Warm-start One-step RT-qPCR by Aptamer-blocking Tth DNA Polymerase Mutant

doi:10.13523/j.cb.2211014

China Biotechnology

2023, Vol. 43

Issue (4): 51-58 DOI: 10.13523/j.cb.2211014

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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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

Received: 08 November 2022 Published: 04 May 2023

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	Ya-qi ZHANG
	Jian ZHANG
	Yu-ting TANG
	Qing-yuan HUANG
	Lu JI
	Chen LU
	Zhi-dan LUO

Cite this article:

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.

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

Table 1 Nucleic acid adapter sequences

Fig.1 Amplification curves of each aptamer with Tth DNA polymerase mutant at 35°C for 1 h (a) TQ21 (b) TQ21-RNA (c) Trnc-21 (d) Trnc-21-RNA (e) TQ21-11 (f) TQ21-11-RNA (g) 6-10 (h) 6-10-RNA

Fig.2 Inhibition rate of DNA polymerase activity of Tth DNA polymerase mutant by each aptamer after reaction for 1 h at four temperatures (a) At 35℃ (b) At 40℃ (c) At 45℃ (d) At 50℃

Fig.3 Results of one-step RT-qPCR (a) (b) qPCR amplification curve after RT at 40℃ (c) (d) qPCR amplification curve after RT at 50℃ (e)(f) qPCR melting curve after RT at 50℃

Fig.4 Best molecular docking poses between each aptamer and Tth DNA polymerase mutant (a) TQ21 (b) TQ21-RNA (c) Trnc-21 (d) Trnc-21-RNA (e) TQ21-11 (f) TQ21-11-RNA (g) 6-10 (h) 6-10-RNA

Table 2 Best docking scores of each aptamer


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