated preliminarily. The thesis is divided into two chapters: in the first chapter, we integrate the split G-tetraad formation sequence into two probes. Only in the presence of transcripts containing specific splicing sites, probes can be assembled together to form a three-way conjugation conformation, thus providing a functional G-tetraad conformation. After binding with hemin monomer, the peroxidase activity of hemin can be greatly enhanced. The detection limit for target gene transcripts by colorimetric assay is 0.063 渭 M, and the system can specifically identify single base mutations in the target sequence (about 3 times the amount of mutation sequence is required for the detection system to obtain the same signal as the target sequence). The target sequence identified by the conjugation probe can be up to 46 bp, long and does not require complex sequence design steps, showing its potential for further construction of a simple and efficient nucleic acid detection system. In the second chapter, we construct a method for nucleic acid target sequence recognition and signal amplification. Nucleic acid amplification is based on chain substitution amplification (strand displacement amplification,SDA), in which two DNA probes do not assemble each other to trigger downstream strand substitution amplification under the action of inhibitory chain (inhibitor). However, in the presence of the target sequence, the probe can release the inhibitory chain through the three-way conjugation structure induced by the target sequence (see chapter I for the core principle), and then trigger the SDA reaction to amplify the C (cytosine)-rich sequence template. This leads to the formation of a series of G-rich (guanine) sequences, which combine with hemin monomers and exhibit strong peroxidase activity. When TMB was used as substrate and the product was determined at the wavelength of 450nm, it had a detection limit of 0.8 pM. The detection system has very low detection background and high selectivity of nucleic acid detection.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R440
，
本文编号：2464700
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