Methodology Of Proteins And Single-stranded Nucleic Acids Interaction By Capillary Electrophoresis

Aptamers,as chemical antibodies,have been the research highlights in biomedical science,environmental and food analysis,nucleic acid drugs,clinical testing and other areas in recent years.In particular,aptamers have broad application prospects in diagnosis and disease treatment.At present,the difficulty of aptamer screening is still the bottleneck of practical application.The existing screening methods,such as magnetic beads,affinity chromatography,nitrocellulose membrane filtration method,require fixed targets or nucleic acids and have long screening cycles.Capillary electrophoresis(CE)is one of the most efficient and rapid microanalysis methods for screening nucleic acid aptamers and a variety of CE-SELEX technologies have been developed by now.Compared with other screening methods,CE-SELEX has a short screening time,high screening efficiency,wide range of screening targets,and can select aptamers with specified kinetic parameters.In addition,capillary electrophoresis can also be used for the rapid characterization of the interactions between targets and single-stranded nucleic acids,which can help to understand the high affinity and specific interaction of targets and nucleic acids,thus laying the foundation for efficient screening of aptamers.In this paper,the interactions of proteins with single-stranded nucleic acids were studied based on variety modes of capillary electrophoresis.This thesis consists of four sections:(1)The development history,instrumental composition and analytical modes of capillary electrophoresis were reviewed.Then,the capillary zone electrophoresis(CZE),the on-line reaction mode of capillary electrophoresis(on-line reaction CE),the research progress of aptamers in capillary electrophoresis,the application of capillary electrophoresis in the screening of aptamers and in the study of the interaction of nucleic acids with targets were summarized.In addition,the properties and applications of several nucleic acid binding proteins used in this study were introduced.Above all,the research content and significance of this paper were introduced.(2)A characterization method of proteins interacting with single-stranded nucleic acids of different lengths was developed based on CE.The interaction of single-stranded DNA-binding protein(SSB),transferrin(TRF)with different lengths of ssDNA and poly(T)and interaction of Taq DNA polymerase with its two aptamers were characterized by capillary electrophoresis equiped with laser-induced fluorescence detection(CE-LIF).It was found that the interaction of the selected proteins with ssDNA or poly(T)were affected by the length of nucleic acids,the ratio of protein to nucleic acids and the formation of secondary structure of ssDNA.And it was pointed out that the length of nucleic acid library,target-nucleic acid library concentration ratio were the directions that can be optimized in aptamer screening.Besides,based on CE-LIF,the interactions between target proteins and ssDNA of different lengths were compared qualitatively.(3)Quantitative analysis of the interactions of SSB,Taq DNA polymerase with ssDNA or poly(T)with different lengths were performed based on kinetic capillary electrophoresis(KCE).The dissociation constant(K_d)of SSB with 15mer/29mer/dT29,Taq polymerase with Apt30/Apt46 were determined,and the influences of the concentration ratio and capillary temperature on the dissociation constant K_d was quantitatively analyzed.In addition,the effect of integral deviation of peak area on the caculation of dissociation constant was analyzed by numerical calculation.The experimental results showed that the method based on KCE had good stability under the present experimental and technical conditions.(4)The interaction model of SSB,TRF,Taq DNA polymerase with nucleic acids,thrombin with its ligands were used to investigate the basic principles and modes of on-line reaction CE.The effects of injection time,capillary temperature and residence time on the on-line reaction CE,the interaction of single targets with multiple ligands and thrombin with multiple ligands were investigated.At last,the theoretical analysis model of on-line reaction CE was established and the controllability of on-line reaction CE was explored.