核酸探针技术用于mRNA检测的研究
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摘要
实时获取RNA合成、转运、表达、定位的信息对分子生物学、病理生理学、药物的开发及疾病的诊断等研究都具有重要的推动作用。核酸荧光分子探针为RNA研究提供了一种强有力的工具,已为我们提供了大量有关RNA的基本信息。但是,如何进一步拓展已有核酸分子探针在生命科学与医学中的应用范围,如何进一步改进或研制新的核酸分子探针来解决生命研究过程中遇到的新问题,如何实时、动态、灵敏、准确地获取生命活动相关信息,仍然是分析化学工作者所面临的重大挑战。
本论文瞄准上述挑战进行研究,结合分子信标和相邻探针技术建立了一系列检测mRNA的新方法,主要内容包括:
1.建立了一种直接以mRNA为模板,基于Reverse分子信标检测单碱基突变的新方法。本文以重要的肿瘤相关基因p53为检测对象,利用T4 DNA连接酶的高特异性、以及RNase H对RNA/DNA杂交体中RNA链的降解作用,结合Reverse分子信标技术,发展了一种快速检测靶基因mRNA单碱基突变的新方法。该方法通过比较分析RNase H作用前后样品中荧光信号的变化,确定是否存在单碱基突变。这种方法探针设计简单,避免了反转录、PCR扩增、电泳分离等步骤,检测结果准确,已应用于细胞总RNA提取物中目标基因的单碱基错配检测。
2.设计合成了超猝灭分子信标。提高分子信标的猝灭效率是优化分子信标检测性能,提高其检测灵敏度的有效途径之一。本文设计、合成、纯化了常规分子信标和超淬灭分子信标,利用基质辅助激光解吸电离飞行时间质谱对产物进行解析,证明其为目标产物。与相同序列的常规分子信标相比,超淬灭分子信标的信背比可达到112,比常规分子信标大约高5倍,信背比的提高主要是通过降低背景信号达到的。研究还表明,与荧光基团相邻的末端为C碱基,或者在探针末端引入与目标互补的序列,也可以提高超猝灭分子信标的检测灵敏度。
3.建立了基于硫代修饰相邻探针直接检测细胞裂解液中mRNA的新方法。常规相邻探针用于生物样品中核酸检测时,有可能被核酸酶降解而产生假阴性信号。本文设计了一种硫代修饰的相邻探针,并用于细胞裂解液中相关mRNA的检测。结果表明,硫代修饰探针可以有效抵御核酸酶的降解作用,降低假阳性信号及假阴性信号。探针设计简单、灵敏度高、与靶分子作用速度快;检测过程中信号呈逐渐增强模式、可免除洗脱及分离等步骤。
4.建立了基于分子信标荧光增强速率检测活细胞内mRNA表达的新方法。常规分子信标在活细胞内mRNA表达水平的研究中,由于细胞内核酸酶对分子信标骨架的降解和破坏作用,常导致假阳性信号的产生,对检测结果的准确性影响较大。本文根据肿瘤抑制基因p21序列设计合成分子信标,通过显微操作将其注入p21表达水平存在差异的两种鼻咽癌细胞内,以活细胞成像方式动态检测了分子信标进入鼻咽癌细胞后荧光信号的变化。p21分子信标注入两种细胞后,荧光信号均逐渐增强,约15 min后达到最大值;注入细胞后4 min内,两种细胞间荧光增强速率存在明显差异,该差异反映了p21 mRNA表达水平的变化趋势,与常规逆转录PCR (RT-PCR)结果相符。在使用分子信标进行活细胞内mRNA表达水平的研究中,通过分析荧光增强速率的变化,可有效降低细胞内的酶的影响,提高检测结果的准确性。
5.建立了基于硫代修饰分子信标检测活细胞内mRNA表达的新方法。本文针对分子信标在活细胞检测中所面临的生物稳定性的关键问题,用可抵御核酸酶切的硫代修饰的DNA设计合成分子信标探针,提高了细胞内检测结果的准确性,同时还可保持常规分子信标高选择性、高灵敏度以及无需对多余探针洗脱的优点。该分子信标已应用于转染GFP基因前后活细胞内靶GFP mRNA的实时检测。
The essential information on RNA synthesis, processing, transport, and localization obtained in real time will offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery, and medical diagnostics. As a powerful tool, nucleic acid probes have been utilized in RNA study and explored wealth of essential information of RNA. However,how to further extend the applications of nucleic acid probes in biology and medicine research, how to develop novel nucleic acid probes to resolve new problems, how to get the dynamic data of these life processes sensitively and accurately in real time are still great challenges to analysts.
In this dissertation, a series of mRNA detection methods based on molecular beacon and adjacent probe have been developed. The main researches of this dissertation are summarized as follows.
1. RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon. A novel RNA-templated single-base mutation detection method was developed and applied to identification of single-base mutation in codon 273 of the p53 gene taking advantage of the high specificity of T4 DNA ligase and the capability of RNase H to digest RNA in RNA/DNA complex. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as ease of design of detection probes; direct discrimination of single-base mismatch of the RNA extracted from cells; no requirement of PCR amplification; and performance of homogeneous detection.
