多重信號放大策略用于循環(huán)microRNA的檢測研究
發(fā)布時間：2018-12-06 09:29
【摘要】：循環(huán)microRNA是目前一個相當活躍的研究領(lǐng)域,它對于探索生命現(xiàn)象、疾病發(fā)生機制、疾病診斷與預(yù)后、基因治療、藥物開發(fā)等方面都具有重大意義。然而傳統(tǒng)的循環(huán)microRNA檢測技術(shù)大多操作復(fù)雜且受靈敏度等方面的限制,往往不能滿足考察循環(huán)microRNA生物功能的需要。環(huán)糊精聚合物不僅保持了環(huán)糊精的包絡(luò)識別能力,且具有穩(wěn)定性增強、溶解度增大、交聯(lián)劑效應(yīng)及多價結(jié)合效應(yīng)等優(yōu)點,我們在前期研究中發(fā)現(xiàn)p-環(huán)糊精聚合物對芘有較強的熒光增強作用,可與其它檢測技術(shù)結(jié)合從而提高分析方法的靈敏度�；诖�,本文基于p-環(huán)糊精聚合物對芘的熒光增強作用并結(jié)合工具酶的優(yōu)點,發(fā)展了快速簡便的高靈敏分析方法用于循環(huán)microRNA的檢測,具體內(nèi)容如下：1.基于p-環(huán)糊精聚合物對芘的熒光增強效果,設(shè)計了以DNA聚合酶與Lambda核酸外切酶為工具酶、3'端標芘且5'端磷酸化的分子信標為報告探針,實現(xiàn)了microRNA-21的高靈敏檢測。在目標microRNA存在時,該體系依次發(fā)生兩步反應(yīng)：(1)DNA聚合酶輔助的等溫鏈置換放大反應(yīng)；(2) Lambda核酸外切酶輔助的循環(huán)酶切放大反應(yīng)。兩步反應(yīng)的結(jié)果生成大量位于單核苷酸上的芘,并嵌入p-環(huán)糊精聚合物的空腔內(nèi),引起熒光信號的顯著增強。反之,在沒有目標microRNA存在時,位于分子信標上的芘由于大的空間位阻難以嵌入β-環(huán)糊精聚合物空腔內(nèi),因而熒光信號很弱。本方法中的分子信標不需要淬滅基團的參與,選擇性和靈敏度均較好,對microRNA的檢測限為0.3 pM。同時可以在人血清中進行檢測,有望在臨床檢驗等方面得到應(yīng)用。2.基于β-環(huán)糊精聚合物對芘的熒光增強效果,設(shè)計了以DNA聚合酶、切刻內(nèi)切酶及核酸外切酶Ⅲ為工具酶、中間標芘的直鏈DNA為報告探針,實現(xiàn)了目標DNA或microRNA的多重信號放大檢測。在目標DNA或microRNA存在時,該體系依次發(fā)生兩步反應(yīng)：(1)切刻內(nèi)切酶介導(dǎo)的等溫鏈置換放大反應(yīng)：(2)核酸外切酶Ⅲ輔助的循環(huán)酶切放大反應(yīng)。反應(yīng)生成位于單核苷酸上的芘就極易嵌入β-環(huán)糊精聚合物的空腔內(nèi),最終引起熒光信號的顯著增強。反之,在沒有目標DNA或microRNA存在時,位于直鏈DNA中間的芘由于大的空間位阻很難嵌入p-環(huán)糊精聚合物空腔內(nèi),因而熒光信號很弱。該方法探針設(shè)計簡便,特異性好,靈敏度高,對DNA檢測下限可達41 fM。同時該方法還用于人血清樣品中microRNA檢測,有望發(fā)展為一種通用的核酸檢測平臺。此外,本文還基于上述方法考察了水環(huán)境污染物對核酸外切酶Ⅲ活性的影響,該結(jié)論有望為核酸外切酶Ⅲ 抑制劑的篩選以及農(nóng)藥毒性評估提供理論依據(jù)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，
本文編號：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，
本文編號：2365822
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