Studies On New Biosensing Methods Based On DNAzyme And Protein-DNA Interaction

With the development of bilogical sciences and the increasing requirementconcerning human health，the application of new analytical methods and technologiesin life sciences and medical sciences is receiving unprecedented attention. Bisensor isan analytical tool or system based on the specific recognition of biological materials,which has been widely used in the biochemical analysis, clinic dignosis, environementtest, drug screening and military science etc.. In the practical application, biosensor issensitive, selective, cost-effective and can be employed in fast real-time andcontinuous assay. Therefore, the development of new biosensing technologies ormethods would be of scientific significance to drive the further research in the relatedarea. In the development of new biosensing method，the introduction of DNAzyme andprotein-DNA interaction brings a novel idea to construct convenient, label-free anduniversal bioanalysis platform. In this dissertation, we focus on developing simple,cost-effective, high sensitive and selective biosensing methods by using severalbological functional proteins, enzymes and biological small molecules as targets. Bythe advantage of the effective catalytic ability of G-quadruplex DNAzyme and thespecific recognition of protein-DNA, our studies on new biosensing methods providesatisfactory results as expected. The colorimetric methods based on G-quadruplexDNAzyme and the bioluminescence and electrochemical methods based on the specificinteraction between protein and DNA are described in the Chapter2to4and in theChapter5to6respectively, more details are as follows:T4polynucleotide kinase (PNK) plays a critical role in various cellular events. InChapter2, we describe a novel colorimetric strategy for estimating the activity ofPNK and screening its inhibitors taking advantage of the efficient cleavage of λexonuclease and the signal amplification of G-quadruplex DNAzyme withperoxidase-mimicking activity. A label-free hairpin DNA with the sequence ofG-quadruplex was utilized in the assay. Th′e5-hydroxyl terminal of the hairpin DNAwas firstly phosphorylated in the presence of PNK and then digested by λ exonuclease.As a result, the blocked G-quadruplex sequence of the hairpin DNA was released dueto the removal of its completely complementary sequence. Using this strategy, theassay for PNK activity was successfully transfered into the detection of G-quadruplexDNAzyme. Because of the completely blocking and efficiently releasing of G-quadruplex DNAzyme, the colorimetric method exhibited an excellent performancein PNK analysis with a low detection limit of0.06U/mL and a wide detection rangefrom0.06to100U/mL. Additionally, the effects of different inhibitors on PNKactivity were also evaluated. The proposed strategy holds great potential in thedevelopment of high-throughput phosphorylation investigation as well as in thescreening of the related drugs.Existing strategies for detecting nicotinamide adenine dinucleotide (NAD+) orother cofactors are commonly cumbersome and moderate sensitive. In Chapter3, wereport a novel DNAzyme-based visual assay strategy for NAD+based onligase-mediated inhibition of the strand displacement amplification (SDA). In thepresence of NAD+, the SDA can be inhibited by the ligase reaction of two primers,which can initiate the SDA reaction in the case of no ligation, resulting in adramatically decreasing yield of the SDA product, a G-quadruplex DNAzyme that canquantitatively catalyze the formation of a colored product. Therefore, the quantitativeanalysis for NAD+can be achieved visually with high sensitivity. The developedstrategy provides a simple colorimetric approach with high selectivity against mostinterferences and a detection limit as low as50pM. It also provides a universalplatform for investigating cofactors or other related small molecules as well asquantifying the activity of DNA ligases.Screening of potential drugs specifically binding to polydeoxyadenosine [poly(dA)]is of great interest in recent studies. In Chapter4, we developed a simple colorimetricstrategy through the mechanism of target induced split G-quadruplex formation fordetecting coralyne, a poly(dA)-binding drug with noticeable antitumor activity. TwoDNA oligonucleotides containing split G-quadruplex sequence and adenine-richsequence are used in our strategy. In the presence of coralyne, the adenine-richsequences of two oligonucleotides are drawn in close proximity, resulting in theformation of a split G-quadruplex DNAzyme that catalyzes the generation of a coloredproduct. The DNAzyme-based colorimetric assay for coralyne has a linear range from0.033to1.667μM with a low detection limit of16nM. The developed method issimple, cost-effective, visible, and holds great potential for applications in drugscreening.DNA methyltransferase (MTase) is a kind of important regulatory factor in variousbiological processes. Current methods to investigate DNA MTase activity are stilllimited in the sensitivity and/or generality. In Chapter5, we develop a newbioluminescence strategy based on methylation-resistant cleavage and protein expression in vitro to detect DNA MTase activity. In the strategy, Dam MTase wasused as a model enzyme and MboI as the methylation-resistant endonuclease, andluciferase reporter DNA (LR-DNA) was used as their action target. As the completelymethylated LR-DNA was able to be expressed as detectable luciferase, Dam MTaseactivity was quantified by measuring the luminescence intensity of the expressedluciferase. The assay provides a very low detection limit (0.08U/mL) as well as a widelinear range from0.2U/mL to100U/mL. Besides, the analysis mode has improvedgenerality and could be extended to the detection of other DNA MTases and thecorresponding inhibitor screening.Quantitative detection of DNA-specific binding protein is significant for thediagnosis of transcription-interrelated diseases and the understanding of thecorresponding regulation mechanism. In Chapter6, a simple, sensitive and selectivemethod to detect human TATA binding protein (TBP) was developed based onelectrochemical impedance spectroscopy (EIS) measurement. In the protocol, a doublestrand DNA probe (TBP-DNA) containing ‘TATA box’ sequence was designed. In thepresence of TBP, the specific combination with TBP-DNA immobilized on the goldelectrode would cause the change of electron transfer resistance (Ret). The change ofelectrochemical impedance was resulted from the interaction of the positively chargedTBP with the negatively charged TBP-DNA. Therefore, a simple and highly sensitivedetection was carried out for TBP and its interaction with TBP-DNA by measuring thechange of Ret. Compared with those of previous studies, the detection limit (4.2ng/mL)of the strategy is lower. The cost-effective strategy is promising to be applied in thebiomedical studies concerning transcriptional regulation and disease diagnosis.