Features of Determining Thermodynamic Parameters of Formation of Nucleic Acid Complexes Using Thermal Denaturation with Fluorimetric Signal Detection
In this work, the possibility of precise physicochemical analysis of formation of nucleic acid complexes has been demonstrated using thermal cyclers with fluorescence signal detection. Individual features of heating blocks of various thermal cyclers have been identified. A nonlinear correlation between fluorescence signal intensity and concentration of fluorescent dyes (5,6)-carboxyfluorescein and Rhodamine B has been identified. Using UV and fluorescence melting, the comparison of melting curves at various concentration of full-complementary and mismatched (A/A and C/A) complexes has been conducted. The experimental values of thermodynamic parameters of complex formation for full-complementary and mismatched complexes have been obtained.
Keywords:fluorimetry thermodynamic analysis melting temperature of DNA complexes oligonucleotides nucleic acid complexes
The authors are grateful to A.N. Sinyakov and V.A. Ryabinin, the Laboratory of Biomedical Chemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, for synthesis and isolation of fluorescently labeled oligonucleotides being part of the complexes DF, DFm, D20F, and D20Fm as well as the HF oligonucleotide.
This work was supported by the Russian Science Foundation (project no. 17-74-10157). The design of the model system of nucleic acid complexes was carried out with the support of the project of basic budget financing of the Program of Fundamental Science Research of the State Academies 2013–2020 no. 0245-2019-0002. The synthesis, isolation, and characterization of model oligonucleotides D and Dm were carried out by I.S. Dovydenko with the support of the Russian Foundation for Basic Research, project no. 18-29-08015.
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of Interest
The authors declare that they have no conflict of interest.
Statement of the Welfare of Animals
This article does not contain any studies involving animals or human participants performed by any of the authors.
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