Issue 13, 2019

Aptamer-induced thermofluorimetric protein stabilization and G-quadruplex nucleic acid staining by SYPRO orange dye

Abstract

SYPRO Orange™ is a commercial dye commonly employed in a thermofluorimetric analysis (TFA) to stain thermally-denatured proteins and obtain their melting temperature (Tm values). Herein, the ability of DNA–aptamer-based ligands to increase protein target melting temperature (Tm) was investigated and a correlation between protein target thermal stability (ΔTm) and DNA–aptamer protein binding affinity (Kd) was found. Also, a serendipitously discovered nucleic acid staining property of SYPRO Orange dye has been revealed while performing the TFA of thrombin. The dye specifically binds to G-quadruplex (GQ) forming nucleic acids, such as the 29-mer thrombin binding aptamer (HD22 or TBA29) and hemin binding aptamer (PS2.M) providing a ‘turn-on’ fluorescence response. Our studies demonstrate that protein stability is proportional to aptamer binding affinity in aptamer–protein complexes and establish the potential of SYPRO Orange for nucleic acid staining applications.

Graphical abstract: Aptamer-induced thermofluorimetric protein stabilization and G-quadruplex nucleic acid staining by SYPRO orange dye

Supplementary files

Article information

Article type
Letter
Submitted
11 Jan 2019
Accepted
27 Feb 2019
First published
05 Mar 2019

New J. Chem., 2019,43, 4994-4997

Aptamer-induced thermofluorimetric protein stabilization and G-quadruplex nucleic acid staining by SYPRO orange dye

P. S. Deore and R. A. Manderville, New J. Chem., 2019, 43, 4994 DOI: 10.1039/C9NJ00188C

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