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 ...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.展开更多
In typical experiments where magnetic tweezers are involved,precise measurement of the magnetic forces is of crucial importance.To achieve this,a widely applied method is to track the bead's Brownian motion trajec...In typical experiments where magnetic tweezers are involved,precise measurement of the magnetic forces is of crucial importance.To achieve this,a widely applied method is to track the bead's Brownian motion trajectory and to calculate the force from its mean-squared-displacement.However,this method does not take into account the fact that the bead-tracking device always has a finite bandwidth,acting effectively as a low-pass filter.The result could be subjected to significant system errors,which overestimates the magnetic force.We analyze the power spectrum of the bead's Brownian motion,and provide a corrected formula to calculate the magnetic force,which is free of system errors induced by limited detection bandwidth.A dsDNA force-extension curve is experimentally measured.The curve is consistent with the WLC model,exhibiting correctness of the new formula.On the other hand,the force given by the traditional method shows significant deviation from the WLC model,which is 3 times larger at most.展开更多
基金Supported by the National Natural Science Foundation of China under Grant No 11574382
文摘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.
基金Supported by the National Natural Science Foundation of China under Grant No 61275192.
文摘In typical experiments where magnetic tweezers are involved,precise measurement of the magnetic forces is of crucial importance.To achieve this,a widely applied method is to track the bead's Brownian motion trajectory and to calculate the force from its mean-squared-displacement.However,this method does not take into account the fact that the bead-tracking device always has a finite bandwidth,acting effectively as a low-pass filter.The result could be subjected to significant system errors,which overestimates the magnetic force.We analyze the power spectrum of the bead's Brownian motion,and provide a corrected formula to calculate the magnetic force,which is free of system errors induced by limited detection bandwidth.A dsDNA force-extension curve is experimentally measured.The curve is consistent with the WLC model,exhibiting correctness of the new formula.On the other hand,the force given by the traditional method shows significant deviation from the WLC model,which is 3 times larger at most.
