非标记信号增强型荧光分子信标的构建研究
摘要
基因检测是指对受检者基因编码序列的测定和定位分析,根据已知的基因片段来确定与检测序列相关的疾病。基因变异与人类许多疾病有关,如心血管疾病、糖尿病等。
分子信标技术(Molecular beacon,MB)是由Tyagi和Kramer等于1996年建立的。分子信标是一个U型发卡结构的单链寡聚核苷酸链,在其内部含有部分序列互补配对形成的茎状部分。分子信标的茎-环结构中,环状部分一般为15-30碱基,并与目标序列互补；茎状部分一般含有5-7碱基,根据碱基互补配对原则相互配对形成茎的结构。探针的两端即5’端标记有荧光素,而3’端标记有淬灭剂。探针与目标序列互补杂交之前,由于荧光素和淬灭剂在空间结构上靠得很近,根据能量共振原理,淬灭剂将荧光素荧光淬灭,不会产生荧光；探针与目标序列完全互补配对后,发卡结构打开,荧光素与淬灭剂分开,荧光恢复；如果探针与目标序列之间存在错配碱基,发卡结构不能打开,也不会产生荧光。因此分子信标可以有效的区分全匹配目标DNA链和存在单碱基错配的目标DNA链。
经典的分子信标需要在寡核苷酸链两端分别标记荧光基团和淬灭基团,实验成本较高,而且操作过程难于操作控制。近年来,许多人改变分子信标结构,构建了许多新型的分子信标来检测DNA。因此,本论文在经典分子信标结构的基础上,构建一种非标记的分子信标来检测DNA。有文献报道,荧光杂环小分子AMND (2-amino-7-methyl-1,8-naphthyridine, AMND)可以通过氢键与DNA双链中未配对的C碱基(胞嘧啶,Cytosine)结合,其荧光发生淬灭。我们拟利用DNA双链中未配对碱基对荧光小分子的荧光淬灭作用制备非标记的分子信标,用于目标DNA链的检测。
本论文分为两部分：第一部分为综述部分,第二部分为研究报告部分。
第一章是综述部分。综述部分介绍分子信标的原理,应用及其研究进展。重点介绍分子信标的结构,同时介绍国内外非标记DNA检测技术,阐明了本论文的研究背景,研究意义,研究目的以及研究内容。
第二部分是研究报告,由两部分组成。第一部分为非标记信号增强型荧光分子信标的构建研究。由于AMND可以特异性的与C碱基(胞嘧啶,Cytosine)结合。因此,本实验选择AMND作为荧光小分子。首先合成荧光杂环小分子AMND(2-氨基-7-甲基-1,8-二氮杂萘,2-amino-7-methyl-1,8-naphthyridine)并对其进行了结构表征。
之后,将合成出的荧光小分子作为信号物质用于构建非标记信号增强型荧光分子信标。利用两段单链DNA杂交形成一个发卡DNA结构,其特点在于发卡DNA的茎状部分形成了一个空缺位点,空缺位点对面是C碱基。荧光杂环小分子AMND通过氢键识别未配对的C碱基并嵌入空缺位点,AMND荧光发生淬灭。当向体系加入与分子信标环状部分互补的目标连时,分子信标环状部分打开,AMND与目标碱基结合的微环境发生变化,AMND被释放出,其荧光强度恢复表明目标链的存在。研究了影响荧光淬灭的因素,优化了实验条件。研究发现,当加入目标DNA链时,AMND荧光强度恢复,而加入含有错配碱基的目标链时,荧光恢复不明显,表明我们的非标记分子信标可以有效的区分完全互补链和单碱基错配。
第三部分是有关汞离子的检测。汞蒸气有高度的扩散性和较大的脂溶性,通过血液循环,进入人体器官,然后被氧化,在人体器官中累积起来,当人体汞离子浓度达到一定浓度时,就会出现各种症状,如头痛、肢体麻木,严重者出现肝炎、肾炎、尿血等症状。因此,对汞离子的检测显得尤为重要。近年来,许多学者报道了基于金纳米粒子比色法来检测汞离子。本实验基于单链DNA与金纳米粒子发生作用,当向体系加入一定浓度的NaCl时,由于单链DNA对金纳米粒子的保护作用,金纳米粒子不发生团聚；然后再加入汞离子,使得单链DNA与汞离子形成“T-Hg2+-T”结构,从而与金纳米粒子分离,金纳米粒子聚沉,颜色由酒红色变为蓝色,从而达到检测汞离子的目的。
本研究利用荧光小分子和双链中未配对碱基通过氢键结合并伴随着小分子荧光淬灭的原理设计了简单、非标记的分子信标,并将其用于目标单链DNA的检测。这一非标记分子信标能够很好地区分全匹配和单碱基错配DNA序列,因而这一非标记分子信标可以用来检测SNPs,将为SNPs分型方法的基础和应用研究提供新思路。此外,我们还研究了利用“T-Hg2+-T”结构的形成,通过比色法检测Hg2+。我们方法的特点在于使用了含T-T错配的双链DNA来稳定金纳米粒子,与通常的单链相比,我们的体系响应速度更快。
The sequence-specific detection of DNA hybridization has attracted considerable interest in a wide range of areas including molecular diagnostics, environmental monitoring, and antibioterrorism. Therefore, there is a continuing demand for sensitive and selective DNA probes. Many kinds of DNA probes have been developed in recent years through various molecular-engineering strategies to meet this demand. Amount these DNA probes, MBs have attract much attentions due in part to their stability, unique functionality and molecular specificity.
