利用核酸酶結合滾輪式擴增法開發針對具子癲前症特異性的微型核糖核酸之恆溫篩選平台

A Highly Specific Isothermal Screening Platform for Pre-eclampsia-associated microRNA Based on Duplex Specific Nuclease and Rolling Circle Amplification

彭宜萱(Yi-Shuang Peng)

指導教授 ： 何佳安

國立臺灣大學/生命科學院/生化科技學系/碩士(2020年)

https://doi.org/10.6342/NTU202004312

摘要

子癲前症 (pre-eclampsia) 是一種以高血壓為特徵的妊娠相關疾病，根據世界衛生組織 (World Health Organization WHO) 統計約有3~8% 的孕產婦受到子癲前症的影響而造成母體損傷及胎兒發育不完全，並且在中低收入的國家中，子癲前症更是導致胎兒早產及死亡的主要原因之一。雖然子癲前症的患者大多在孕期20週後才出現不適的症狀，但許多研究也指出子癲前症可能在懷孕的初期就已發生，因此開發能夠早期偵測並診斷子癲前症的檢測方法是必要的。在本實驗中，我們選擇 miR-210 作為偵測目標並且結合雙股專一性核酸酶 (duplex specific nuclease, DSN)以及核酸增幅技術 (DNA amplification method) 並以96孔盤作為反應平台設計一種恆溫且類酵素連結免疫分析法 (Enzyme-linked immunoassay)的檢測方法。首先，目標 miRNA會被修飾在96孔盤底部的髮夾狀探針 (Hairpin probe) 辨識形成RNA-DNA雙股，接著透過DSN的專一性作用來產生附著於盤底的單股DNA，而此DNA可作為引子進而啟動下一階段的滾環式擴增法 (Rolling circle amplification, RCA) 使訊號得以被放大。在此RCA的反應中，核酸聚合酶可利用環狀探針 ( circular probe) 為模板合成出G-四聯體 (G-quadruplex) ，並且在加入氯化血紅素 (Hemin) 時形成具有辣根過氧化物酶 (Horseradish peroxidase, HRP) 活性的 DNAzyme。當環境中有3,3',5,5' –四甲基聯苯 (3,3',5,5'-Tetramethyl benzidine, TMB) 時，具HRP 活性的DNAzyme 便可催化過氧化氫的還原，進而使溶液由透明轉為藍色，透過分析顏色變化便可計算出樣品中 miR-210的含量。利用此設計，我們期望針對 miR-210開發出一套恆溫、高專一性、高靈敏性且操作門檻較低並有潛力診斷子癲前症的檢測平台。

關鍵字

子癲前症 ； miR-210 ； 恆溫檢測平台 ； 雙股專一性核酸酶 ； 滾環式擴增法 ； G-四聯體 ； 去氧核醣核酸酶 ； 3,3',5,5' –四甲基聯苯

並列摘要

Pre-eclampsia is a pregnancy-related disease characterized by high blood pressure, affecting 3-8% of pregnancies. It is also one of the leading causes to higher risks of maternal damage and fetal growth restriction. In low- and middle-income countries, pre-eclampsia is a common cause of preterm delivery and fetal death. The symptoms of pre-eclampsia are often apparent at late second to early third trimester, but the abnormal interaction occurred much earlier in pregnancy. Therefore, a early detection of pre-eclampsia is an urgent need. We herein choose miR-210 as a target, designed an Enzyme-linked immunoassay (ELISA)-like detection assay coupled with isothermal signal amplification methods that combined duplex specific nuclease (DSN) specific miRNA cleavage and nucleic acid amplification. We first modified the hairpin probe on the wells of ELISA microplate. It was followed by the addition of the sample containing miR-210. After proper mixing, the hybridization occurred between the hairpin probe and the target to form RNA-DNA duplex, which can be further recognized by DSN. After the specific cleavage with DSN, a residual hairpin probe remained attached to the bottom of the well them served as the primer for subsequent rolling circle amplification (RCA). In the RCA, the G-quadruplex was generated via nucleic acid polymerase using a circular probe as a template. Horseradish peroxidase (HRP)-mimicking DNAzyme was formed, upon addition of hemin. With the presence of 3,3’,5,5’-Tetramethyl benzidine (TMB), Hemin/G-quadruplex HRP-mimicking DNAzyme catalyzed the reduction of hydrogen peroxide, leading to the colorimetric detection of miRNA. This sensing platform was anticipated to achieve a sensitive identification of miR-210 in blood samples collected from patients with pre-eclampsia.

並列關鍵字

Pre-eclampsia ； miR-210 ； Isothermal amplification ； Duplex specific nuclease (DSN) ； RCA ； G-quadruplex ； DNAzyme ； 3,3’,5,5’-Tetramethyl benzidine

參考文獻

1. Brown, M. A.; Magee, L. A.; Kenny, L. C.; Karumanchi, S. A.; McCarthy, F. P.; Saito, S.; Hall, D. R.; Warren, C. E.; Adoyi, G.; Ishaku, S.; Int Soc Study Hypertension, P., The hypertensive disorders of pregnancy: ISSHP classification, diagnosis management recommendations for international practice. Pregnancy Hypertension-an International Journal of Womens Cardiovascular Health 2018, 13, 291-310.

2. Duley, L., The Global Impact of Pre-eclampsia and Eclampsia. Semin. Perinatol. 2009, 33 (3), 130-137.

3. Bellamy, L.; Casas, J.-P.; Hingorani, A. D.; Williams, D. J., Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis. BMJ 2007, 335 (7627), 974.

4. McDonald, S. D.; Malinowski, A.; Zhou, Q.; Yusuf, S.; Devereaux, P. J., Cardiovascular sequelae of preeclampsia/eclampsia: a systematic review and meta-analyses. Am. Heart J. 2008, 156 (5), 918-930.

5. Brown, M. C.; Best, K. E.; Pearce, M. S.; Waugh, J.; Robson, S. C.; Bell, R., Cardiovascular disease risk in women with pre-eclampsia: systematic review and meta-analysis. Eur. J. Epidemiol. 2013, 28 (1), 1-19.