Effects of Fluorescent Pair on the Kinetics of DNA Strand Displacement Reaction

Key Laboratory of Precision and Intelligent Chemistry, School of Chemistry and Materials Science, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, China

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Corresponding author: E-mail: xiaosy@ustc.edu.cn; hjliang@ustc.edu.cn

摘要

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Abstract: Fluorescent labels are widely used in the characterizations of DNA-based reaction network operations. We systematically studied the effects of commonly used fluorescent pairs on thermal stabilities of signal-substrate duplex and the strand displacement kinetics. It is demonstrated that the modifications of duplex with fluorescent pairs stabilize DNA duplex by up to 3.5 °C, and the kinetics of DNA strand displacement circuit is also evidently slowed down. These results highlight the importance of fluorescent pairs towards the kinetic modulation in designing nucleic acid probes and complex DNA dynamic circuits.
- DNA strand displacement /
- Fluorescent label /
- Kinetics /
- Thermodynamic property
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Figure 1. Schematic diagram of two-step DNA circuit based on TMSDR. The 5' end of signal strand (Sig) and the 3' end of substrate strand (S) are labelled with different fluorophore dyes and quenchers, respectively. The invading toehold domain a* (marked in red) has 5 bases. The solid arrows represent steric hindrance brought by labels and the dotted arrow represents the tendency of these fluorophore dyes and quenchers to bind to each other.

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Figure 2. Real-time fluorescent kinetic curves of the circuit in FIG. 1. (A) The 5' end of the signal strand (Sig) is labelled with fluorophore dye, while the 3' end of the substrate strand (S) is modified with the corresponding quencher. (B) The 3' end of the substrate strand (S) is labelled with fluorophore dye, while the 5' end of the signal strand (Sig) is modified with its corresponding quencher. The experimental conditions were T = 25 °C, [Invader] = 10 nmol/L, [S-Sig] = 10 nmol/L, [R-Q] = 20 nmol/L. Three parallel experiments were performed for each system.

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Figure 3. (A) Melting temperatures of signal-substrate duplexes with fluorophore dyes labelled at the 5' end of signal strand, and quenchers labelled at the 3' end of substrate strand. (B) Melting temperatures of signal-substrate duplexes with quenchers labelled at the 5' end of signal strand, and fluorophore dyes labelled at the 3' end of substrate strand. Error bars show the standard deviation on the mean. The concentrations of signal-substrate duplexes were 1 μmol/L. Three parallel experiments were performed for each system.

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Figure 4. (A) Free energy changes of dual-labelled signal-substrate duplexes with fluorophore dyes labelled at the 5' end of signal strand, and quenchers labelled at the 3' end of substrate strand. (B) Free energy changes of dual-labelled signal-substrate duplexes with quenchers labelled at the 5' end of signal strand, and fluorophore dyes labelled at the 3' end of substrate strand. The change of free energy caused by the fluorescent labels combination was calculated via $\Delta {\Delta }{G}_{T}^{0}=({{\Delta }{G}_{T}^{0})}_{\rm{labelled\ duplex}}-{\left({\Delta }{G}_{T}^{0}\right)}_{\rm{no\ label}}$. In all calculations, the temperature was chosen as T = 25 °C in line with the experimental temperature.

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