Research Of Biosensing Technology On Small Molecule-Protein Interaction

Protein information has long been of significance in biological research, of which the interactions between small molecules and proteins attracted increasingly attentions in recent years. Small molecules are essentially involved in protein function processes; therefore, understanding of small molecules-protein interactions could be of substantial importance to the discovery and development of biology. With its diversity, active small molecules can be designed to selectively target proteins and explore protein functions, which make small molecues as valuable probes for markers detection of disease diagnosis. Hence, identification and quantification of affinity between small molecules-protein in complex biological system is a major goal of analytical chemistry, biochemistry, and clinical chemistry.This research paper is concerned with a number of problems for the current development of small molecules-protein biosensing. The detailed materials are described as follows:(1) A novel fluorescence assay strategy for identifying small molecule-protein interactions based on a smart DNA-cleaving molecular machine that is regulated with protein binding has been proposed. The molecular machine is readily constructed via the assembly of two Fok I endonuclease molecules on a DNA heteroduplex with a small-molecule label at a specific site. It is discovered that DNA-cleaving rate of the molecular machine is quantitatively inhibited by the interactions between the small molecules and their binding proteins with wide-ranging affinities (dissociation constants ranging from micromolar to sub-nanomolar). Based on this finding, we propose a novel homogeneous fluorescence assay strategy for sensitive detection of small molecule-protein interactions. The developed strategy is demonstrated using two small-molecule compounds, folate and biotin, with their binding proteins. The results reveal that the strategy creates a sensitive, specific and robust platform for the assays of small molecule-binding proteins or competitive small-molecule binders of the protein targets, implying the wide application spectrum of the developed DNA-cleaving machine for small molecule-protein interaction assays.(in Chapter 2)(2) A sensitive, selective and robust chemiluminescence biosensor based on DNAzyme amplification and hemin aptamer chemiluminescence has been developed. A DNA sequence containing Nt.BstNBI nicking site and hemin aptamer complementary region and intramolecular loop has been designed, in the presence of hemin aptamer, luminol and H₂O₂, strong chemiluminescence signal will be detected. While small molecule binding protein is conjugated with the DNA, chemiluminescence signal decreased. The experiment results demonstrated that the developed strategy signal was a sensitive and specific platform for identifying small molecule-protein interaction (in Chapter 3).