| The conformation and dynamic of biomacromolecules are building blocks of all life phenomena.The study of biomacromolecule structure and function at the single molecular level has great significance in explaining the essence of life phenomenon and improving cognition of the world and mankind.In this thesis,I systematically investigated different optical tweezers strategies of single molecule study,and applied them into different molecular biology systems.The main achievements are as follows:1.Improvement of stability and measurement accuracy of optical tweezers setup for single molecule stretching.The measurement accuracy of displacement and external force and the stability of the system determine the suitability of the optical tweezers setup.In this work,a detection light is introduced to improve the measurement accuracy,and the microspheres',image recognition is employed to construct a feedback compensation system against mechanical drift.With all these attempts,an optical tweezers setup with excellent stability was obtained.Furthermore,I developed single-molecule sample preparation,biomolecule conjugation technology for the single-molecule measurements.2.Study of rpoS RNA self-inhibition stem-loop conformation.The"force-extension" curve of rpoS RNA was obtained by stretching the self-inhibitory stem loop of rpoS RNA with optical tweezers.I studied the dynamic unfolding/folding process of rpoS RNA self-inhibition stem-loop through"force-extension" curves obtained by optical tweezers,confirmed its three-way-junction structure,and calculated the free energy of the RNA subunits.I found that magnesium ions significantly enhanced the strength of RNA junction and therefore compressed RNA conformation,change the spatial orientation of stem loop.This conformational change objectively shortens the distance between D1 and D2 stem loops,facilitates the sRNA recruited by Hfq protein to bind to the SD region and thereby activate the gene expression.3.The study on the mechanism of GSK and syntelin inhibiting CENP-E.I characterized the dynamic of CENP-E walking along microtubulewith optical tweezers and found the average speed of CENP-E was lower than that of kinesin-1.Experimental results indicated that GSK blocked ATP hydrolysis and inhibited CENP-E walking.Therefore,I hypothesize that syntelin inhibits CENP-E motion by preventing CENP-E·ADP dissociation from microtubules.4.The study of interaction between CENP-T and CENP-W protein.In this work,a method of coupling CENP-T or CENP-W to microspheres via DNA handle is presented.Using this strategy,I attempted to measure the breaking force between CENP-T/W and directly study the effect of phosphorylation on the binding strength of CENP-T/W protein at the single molecule level.5.A single pixel phase imaging method is proposed to realize the dynamic phase imaging of mobile cells in this paper.Based on complex Fourier spectrum of an object which is acquired by dynamically loading plane waves with different wave vectors,and the amplitude and phase distributions of the object are reconstructed by reverse Fourier transform.The binary phase distribution and the continuous phase distribution are tested experimentally,and both of them have good reconstruction results.In this dissertation,a high-precise and ultra-stable optical tweezers setup for single molecule studywas established,and its value in life science has been confirmed through a series of biophysical interdisciplinary projects.The results obtained in this study contributed to the fundamental concerns of life science and medical applications,and established a foundation for further optical tweezers study of molecular biology. |
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