The conventional MBs are single-stranded oligonucleotide probes that possess a stem-and-loop structure. The loop portion of an MB is complementary to a target single-stranded DNA, while the stem is formed by 5 to 7 bp from two complementary arm sequences that are on either end of the MB. A fluorophore is attached to the end of one arm, while a quencher is attached to the end of the other arm. The stem maintains a close proximity of the two moieties, causing fluorescence to be quenched by fluorescence resonance energy transfer (FRET). When an MB hybridizes with its complementary DNA (cDNA), the MB undergoes a spontaneous conformational reorganization with the opening of the stem, leading to a fluorescence restoration. The unique target recognition and signal transduction capabilities of MBs have led to their application in many biochemical and biological assays including quantitative PCR, protein-DNA interactions, multiplex genetic analysis, and the detection of mRNA in living cells. As part of a general program aimed at developing free-labeled MBs, we want to investigate whether the non-covalently bind small fluorescence molecules can be a signal molecules for MBs.
The paper includes two parts, review and study.
The first chapter is part of the overview section to describe the principle of molecular beacons, application and research. Focuses on the structure of molecular beacons, we also introduced domestic and foreign non-labeled DNA testing technology, explained the background of this thesis, and to study the meaning, purpose and research content.
The second chapter is research report:First, we synthesit the fluorescent small molecules AMND (2-amino-7-methyl-1,8-naphthyridine) as it has been reported that AMND can bind to cytosine bases specifically.
And then, a free-labeled molecular beacon (MB) based on non-covalent binding of a fluorescence molecule,2-amino-7-methyl-1,8-naphthyridin-(AMND), to the intentional gap site in the stem moiety of a hairpin DNA has been developed. When the free-labeled MB was under a closed state, a gap site was formed in the stem moiety of a hairpin DNA. The fluorescence of the small fluorescence molecule was significantly quenched by binding to the unpaired base at the gap site through hydrogen bond, and thus the free-labeled MB shows almost no fluorescence. Upon hybridization with a complementary target ss-DNA (cDNA), the free-labeled MB undergoes a conformational change to take an open state, resulting in an effective fluorescence enhancement owing to a release of the small fluorescence molecules from the gap site. Fluorescence titration and circular dichroism (CD) spectra were used to demonstrate that the binding of AMND to cytosine base at gap site was strong and do not induce a significant conformational change of the hairpin DNA. Under the optimal conditions, the fluorescence intensity of the free-labeled MB increased with an increase of the concentration of cDNA and a detection limit of 9×10-11 M cDNA was achieved. Single mismatched target ss-DNA can be effectively discriminated from complementary target ss-DNA. There may be some advantages in utilizing this type of free-labeled MB over more-traditional ones. First, both ends of the free-labeled MB can be left free to introduce other useful functionalities. Second, our free-labeled MB synthesis is relatively simple and inexpensive because no label is required.
The third part is colorimetric detection method for Hg2+ ions based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing system. Mercury is a widespread pollutant with distinct toxicological profiles, and it exists in a variety of different forms (metallic, ionic, and as part of organic and inorganic salts and complexes). Solvated mercuric ion (Hg2+), one of the most stable inorganic forms of mercury, is a caustic and carcinogenic material with high cellular toxicity. Here, we report a simple and sensitive colorimetric detection method for Hg2+ions based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing system. Complementary DNA strands with T-T mismatches could effectively protect AuNPs from salt induced aggregation. While in the presence of Hg2+ions T-Hg2+-T coordination chemistry leads to the formation of DNA duplexes, and AuNPs are less well protected thus aggregate at the same salt concentration, accompanying by color change from red to blue. Employing duplex oligonucleotides with T-T mismatches in the sensing system, a sensitive linear range for Hg2+ ions from 0.05 to 2μM and a detection limit of 17 nM are obtained.
