An Improvement on the Combination of Magnetic Trap and Fluorescent Resonant Energy Transfer
Da-guan Nong1,2, Ming Li1,2**
1Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 2University of Chinese Academy of Sciences, Beijing 100049
Abstract：The combination of magnetic trap (MT) and fluorescence resonant energy transfer (FRET) allows for nanoscale measurements of configurational changes of biomolecules under force. However, the magnetic bead involved in MT experiments introduces a substantial amount of background fluorescence which reduces the signal-to-noise ratio (SNR) of FRET significantly. Moreover, the short lifetime of the dye used in FRET limits the total sampling time when combined with MT. Here we use a moveable tube lens to adjust the wave front in the light pathway of MT so that both images of the magnetic bead and the fluorescent signals can be detected when long DNA handles are used to reduce the auto-fluorescence of the magnetic bead. We utilize the internal trigger of an electron multiplying charge-coupled device camera to control a shutter so that the dye can be excited intermittently when long time measurement of FRET is needed. As a demonstration of the hybrid technique, we observe the unfolding/refolding dynamics of a DNA hairpin and measure the DNA unwinding activity of the saccharomyces cerevisiae Pif1 (Pif1). Our results show that the unwinding burst of Pif1 under external force is different from that without the force. In addition, the improvement provides a better SNR and a longer sampling time in experiments in the MT-FRET assay.
(Single molecule manipulation of proteins and other biological molecules)
引用本文:
. [J]. 中国物理快报, 2019, 36(3): 38701-.
Da-guan Nong, Ming Li. An Improvement on the Combination of Magnetic Trap and Fluorescent Resonant Energy Transfer. Chin. Phys. Lett., 2019, 36(3): 38701-.
2019, Vol. 36 
