The Design Of Novel Molecular Probes And Their Applications In Real Time Assays For Methylase & Restriction Endonuclease And Drug-screening

The nucleic acid and protein are very important biomacromolecules in life and the interaction of them is the basis of varied life processes such as generation, development and reproduction. The study of interaction of DNA and protein in molecular level is the hotspots in life science now. However, it is difficult to acquire the real time information of the interaction of biomolecules by traditional methods, which hampers the progress of related study. Therefore, it is a great challenge to obtain the real time information of life process, especially the detail of interaction between nucleic acid and protein. In this thesis, the real time monitoring of the methylation and enzymatic cleavage has been investigated based on the design of new hairpin probe and the utilization of specific function of methylases and restriction endonucleases. New fluorescence assays are established for the detection of methylases and restriction endonucleases.The effects of some drugs on the activity of these enzymes are also investigated. The main researches included in this thesis are presented as following: 1. The assay of DNA methylase based on DH-MB and its application for screening drugs. A novel double hairpin molecular beacon that has more stable structure than traditional molecular beacon has been designed. Its hairpin structure will not fully open when it hybridizes to cDNA but the hybrid can be cut by restriction endonucleases, which leads to destroy of its structure and restore the fluorescence. Because their recognition sites usually are double stranded sequences, the double hairpin molecular beacon is more suitable to analyze the restriction endoneuclease and methylase than traditional molecular beacon. In this chapter, a DH-MB is designed to contain the AluI recognition site. A quick, accurate assay of the AluI methylase has been established and the effects of some drugs on the activity of methylase are also investigated. 2. Real time monitoring of DNA methylation using hairpin probe and its application on screening drugs. The Dam methylase recognition site is designed in the stem of a new hairpin probe. Based on the coupled enzyme reaction of hairpin probe with Dam methylase and DpnI endonuclease, a quick and simple assay for Dam methylase is established. Using this method, the DNA methylation process is monitored in real time and the effects of some drugs on Dam methyalse also have been investigated. It has the potential for the application of screening the inhibitor or activator of Dam methylase. 3. A continuous assay of single-stranded DNA cleavage by restriction endonulease using molecular beacon. Based on the fact that single-stranded molecular beacon digested by restriction endonuclease can restore the fluorescence in real time, a sensitive and simple fluorescence assay of restriction endonuclease has been proposed. The mechanism of single-stranded DNA cleavage by endonuclease is discussed, which confirms the formation of recognition site on the instantaneous dimmer molecular beacon. In this thesis, the processes of DNA methylation and enzymatic cleavage have been monitored in real time by taking full advantage of the novel molecular probes. The application of molecular probe in the screening of drugs is also discussed. A new approach is provided for the study of the real time interaction between biomicromolecules and drug exploitation.