多重信号放大策略用于循环microRNA的检测研究
发布时间：2018-12-06 09:29
【摘要】：循环microRNA是目前一个相当活跃的研究领域,它对于探索生命现象、疾病发生机制、疾病诊断与预后、基因治疗、药物开发等方面都具有重大意义。然而传统的循环microRNA检测技术大多操作复杂且受灵敏度等方面的限制,往往不能满足考察循环microRNA生物功能的需要。环糊精聚合物不仅保持了环糊精的包络识别能力,且具有稳定性增强、溶解度增大、交联剂效应及多价结合效应等优点,我们在前期研究中发现p-环糊精聚合物对芘有较强的荧光增强作用,可与其它检测技术结合从而提高分析方法的灵敏度。基于此,本文基于p-环糊精聚合物对芘的荧光增强作用并结合工具酶的优点,发展了快速简便的高灵敏分析方法用于循环microRNA的检测,具体内容如下：1.基于p-环糊精聚合物对芘的荧光增强效果,设计了以DNA聚合酶与Lambda核酸外切酶为工具酶、3'端标芘且5'端磷酸化的分子信标为报告探针,实现了microRNA-21的高灵敏检测。在目标microRNA存在时,该体系依次发生两步反应：(1)DNA聚合酶辅助的等温链置换放大反应；(2) Lambda核酸外切酶辅助的循环酶切放大反应。两步反应的结果生成大量位于单核苷酸上的芘,并嵌入p-环糊精聚合物的空腔内,引起荧光信号的显著增强。反之,在没有目标microRNA存在时,位于分子信标上的芘由于大的空间位阻难以嵌入β-环糊精聚合物空腔内,因而荧光信号很弱。本方法中的分子信标不需要淬灭基团的参与,选择性和灵敏度均较好,对microRNA的检测限为0.3 pM。同时可以在人血清中进行检测,有望在临床检验等方面得到应用。2.基于β-环糊精聚合物对芘的荧光增强效果,设计了以DNA聚合酶、切刻内切酶及核酸外切酶Ⅲ为工具酶、中间标芘的直链DNA为报告探针,实现了目标DNA或microRNA的多重信号放大检测。在目标DNA或microRNA存在时,该体系依次发生两步反应：(1)切刻内切酶介导的等温链置换放大反应：(2)核酸外切酶Ⅲ辅助的循环酶切放大反应。反应生成位于单核苷酸上的芘就极易嵌入β-环糊精聚合物的空腔内,最终引起荧光信号的显著增强。反之,在没有目标DNA或microRNA存在时,位于直链DNA中间的芘由于大的空间位阻很难嵌入p-环糊精聚合物空腔内,因而荧光信号很弱。该方法探针设计简便,特异性好,灵敏度高,对DNA检测下限可达41 fM。同时该方法还用于人血清样品中microRNA检测,有望发展为一种通用的核酸检测平台。此外,本文还基于上述方法考察了水环境污染物对核酸外切酶Ⅲ活性的影响,该结论有望为核酸外切酶Ⅲ 抑制剂的筛选以及农药毒性评估提供理论依据和新的方法。
[Abstract]:Circulating microRNA is a very active research field at present. It is of great significance in exploring life phenomena, disease pathogenesis, disease diagnosis and prognosis, gene therapy, drug development and so on. However, most of the traditional cyclic microRNA detection techniques are complex and sensitive, and can not meet the needs of investigating the biological functions of circular microRNA. The cyclodextrin polymer not only keeps the envelope recognition ability of cyclodextrin, but also has the advantages of increasing stability, increasing solubility, crosslinking agent effect and multivalent binding effect, etc. In previous studies, we found that p-cyclodextrin polymer has a strong fluorescence enhancement effect on pyrene, which can be combined with other detection techniques to improve the sensitivity of analytical methods. Based on the fluorescence enhancement effect of p-cyclodextrin polymer on pyrene and the advantages of instrumental enzyme, a rapid and simple method for the detection of cyclic microRNA was developed in this paper. The main contents are as follows: 1. Based on the fluorescence enhancement effect of pcyclodextrin polymer on pyrene, the high sensitivity detection of microRNA-21 was realized by using DNA polymerase and Lambda nucleic acid exonuclease as tool enzyme and 3 'end labeled pyrene and 5' terminal phosphorylation molecular beacon as report probe. In the presence of target microRNA, the system had two steps in turn: (1) DNA polymerase assisted isothermal chain replacement amplification reaction; (2) Lambda nucleic acid exonuclease assisted circular enzyme digestion amplification reaction. The results of the two-step reaction resulted in a large amount of pyrene located on a single nucleotide, which was embedded in the cavity of the pcyclodextrin polymer, resulting in a significant enhancement of the fluorescence signal. On the other hand, when there is no target microRNA, the fluorescence signal of pyrene on the molecular beacon is very weak because the large steric hindrance is difficult to be embedded into the 尾 -cyclodextrin polymer cavity. The molecular beacons in this method do not require the participation of quenched groups. The selectivity and sensitivity of the beacons are good. The detection limit for microRNA is 0.3 pM.. At the same time, it can be detected in human serum, which is expected to be used in clinical examination. 2. 2. Based on the fluorescence enhancement effect of 尾 -cyclodextrin polymer on pyrene, DNA polymerase, endonuclease and nucleic acid exonuclease 鈪，
本文编号：2365822
[Abstract]:Circulating microRNA is a very active research field at present. It is of great significance in exploring life phenomena, disease pathogenesis, disease diagnosis and prognosis, gene therapy, drug development and so on. However, most of the traditional cyclic microRNA detection techniques are complex and sensitive, and can not meet the needs of investigating the biological functions of circular microRNA. The cyclodextrin polymer not only keeps the envelope recognition ability of cyclodextrin, but also has the advantages of increasing stability, increasing solubility, crosslinking agent effect and multivalent binding effect, etc. In previous studies, we found that p-cyclodextrin polymer has a strong fluorescence enhancement effect on pyrene, which can be combined with other detection techniques to improve the sensitivity of analytical methods. Based on the fluorescence enhancement effect of p-cyclodextrin polymer on pyrene and the advantages of instrumental enzyme, a rapid and simple method for the detection of cyclic microRNA was developed in this paper. The main contents are as follows: 1. Based on the fluorescence enhancement effect of pcyclodextrin polymer on pyrene, the high sensitivity detection of microRNA-21 was realized by using DNA polymerase and Lambda nucleic acid exonuclease as tool enzyme and 3 'end labeled pyrene and 5' terminal phosphorylation molecular beacon as report probe. In the presence of target microRNA, the system had two steps in turn: (1) DNA polymerase assisted isothermal chain replacement amplification reaction; (2) Lambda nucleic acid exonuclease assisted circular enzyme digestion amplification reaction. The results of the two-step reaction resulted in a large amount of pyrene located on a single nucleotide, which was embedded in the cavity of the pcyclodextrin polymer, resulting in a significant enhancement of the fluorescence signal. On the other hand, when there is no target microRNA, the fluorescence signal of pyrene on the molecular beacon is very weak because the large steric hindrance is difficult to be embedded into the 尾 -cyclodextrin polymer cavity. The molecular beacons in this method do not require the participation of quenched groups. The selectivity and sensitivity of the beacons are good. The detection limit for microRNA is 0.3 pM.. At the same time, it can be detected in human serum, which is expected to be used in clinical examination. 2. 2. Based on the fluorescence enhancement effect of 尾 -cyclodextrin polymer on pyrene, DNA polymerase, endonuclease and nucleic acid exonuclease 鈪，
本文编号：2365822
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