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 (T_m values). Herein, the ability of DNA–aptamer-based ligands to increase protein target melting temperature (T_m) was investigated and a correlation between protein target thermal stability (ΔT_m) and DNA–aptamer protein binding affinity (K_d) 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.