2. Design and synthesis of the superquencher molecular beacon. Improving the quenching efficiency is one of the useful strategies to improve the signal-to-background ratio and to optimize the sensitivity of the molecular beacon. In this work, normal molecular beacon and superquencher molecular beacon were synthesized and purified using the DNA autosynthesizer and liquid chromatogram.
The products were identified by MALDI-TOF-MS. Our results showed that, the signal-to-background ratio of superquencher molecular beacon was 112, which is 5 times higher than that of normal molecular beacon with the same sequence. The significantly increased signal-to-background ratio of the superquencher molecular beacon was due to the decreasing of the background signal of the probe. Choosing cytidine as the final nucleotide base next to the fluorophore, or let one arm of molecular beacon be complementary to the target can further increase the signal-to-background ratio of the superquencher molecular beacon.
3. Detection of nucleic acid in cell lysate using phosphorothioate-modified adjacent probe. In order to eliminate the false-negative signals resulting from nuclease degradation of the convenient adjacent probe in biological sample analysis, phosphorothioate modified adjacent probe was designed and synthesized for mRNA detection in cell lysate. Results showed that phosphorothioate modified adjacent probe can efficiently reduce the false-positive and false-negative signals resulting from nuclease degradation, thus, improving the accuracy in mRNA detection in biological samples. Phosphorothioate modified adjacent probes are easy to design, very sensitive and selective in monitoring nucleic acid targets, easy to hybrid to the target sequence, and allow rapid detection in complex sample matrix. As the light-up signaling approach is used to detect the presence of target sequence, removing of unbound probes are not necessary and tedious washing and separating procedures are avoided.
4. Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate. The normal molecular beacons tend to generate false positive signals due to nuclease degradation, when used for intracellular mRNA expression level analysis. A specific molecular beacon (p21 MB) for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after injecting the p21 MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell. In both cases, the fluorescence intensities achieved maximum in about 15 minute. However, the fluorescence increasing rate within 4 minutes right after microinjection was significantly different between these two cell lines, indicating different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the nuclease degradation and improve the accuracy in living cell mRNA detection.
5. mRNA detection in living cell using phosphorothioate-modified molecular beacon. In order to resolve the intracellular biostability problem of molecular beacons, phosphorothioate modified DNA, which can efficiently improve the nuclease resistance of the backbone of the DNA, were chosen to design and synthesize molecular beacons. Phosphorothioate modified molecular beacon can efficiently improve the accuracy in living cell mRNA detection while keeping the advantages of molecular beacons, such as excellent selectivity, high sensitivity, and without-separation detection, and has been applied in real time detection of GFP mRNA in COS-7 cell and GFP tansfected COS-7 cell.
本论文瞄准上述挑战进行研究,结合分子信标和相邻探针技术建立了一系列检测mRNA的新方法,主要内容包括:
1.建立了一种直接以mRNA为模板,基于Reverse分子信标检测单碱基突变的新方法。本文以重要的肿瘤相关基因p53为检测对象,利用T4 DNA连接酶的高特异性、以及RNase H对RNA/DNA杂交体中RNA链的降解作用,结合Reverse分子信标技术,发展了一种快速检测靶基因mRNA单碱基突变的新方法。该方法通过比较分析RNase H作用前后样品中荧光信号的变化,确定是否存在单碱基突变。这种方法探针设计简单,避免了反转录、PCR扩增、电泳分离等步骤,检测结果准确,已应用于细胞总RNA提取物中目标基因的单碱基错配检测。
2.设计合成了超猝灭分子信标。提高分子信标的猝灭效率是优化分子信标检测性能,提高其检测灵敏度的有效途径之一。本文设计、合成、纯化了常规分子信标和超淬灭分子信标,利用基质辅助激光解吸电离飞行时间质谱对产物进行解析,证明其为目标产物。与相同序列的常规分子信标相比,超淬灭分子信标的信背比可达到112,比常规分子信标大约高5倍,信背比的提高主要是通过降低背景信号达到的。研究还表明,与荧光基团相邻的末端为C碱基,或者在探针末端引入与目标互补的序列,也可以提高超猝灭分子信标的检测灵敏度。
3.建立了基于硫代修饰相邻探针直接检测细胞裂解液中mRNA的新方法。常规相邻探针用于生物样品中核酸检测时,有可能被核酸酶降解而产生假阴性信号。本文设计了一种硫代修饰的相邻探针,并用于细胞裂解液中相关mRNA的检测。结果表明,硫代修饰探针可以有效抵御核酸酶的降解作用,降低假阳性信号及假阴性信号。探针设计简单、灵敏度高、与靶分子作用速度快;检测过程中信号呈逐渐增强模式、可免除洗脱及分离等步骤。
4.建立了基于分子信标荧光增强速率检测活细胞内mRNA表达的新方法。常规分子信标在活细胞内mRNA表达水平的研究中,由于细胞内核酸酶对分子信标骨架的降解和破坏作用,常导致假阳性信号的产生,对检测结果的准确性影响较大。本文根据肿瘤抑制基因p21序列设计合成分子信标,通过显微操作将其注入p21表达水平存在差异的两种鼻咽癌细胞内,以活细胞成像方式动态检测了分子信标进入鼻咽癌细胞后荧光信号的变化。p21分子信标注入两种细胞后,荧光信号均逐渐增强,约15 min后达到最大值;注入细胞后4 min内,两种细胞间荧光增强速率存在明显差异,该差异反映了p21 mRNA表达水平的变化趋势,与常规逆转录PCR (RT-PCR)结果相符。在使用分子信标进行活细胞内mRNA表达水平的研究中,通过分析荧光增强速率的变化,可有效降低细胞内的酶的影响,提高检测结果的准确性。
5.建立了基于硫代修饰分子信标检测活细胞内mRNA表达的新方法。本文针对分子信标在活细胞检测中所面临的生物稳定性的关键问题,用可抵御核酸酶切的硫代修饰的DNA设计合成分子信标探针,提高了细胞内检测结果的准确性,同时还可保持常规分子信标高选择性、高灵敏度以及无需对多余探针洗脱的优点。该分子信标已应用于转染GFP基因前后活细胞内靶GFP mRNA的实时检测。
