Building And Application Of Single-molecule Fluorescence Resonance Energy Transfer Microsocopy

Many biological reactions are too complex to be fully comprehended through conventional ensemble techniques.At the most fundamental level,all biological reactions occur via the action of single enzyme,DNA molecule,or RNA molecule.Studying one biological macromolecule at a time can provide us with extraordinarily clear and often surprising views of these molecules in action.Two broadly defined categories of single-molecule methods are fluorescence-based imaging and spectroscopy and force-based manipulation and detection.The fluorescence-based imaging and spectroscopy can be performed either in vitro or in vivo.The force-based manipulation and detection includes optical trap,magnetic trap,atomic force microscope,and nanopores.In ensemble FRET(fluorescence resonance energy transfer)measurements,it is often very difficult to synchronize the conformational changes of biological molecules and not feasible to detect the short-lived conformers.smFRET(single-molecule fluorescence resonance energy transfer)opens up new opportunities by probing the structural changes of individual biological molecules in real time.It readily determines the distribution of several conformations,not just the average of them.This makes it possible to directly identify rarely visited and short-lived states.This thesis focuses primarily on single-molecule fluorescence resonance energy transfer based on total internal reflection microscopy.We first discussed the principle of smFRET including total internal reflection microscope,fluorescence resonance energy transfer and flowing chamber preparing.Then we presented how we built the hardware platform.The sm-FRET hard platform bases on OLYMPUS IX81 microscope.In order to flow samples easily,we designed special flowing stage.Next,we prestend how we established the data acquisition and analysis procedures using ImageJ,IDL and MATLAB.Finally using smFRET to study RNA unwinding by Dedl protein was discussed.We find ATP non-hydrolytic analogues AMP-PNP can not facilitate chimeric DNA-RNA duplexes unwinding by Dedlp.