分子信标技术(Molecular beacon,MB)是由Tyagi和Kramer等于1996年建立的。分子信标是一个U型发卡结构的单链寡聚核苷酸链,在其内部含有部分序列互补配对形成的茎状部分。分子信标的茎-环结构中,环状部分一般为15-30碱基,并与目标序列互补；茎状部分一般含有5-7碱基,根据碱基互补配对原则相互配对形成茎的结构。探针的两端即5’端标记有荧光素,而3’端标记有淬灭剂。探针与目标序列互补杂交之前,由于荧光素和淬灭剂在空间结构上靠得很近,根据能量共振原理,淬灭剂将荧光素荧光淬灭,不会产生荧光；探针与目标序列完全互补配对后,发卡结构打开,荧光素与淬灭剂分开,荧光恢复；如果探针与目标序列之间存在错配碱基,发卡结构不能打开,也不会产生荧光。因此分子信标可以有效的区分全匹配目标DNA链和存在单碱基错配的目标DNA链。
经典的分子信标需要在寡核苷酸链两端分别标记荧光基团和淬灭基团,实验成本较高,而且操作过程难于操作控制。近年来,许多人改变分子信标结构,构建了许多新型的分子信标来检测DNA。因此,本论文在经典分子信标结构的基础上,构建一种非标记的分子信标来检测DNA。有文献报道,荧光杂环小分子AMND (2-amino-7-methyl-1,8-naphthyridine, AMND)可以通过氢键与DNA双链中未配对的C碱基(胞嘧啶,Cytosine)结合,其荧光发生淬灭。我们拟利用DNA双链中未配对碱基对荧光小分子的荧光淬灭作用制备非标记的分子信标,用于目标DNA链的检测。
本论文分为两部分：第一部分为综述部分,第二部分为研究报告部分。
第一章是综述部分。综述部分介绍分子信标的原理,应用及其研究进展。重点介绍分子信标的结构,同时介绍国内外非标记DNA检测技术,阐明了本论文的研究背景,研究意义,研究目的以及研究内容。
第二部分是研究报告,由两部分组成。第一部分为非标记信号增强型荧光分子信标的构建研究。由于AMND可以特异性的与C碱基(胞嘧啶,Cytosine)结合。因此,本实验选择AMND作为荧光小分子。首先合成荧光杂环小分子AMND(2-氨基-7-甲基-1,8-二氮杂萘,2-amino-7-methyl-1,8-naphthyridine)并对其进行了结构表征。
之后,将合成出的荧光小分子作为信号物质用于构建非标记信号增强型荧光分子信标。利用两段单链DNA杂交形成一个发卡DNA结构,其特点在于发卡DNA的茎状部分形成了一个空缺位点,空缺位点对面是C碱基。荧光杂环小分子AMND通过氢键识别未配对的C碱基并嵌入空缺位点,AMND荧光发生淬灭。当向体系加入与分子信标环状部分互补的目标连时,分子信标环状部分打开,AMND与目标碱基结合的微环境发生变化,AMND被释放出,其荧光强度恢复表明目标链的存在。研究了影响荧光淬灭的因素,优化了实验条件。研究发现,当加入目标DNA链时,AMND荧光强度恢复,而加入含有错配碱基的目标链时,荧光恢复不明显,表明我们的非标记分子信标可以有效的区分完全互补链和单碱基错配。
第三部分是有关汞离子的检测。汞蒸气有高度的扩散性和较大的脂溶性,通过血液循环,进入人体器官,然后被氧化,在人体器官中累积起来,当人体汞离子浓度达到一定浓度时,就会出现各种症状,如头痛、肢体麻木,严重者出现肝炎、肾炎、尿血等症状。因此,对汞离子的检测显得尤为重要。近年来,许多学者报道了基于金纳米粒子比色法来检测汞离子。本实验基于单链DNA与金纳米粒子发生作用,当向体系加入一定浓度的NaCl时,由于单链DNA对金纳米粒子的保护作用,金纳米粒子不发生团聚；然后再加入汞离子,使得单链DNA与汞离子形成“T-Hg2+-T”结构,从而与金纳米粒子分离,金纳米粒子聚沉,颜色由酒红色变为蓝色,从而达到检测汞离子的目的。
本研究利用荧光小分子和双链中未配对碱基通过氢键结合并伴随着小分子荧光淬灭的原理设计了简单、非标记的分子信标,并将其用于目标单链DNA的检测。这一非标记分子信标能够很好地区分全匹配和单碱基错配DNA序列,因而这一非标记分子信标可以用来检测SNPs,将为SNPs分型方法的基础和应用研究提供新思路。此外,我们还研究了利用“T-Hg2+-T”结构的形成,通过比色法检测Hg2+。我们方法的特点在于使用了含T-T错配的双链DNA来稳定金纳米粒子,与通常的单链相比,我们的体系响应速度更快。
The sequence-specific detection of DNA hybridization has attracted considerable interest in a wide range of areas including molecular diagnostics, environmental monitoring, and antibioterrorism. Therefore, there is a continuing demand for sensitive and selective DNA probes. Many kinds of DNA probes have been developed in recent years through various molecular-engineering strategies to meet this demand. Amount these DNA probes, MBs have attract much attentions due in part to their stability, unique functionality and molecular specificity.
