Amino Acids

, Volume 47, Issue 4, pp 729–734 | Cite as

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid

Original Article

Abstract

Protein–nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein–DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein–DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.

Keywords

Protein–DNA interaction Unnatural amino acids Fluorescence resonance energy transfer 

Notes

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2014003870), and the Sogang University Research Grant (201010048.01). We would like to thank P. G. Schultz for providing us the plasmids.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2014_1900_MOESM1_ESM.docx (67 kb)
Supplementary material 1 (DOCX 67 kb)

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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  1. 1.Department of ChemistrySogang UniversitySeoulRepublic of Korea

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