The essential information on RNA synthesis, processing, transport, and localization obtained in real time will offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery, and medical diagnostics. As a powerful tool, nucleic acid probes have been utilized in RNA study and explored wealth of essential information of RNA. However,how to further extend the applications of nucleic acid probes in biology and medicine research, how to develop novel nucleic acid probes to resolve new problems, how to get the dynamic data of these life processes sensitively and accurately in real time are still great challenges to analysts.
In this dissertation, a series of mRNA detection methods based on molecular beacon and adjacent probe have been developed. The main researches of this dissertation are summarized as follows.
1. RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon. A novel RNA-templated single-base mutation detection method was developed and applied to identification of single-base mutation in codon 273 of the p53 gene taking advantage of the high specificity of T4 DNA ligase and the capability of RNase H to digest RNA in RNA/DNA complex. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as ease of design of detection probes; direct discrimination of single-base mismatch of the RNA extracted from cells; no requirement of PCR amplification; and performance of homogeneous detection.
2. Design and synthesis of the superquencher molecular beacon. Improving the quenching efficiency is one of the useful strategies to improve the signal-to-background ratio and to optimize the sensitivity of the molecular beacon. In this work, normal molecular beacon and superquencher molecular beacon were synthesized and purified using the DNA autosynthesizer and liquid chromatogram.
The products were identified by MALDI-TOF-MS. Our results showed that, the signal-to-background ratio of superquencher molecular beacon was 112, which is 5 times higher than that of normal molecular beacon with the same sequence. The significantly increased signal-to-background ratio of the superquencher molecular beacon was due to the decreasing of the background signal of the probe. Choosing cytidine as the final nucleotide base next to the fluorophore, or let one arm of molecular beacon be complementary to the target can further increase the signal-to-background ratio of the superquencher molecular beacon.
3. Detection of nucleic acid in cell lysate using phosphorothioate-modified adjacent probe. In order to eliminate the false-negative signals resulting from nuclease degradation of the convenient adjacent probe in biological sample analysis, phosphorothioate modified adjacent probe was designed and synthesized for mRNA detection in cell lysate. Results showed that phosphorothioate modified adjacent probe can efficiently reduce the false-positive and false-negative signals resulting from nuclease degradation, thus, improving the accuracy in mRNA detection in biological samples. Phosphorothioate modified adjacent probes are easy to design, very sensitive and selective in monitoring nucleic acid targets, easy to hybrid to the target sequence, and allow rapid detection in complex sample matrix. As the light-up signaling approach is used to detect the presence of target sequence, removing of unbound probes are not necessary and tedious washing and separating procedures are avoided.
4. Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate. The normal molecular beacons tend to generate false positive signals due to nuclease degradation, when used for intracellular mRNA expression level analysis. A specific molecular beacon (p21 MB) for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after injecting the p21 MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell. In both cases, the fluorescence intensities achieved maximum in about 15 minute. However, the fluorescence increasing rate within 4 minutes right after microinjection was significantly different between these two cell lines, indicating different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the nuclease degradation and improve the accuracy in living cell mRNA detection.
5. mRNA detection in living cell using phosphorothioate-modified molecular beacon. In order to resolve the intracellular biostability problem of molecular beacons, phosphorothioate modified DNA, which can efficiently improve the nuclease resistance of the backbone of the DNA, were chosen to design and synthesize molecular beacons. Phosphorothioate modified molecular beacon can efficiently improve the accuracy in living cell mRNA detection while keeping the advantages of molecular beacons, such as excellent selectivity, high sensitivity, and without-separation detection, and has been applied in real time detection of GFP mRNA in COS-7 cell and GFP tansfected COS-7 cell.
引文
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