Restriction Endonuclease-based Fluorescence Biosensors For The Detection Of Transcription Factors

Transcription factors (TFs), as a class of sequence specific DNA-binding proteins, can regulate cell development, differentiation and growth, playing important roles in the gene expression regulation. Studies have demonstrated that the expression levels of TFs are closely associated with a variety of diseases, such as cancers, inflammation, abnormal hormone responses and developmental disorders. Now, TFs have become important biomarkers for disease diagnosis and drug development. Thus, the specific, accurate and sensitive detection of TFs is very essential. The enzyme-based fluorescence methods have become increasing popular in the detection of TFs due to their high reaction efficiency and flexible design. However, they have some intrinsic shortcomings, such as the narrow application range and the low reliability.Restriction enzymes are a kind of sequence specific nucleases. They can only recognize and cleave the specific DNA sequences. Due to their high selectivity and precision, they hold great promises for the development of specific and accurate platforms for biologic analysis.Sensitivity is another important parameter to evaluate the performance of an assay. In order to improve the detection sensitivity, signal amplification strategies are widely used because one target can produce multiple signals after the amplification. DNA three-way junction is a branched Y-shaped DNA structure, which is composed of three DNA strands hybridized with each other specifically. DNA three-way junction-induced signal amplification strategies have attracted great attention because of their specificity and sensitivity. Considering the limitation of enzyme-based fluorescence methods in TF detection, we developed a novel strategy for the specific and accurate detection of TFs based on restriction enzyme Fok I. And then, we constructed a sensitive TF detection method on the basis of DNA three-way junction-induced signal amplification. The main contents of this dissertation are as follows: Chapter one is an introduction to summarize the background information about TF detection, including the significance, traditional methods and fluorescence methods which can be mainly divided into imolecular proximity assay, structure-switching assay, competitive binding assay and steric effect assay. The developments of signal amplification strategies based on endonuclease, exonuclease and polymerase are also introduced.In chapter two, a novel Fok I cleavage-inhibition strategy has been developed for the specific and accurate detection of TFs. This strategy is based on the TF binding-induced steric effect and Fok I catalytic properties. In the method, the specific Fok I-DNA interaction can accurately convert the detection of TFs to the detection of DNA, ensuring the reliability of detection results. Moreover, Fok I has no requirement for the sequence of its cleavage site, enhancing the versatility of the strategy. Nuclear factor-kappa B p50 (NF-κB p50) was chosen as a model. The fluorescence response increased proportionally to the concentration of NF-κB p50 within the range from 1.0 ×10-8 to 1.3×10-7 mol L-1, and the limit of detection is 7.9×10-9 mol L-1,which was comparable to or better than those of previously reported systems. This method also showed high selectivity and low matrix effect. Moreover, this method can be used to measure NF-κB p50 in HeLa cell nuclear extracts.In chapter three, we combined the TF binding-induced inhibition effect with DNA three-way junction-induced signal amplification to construct a specific, accurate and sensitive method for TF detection. Besides the accuracy of the conversion of protein detection to DNA detection with restriction enzyme, this method possesses the detection specificity with DNA three-way junction and the detection sensitivity with rolling circle amplification strategy. Additionally, N-methylmesoporphyrin IX was also used to generate the label-free fluorescence signal. The optimal rolling circle amplification mode was selected and the feasibility of this method for detection NF-κB p50 was verified.