Single-molecule FRET (smFRET) setup

The smFRET study was carried out with a home-built objective-type total internal reflection fluorescence microscopy (27). Cy3 was excited by a 532 nm sapphire laser (Coherent Inc., USA). An oil immersion objective (100×, N.A. 1.49) was used to generate an evanescent field of illumination. The fluorescence signals from Cy3 and Cy5 were split by a dichroic mirror and collected by an electron-multiplying charge-coupled-device camera (iXON, Andor Technology, South Windsor, CT, USA). The fluorescence imaging process was controlled and recorded by MetaMorph (Molecular Devices, CA, USA).