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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (4): 51-58    DOI: 10.13523/j.cb.2211014
技术与方法     
利用核酸适配体封闭Tth DNA聚合酶突变体实现温启动一步法RT-qPCR*
张雅琪,张建,唐雨婷,黄庆媛,季璐,卢辰,罗志丹()
江苏海洋大学江苏省海洋生物资源与环境重点实验室 江苏省海洋生物产业技术协同创新中心 连云港 222005
Warm-start One-step RT-qPCR by Aptamer-blocking Tth DNA Polymerase Mutant
ZHANG Ya-qi,ZHANG Jian,TANG Yu-ting,HUANG Qing-yuan,JI Lu,LU Chen,LUO Zhi-dan()
Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005, China
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摘要：

目的:Tth DNA聚合酶是一种同时具备DNA聚合酶和逆转录酶活性的耐热型工具酶。为消除其在一步法逆转录-实时荧光定量PCR(reverse transcription-quantitative real-time PCR,RT-qPCR)反应时的非特异性扩增,筛选可在较低温度下抑制其活性的核酸适配体,从而实现温启动一步法RT-qPCR。方法:在前期获得不依赖Mn2+的Tth DNA聚合酶突变体的基础上,检验四种DNA适配体及其对应的RNA适配体对Tth DNA聚合酶突变体的DNA聚合酶活性和逆转录酶活性的封闭效果以及解离温度,并通过分子对接模拟适配体与聚合酶的结合模式。结果:TQ21-11和TQ21-11-RNA两种适配体在40℃可有效抑制Tth DNA聚合酶的DNA聚合酶活性和逆转录酶活性,抑制率达到97%以上;两种适配体在50℃可几乎完全解离。这两种适配体均可用于温启动一步法RT-qPCR,其中TQ21-11-RNA的非特异性扩增更低。结论:利用TQ21-11和TQ21-11-RNA两种适配体在室温下封闭Tth DNA聚合酶突变体活性,可以实现温启动一步法RT-qPCR。
关键词： 核酸适配体;  Tth;  DNA聚合酶突变体;  温启动;  一步法RT-qPCR    
Abstract:

Objective: Tth DNA polymerase (Tth pol) is a thermostable polymerase with both DNA polymerase and reverse transcriptase activities. To control the non-specific amplification in the one-step reverse transcription-quantitative real-time PCR (RT-qPCR), the aptamers that inhibit the activity of Tth pol mutant at moderate temperature were screened to perform warm-start one-step RT-qPCR. Methods: Based on the Mn2+-independent Tth pol mutant obtained in previous study, the effects of four DNA aptamers and their corresponding RNA aptamers on the inhibition of DNA polymerase and reverse transcriptase activity of this Tth pol mutant and the dissociation temperature were examined. The binding poses between these aptamers and Tth pol mutant were simulated by molecular docking. Results: Two aptamers TQ21-11 and TQ21-11-RNA can block two distinct activities of Tth pol at 40℃ with inhibition rates of over 97% and all of them can be almost completely dissociated at 50℃. Both aptamers were validated for warm-start one-step RT-qPCR, with lower non-specific amplification of TQ21-11-RNA. Conclusion: The aptamers TQ21-11 and TQ21-11-RNA were able to effectively block the activity of Tth pol mutant at moderate temperature and perform warm-start one-step RT-qPCR well.
Key words: Aptamer    Tth    DNA polymerase mutant    Warm-start    One-step RT-qPCR
收稿日期: 2022-11-08 出版日期: 2023-05-04
ZTFLH:  Q78  
通讯作者: **电子信箱:lzd@jou.edu.cn   
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张雅琪
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黄庆媛
季璐
卢辰
罗志丹

引用本文:

张雅琪, 张建, 唐雨婷, 黄庆媛, 季璐, 卢辰, 罗志丹. 利用核酸适配体封闭Tth DNA聚合酶突变体实现温启动一步法RT-qPCR*[J]. 中国生物工程杂志, 2023, 43(4): 51-58.

ZHANG Ya-qi, ZHANG Jian, TANG Yu-ting, HUANG Qing-yuan, JI Lu, LU Chen, LUO Zhi-dan. Warm-start One-step RT-qPCR by Aptamer-blocking Tth DNA Polymerase Mutant. China Biotechnology, 2023, 43(4): 51-58.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2211014    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I4/51

名称 序列(5'-3') 长度/nt 参考文献
TQ21 TTCTCGGTTGGTCTCTGGCGGAGCGATCATCTCAGAGCATTCTTAGCGTTTTGTTCTTGTGTAT
GATTCGCTTTTCCC		 78 [12]
TQ21-RNA UUCUCGGUUGGUCUCUGGCGGAGCGAUCAUCUCAGAGCAUUCUUAGCGUUUUGUUCUUGU
GUAUGAUUCGCUUUUCCC		 78 -
Trnc-21 TGGCGGAGCGATCATCTCAGAGCATTCTTAGCGTTTTGTTCTTGTGTATGA 51 [15]
Trnc-21-RNA UGGCGGAGCGAUCAUCUCAGAGCAUUCUUAGCGUUUUGUUCUUGUGUAUGA 51 -
TQ21-11 GCGGTCGGCTCGGGGCATTCTTAGCGTTTTGCCCCGAGCCGACCGC 46 [16]
TQ21-11-RNA GCGGUCGGCUCGGGGCAUUCUUAGCGUUUUGCCCCGAGCCGACCGC 46 -
6-10 CAAGACGGGCGGGTGTGGTAGGCGCCCGTG 30 [17]
6-10-RNA CAAGACGGGCGGGUGUGGUAGGCGCCCGUG 30 -
表1  所用核酸适配体序列
图1  各适配体与Tth DNA聚合酶突变体在35℃反应1 h的扩增曲线
图2  各适配体在四种温度下反应1 h后对Tth DNA聚合酶突变体DNA聚合酶活性的抑制率
图3  一步法逆转录-实时荧光定量PCR扩增结果
图4  各适配体与Tth DNA聚合酶突变体分子对接结果最优姿态
适配体名称 HDock最佳对接分数
TQ21 -313.34
TQ21-RNA -367.85
Trnc-21 -289.71
Trnc-21-RNA -340.16
TQ21-11 -299.94
TQ21-11-RNA -362.13
6-10 -339.72
6-10-RNA -344.27
表2  各适配体最佳对接分数
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