Molecular Simulation-based Continuous Optimization of Nucleic-acid Aptamers Against Tetrodotoxin

doi:10.13523/j.cb.2205010

China Biotechnology

2022, Vol. 42

Issue (8): 1-12 DOI: 10.13523/j.cb.2205010

Molecular Simulation-based Continuous Optimization of Nucleic-acid Aptamers Against Tetrodotoxin

YAN Zhi-chao,SONG Meng-hua,LIU Jian-ping,HUANG Qiang^**(

)

School of Life Sciences, Fudan University, Shanghai 200438, China

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Abstract

Tetrodotoxin (TTX) is an alkaloid neurotoxin, and its poisoning cases occurr worldwide and therefore seriously threat human health. However, there is no specific antidote yet for TTX, so the detection of TTX is of great importance in the field of food safety. To obtain a more efficient TTX recognition element, guided by molecular simulations, a DNA aptamer (TTX-27) previously discovered by SELEX screening was continuously optimized. First, the stem-loop structure, which hinders the TTX binding, was replaced with a mini-hairpin structure to make the TTX bind more easily to the truncated aptamer; next, T39 and C40 bases were mutated to C and T bases, respectively, and C39 was also modified with 2'-OH to enhance the hydrogen bonding and van der Waals interactions of the bases with TTX. Microscale thermophoresis (MST) experiments confirmed that the affinities of the aptamer variants were increased by the truncation, base mutation and chemical modification. The dissociation equilibrium constant K_d of the binding of the chemically modified variant TTX-D2-X-R to TTX was 1.08 nmol/L, which increased 75.5 times compared to that of TTX-27. Thus, this study demonstrates that the molecular simulation-based truncation-mutation-chemical modification is an effective approach to the post-SELEX optimization of nucleic-acid aptamers, and the resulting aptamer variant TTX-D2-X-R has potential applications in the field of TTX detection.

Key words： Tetrodotoxin Aptamer Molecular simulation Base truncation Base mutation Chemical modification

Received: 05 May 2022 Published: 07 September 2022

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Corresponding Authors: Qiang HUANG E-mail: huangqiang@fudan.edu.cn

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	Zhi-chao YAN
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Cite this article:

YAN Zhi-chao,SONG Meng-hua,LIU Jian-ping,HUANG Qiang. Molecular Simulation-based Continuous Optimization of Nucleic-acid Aptamers Against Tetrodotoxin. China Biotechnology, 2022, 42(8): 1-12.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2205010 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I8/1

Fig.1 2D structure(a) and 3D structure(b) of TTX-27 Cyan: G4-C24 bases; Pink: Binding region bases; Earthy yellow: T47-C67 bases

Table 1 Sequences of nucleic acid aptamers.

Fig.2 MD RMSDs of the TTX-27∶TTX complex(a) and spontaneous association of TTX with TTX-27(b) Blue: G4-C24 bases; Yellow: T47-C67 bases; Green: The bases not directly associated with TTX binding; Red: The bases associated with TTX binding

Fig.3 Residue-based interactions of TTX-27 before and after the truncation optimization (a) TTX-27 (b) TTX-D2 Light: Van der Waals interaction probabilities of aptamer bases with TTX; Dark: Average H-bond numbers between TTX and the bases

Table 2 Docking binding free energies of TTX-27 variants

Fig.4 Analysis of MD and MST experiments for TTX-27 and its variants (a) RMSDs of the TTX-D2∶TTX complex (b) Time-dependent distances of TTX to TTX-27 and TTX-D2 binding regions (c) Comparison of binding free energies of TTX-27∶TTX and TTX-D2∶TTX (d) MST experiments for four nucleic acid aptamers. TTX-27∶ K_d =(81.52 ± 0.28) nmol/L; TTX-D2: K_d =(38.14 ± 0.23) nmol/L; TTX-D2-X: K_d =(12.36 ± 0.48) nmol/L; TTX-D2-X-R: K_d =(1.08 ± 0.64) nmol/L, determined by the average of 4 independent measurements, average ± S.E.

Table 3 Binding free energies of multiple rounds of mutations

Fig.5 Residue-based interactions of TTX-D2-X before and after chemical modification (a) TTX-D2-X (b) TTX-D2-X-R Light: Van der Waals interaction probability of aptamer bases with TTX; Dark: Average H-bond numbers between TTX and the bases

Table 4 The distances from the base deoxyribose C-2 of the binding region to TTX

Fig.6 Intermolecular interaction of TTX-27 with TTX before and after continuous optimization (a) TTX-27 (b) TTX-D2-X-R


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