Development Of New Flourescence Assay For Protein Based On Nucleic Acid Molecule Probe

DNA is vulnerable to damage from a variety of sources, both extrinsic and intrinsic to cells, and DNA damage can threaten the genetic integrity by increasing the frequency of mutations and exacerbating replication errors. T4 polynucleotide kinase (PNK) and Human 8-oxoG DNA glycosylase 1 (hOGG1) were able to repair DNA with 5’-hydroxyl termini and repair damaged DNA though the base excision repair (BER) pathway to maintain the integrity of the gene. Nucleic acid molecule probe fluorescence sensor has achieved a rapid and lasting development because of simple, high sensitivity and selectivity. In this paper, through the ingenious design of fluorescent molecular probes to build a simple, novel DNA biosensor for amplified detection of enzymes.There are three chapters in this thesis:1. Taking advantage of the efficient enzyme reactions, namely the phosphorylation of hairpin probe (HP) by PNK and the λ exonuclease cleavage reaction, the trigger DNA fragment (tDNA) can be released from HP and is used to trigger the catalytic assembly of bimolecular beacons (bi-MBs), resulting in a remarkably amplified fluorescence signal toward PNK activity detection. The detection limit of this method toward PNK was obtained as 1 mU mL-1, which was superior or comparable with the reported methods. Furthermore, the facile and sensitive method can also be used to screen the inhibition effects toward several common inhibitors. It provides a promising platform for sensitive determination of nucleotide kinase activity and inhibition, and also shows great potential for biological process research, drug discovery, and clinic diagnostics.2. A novel strategy for the detection of PNK activity and its inhibition has been proposed, which combines exonuclease enzyme reaction and nicking enzyme assisted fluorescence signal amplification. Through recycling cleavage of DNA fluorescence probe for signal amplification, a highly sensitive PNK sensing platform is developed, and a very low detection limit of 0.05 mU mL-1 is achieved. The present approach adopts a simple separation-free procedure in which the enzyme assay is conducted in homogeneous solutions. Additionally, the inhibitory effects of several known kinase inhibitors on PNK have been successfully detected.3. A novel and highly sensitive fluorescence biosensing platform for hOGG1 activity detection has been constructed based on autonomous Exo Ⅲ-assisted signal amplification. Two hairpin probes (HP1 and HP2) are ingeniously designed. In the presence of hOGG1, HP1 is cleaved at the 8-oxo-7,8-dihydroguanine (8-oxoG) site and the stem is subsequently digested by Exo III, releasing the trigger DNA fragment (tDNA1). Successively, tDNA1 partially hybridizes with HP2 to initiate the Exo Ⅲ-assisted cycling cleavage to release another trigger DNA fragment (tDNA2), which in turn triggers the cycling cleavage of DNA fluorescence probe (FP). Therefore, large amount of fluorophore fragments are released, leading to a significantly amplified fluorescence signal toward hOGG1 activity detection. A directly measured detection limit down to 0.001 U mL-1 is obtained, which is much lower than that of the approaches reported in literature. Furthermore, the Exo Ⅲ-assisted autonomous cycling cleavage approach we proposed here is a universal sensing strategy, and has great potential in assays of many other biological analytes.