Development Of Single Molecule Fluorescence Microscopy And Its Application In Folding Dynamics Analysis And Detection

Single molecule techniques not only realize the action measurements on biological macromolecules,but also provide information about their structures and functions.There are two main types of single molecule techniques based on mechanics and optics,respectively.In this study,based on the optical single molecule techniques,single molecule fluorescence microscopic imaging platform was developed,and its applications in ydaO riboswitch mechanism study and point of care testing applications were discussed in details.First,single molecule fluorescence resonance energy transfer(smFRET)system based on the total internal reflection microscopy was constructed,and it was then adopted for folding kinetics study on the ydaO riboswitch in order to explore its functions.Next,a miniaturized single molecule fluorescence detection system was developed to realize precise mercury ion(Hg2+)measurements by tracking the fluorescent signals of magnetic beads,indicating that the miniaturized single molecule fluorescence detection system is a potential tool for on-site detections.smFRET enables analysis on protein and RNA conformations with high precision,unfortunately,there is still no commercial smFRET system.In order to study folding kinetics of the ydaO riboswitch,in this thesis,smFRET system based on total internal reflection microscopy was constructed combining with the microfluidic channel fabrication,the single molecule fluorescence microscopic imaging system construction and the single molecule fluorescent signal analysis software development.The smFRET system could collect high-quality fluorescent signals in dual-wavelengths simultaneously with high resolution of 1.74 ?m close to the diffraction limit and high signal to noise ratio of 26.8 dB with the acquisition rate of 10 frame per second.Additionally,calibration of dual fluorescence fields of view and analysis on single molecule ttemporal signals were automatically realized by the single molecule fluorescent signal analysis software.The successful construction of the smFRET system can provide an important technical basis for the study of folding kinetics of ydaO riboswitch.Based on the smFRET system,this thesis studied folding kinetics of the ydaO riboswitch from Bacillus subtilis(B.subtilis).First,combining with the fluorescence labeling site determination,RNA fragment purification and ligation,the full-length double-labeled ydaO riboswitch was generated.Then,according to the smFRET data processing,conformational changes of three key structural domains in ydaO riboswitch(two cyclic diadenosine monophosphate(c-di-AMP)binding pockets and a pseudoknot domain)depending on Mg2+,c-di-AMP and ligand analogue ATP were quantitatively studied,showing that there are four key conformations in ligand-free,ligand-bound and ligand-analogue-bound conditions.Additionally,with further kinetic analysis,it is found that the binding pocket site 1 serves as a scaffold available for the first ligand binding,and then supports the second c-di-AMP recognition by binding pocket 2.Additionally,high concentrations of ligand promote the intermedium FRET state formation during pesudoknot formation.The controversial c-di-AMP analogue,ATP,induces fast transitions(less than 1 second)between different RNA conformations,indicating that ATP can bind this riboswitch motif but still not the primary ligand.The study on folding kinetics of the ydaO riboswitch provides important references to explore the mechanism of ydaO riboswitch functions.Single molecule techniques not only can realize quantitative analysis on the biological macromolecules such as riboswitches,but also can provide potential tools in detections due to the advantages as high sensitivity,good specificity,less sample consumption and high accuracy.In order to extend the application scopes of the single molecule techniques in on-site detections,fluorescence sensing based portable single molecule detecting system for mercury ion(Hg2+)testing composing of smartphone microscope for fluorescence signal collection and smartphone application for fluorescence signal collection analysis was developed in this thesis.According to the fluorescence imaging on magnetic beads,the single molecule detecting system not only has good Hg2+detection speci6cally,but also single moiecuie oetecting system not oruy nas gooa ig aetection speciricaluy,nut also realizes precise Hg2+detection with a limit of detection of 1 nM due to the linear relation between Hg2+concentration and average fluorescent signal intensity in a wide range between 1 nM and 1 ?M,proving that the fluorescence sensing based portable single molecule detecting system can realize quantitative Hg2+measurement in high precision and high sensitivity.In addition,combined with 3D printing,the size of the detection system is only 170 mm(length)× 113 mm(width)× 168 mm(height),besides,no external power is needed.Therefore,the portable rapid single molecule detection system is expected to be used in the on-site detection.Based on the development of the single molecule fluorescence microscopic imaging platform,its applications in ydaO riboswitch mechanism study and point of care testing applications are also discussed.This thesis can provide useful references for the development of fluorescence based single molecule technques as well as their applications in the biological macromolecular mechanism studies and detections.