The conventional MBs are single-stranded oligonucleotide probes that possess a stem-and-loop structure. The loop portion of an MB is complementary to a target single-stranded DNA, while the stem is formed by 5 to 7 bp from two complementary arm sequences that are on either end of the MB. A fluorophore is attached to the end of one arm, while a quencher is attached to the end of the other arm. The stem maintains a close proximity of the two moieties, causing fluorescence to be quenched by fluorescence resonance energy transfer (FRET). When an MB hybridizes with its complementary DNA (cDNA), the MB undergoes a spontaneous conformational reorganization with the opening of the stem, leading to a fluorescence restoration. The unique target recognition and signal transduction capabilities of MBs have led to their application in many biochemical and biological assays including quantitative PCR, protein-DNA interactions, multiplex genetic analysis, and the detection of mRNA in living cells. As part of a general program aimed at developing free-labeled MBs, we want to investigate whether the non-covalently bind small fluorescence molecules can be a signal molecules for MBs.
The paper includes two parts, review and study.
The first chapter is part of the overview section to describe the principle of molecular beacons, application and research. Focuses on the structure of molecular beacons, we also introduced domestic and foreign non-labeled DNA testing technology, explained the background of this thesis, and to study the meaning, purpose and research content.
The second chapter is research report:First, we synthesit the fluorescent small molecules AMND (2-amino-7-methyl-1,8-naphthyridine) as it has been reported that AMND can bind to cytosine bases specifically.
And then, a free-labeled molecular beacon (MB) based on non-covalent binding of a fluorescence molecule,2-amino-7-methyl-1,8-naphthyridin-(AMND), to the intentional gap site in the stem moiety of a hairpin DNA has been developed. When the free-labeled MB was under a closed state, a gap site was formed in the stem moiety of a hairpin DNA. The fluorescence of the small fluorescence molecule was significantly quenched by binding to the unpaired base at the gap site through hydrogen bond, and thus the free-labeled MB shows almost no fluorescence. Upon hybridization with a complementary target ss-DNA (cDNA), the free-labeled MB undergoes a conformational change to take an open state, resulting in an effective fluorescence enhancement owing to a release of the small fluorescence molecules from the gap site. Fluorescence titration and circular dichroism (CD) spectra were used to demonstrate that the binding of AMND to cytosine base at gap site was strong and do not induce a significant conformational change of the hairpin DNA. Under the optimal conditions, the fluorescence intensity of the free-labeled MB increased with an increase of the concentration of cDNA and a detection limit of 9×10-11 M cDNA was achieved. Single mismatched target ss-DNA can be effectively discriminated from complementary target ss-DNA. There may be some advantages in utilizing this type of free-labeled MB over more-traditional ones. First, both ends of the free-labeled MB can be left free to introduce other useful functionalities. Second, our free-labeled MB synthesis is relatively simple and inexpensive because no label is required.
The third part is colorimetric detection method for Hg2+ ions based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing system. Mercury is a widespread pollutant with distinct toxicological profiles, and it exists in a variety of different forms (metallic, ionic, and as part of organic and inorganic salts and complexes). Solvated mercuric ion (Hg2+), one of the most stable inorganic forms of mercury, is a caustic and carcinogenic material with high cellular toxicity. Here, we report a simple and sensitive colorimetric detection method for Hg2+ions based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing system. Complementary DNA strands with T-T mismatches could effectively protect AuNPs from salt induced aggregation. While in the presence of Hg2+ions T-Hg2+-T coordination chemistry leads to the formation of DNA duplexes, and AuNPs are less well protected thus aggregate at the same salt concentration, accompanying by color change from red to blue. Employing duplex oligonucleotides with T-T mismatches in the sensing system, a sensitive linear range for Hg2+ ions from 0.05 to 2μM and a detection limit of 17 nM are obtained.
引文
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