Construction And Application Of Nuclease Monitoring System Based On Graphene Oxide Quenched Hairpin Probe

Nucleic acids including DNA and RNA are the most important biological materials,which play central roles in the life systems.Thus,keeping the stability of the genome is essential for maintaining biological functions and also play important roles in living organisms.However,mismatched base pairs in DNA replication often appeared due to several factors including biophysical and others.One of them is the direct misincorporation of bases,which can cause DNA mutations.In order to keep genetic integrity,many different types of nucleases involved in repairing these mismatch of living organisms.These enzymes can catalyze DNA and RNA into single nucleotide or oligonucleotides to ensure high fidelity of the nucleic acid replication process and to effectively prevent disease caused by mismatches or errors.Until now,the study of nuclease activity is still the focus of life science.However,the simple and rapid methods for nuclease is still in scarce.In this dissertation,a new kind of nanomaterials—graphene oxide was introduced into designing of biosensors.By using the adsorption capacity of ssDNA and high fluorescence quenching efficiency of graphene oxide,we have developed a series of simple,rapid and sensitive biosensor systems for the detection of nuclease activity.The main contents are described as follows:(1)A novel fluorescence method to detect DNase I activity based on GO quenched hairpin probe was developed.The effects of Mg2+,temperature and pH on the detection of DNase I are systematically investigated.The method with a detection limit of 0.005 U mL-1 under the optimal conditions.It was then applied to investigate the effects of external factors including antibiotics and heavy metal ions on DNase I.The results indicated that gentamicin sulfate was a strong inhibitor with an IC50 value of 0.57±0.12 ?M.The investigated heavy metal ions showed an inhibitory effect on DNase I activity with IC50 values of 0.04 ?g/mL(Hg2+),0.10?g/mL(Pb2+),1.35 ?g/mL(Cd2+),1.20 ?g/mL(As3+),and 1.80 ?g/mL(Cu2+).Finally,the new method was applied to detect DNase I levels in complicated tumor tissue and cell samples and the results showed that DNase levels increased in tumor tissues compared with that of adjacent tissue.Moreover,we conclude that the method can be widely used for high-throughput assay of DNase I in biological samples as well as drug screening in vitro.(2)Based on the DNase I assay method established in the previous chapter,we further use it to study the interaction between natural drugs and DNase I in vitro and cellular levels.First,a total of 11 small molecule drugs were isolated from leaves of Cyclocarya paliurus by extraction and then investigated the effect of small molecule drugs on DNase I activity by using the established method.The results indicated that 7 drugs can promote the function of enzymatic activity in different degrees and 4 drugs can inhibit DNase I activity.It was then applied to investigate the effect of three drugs including two activators and one inhibitor on DNase I at the cellular level.The results showed that 20 ?M of Schisanlactone E and SBB-L-1 can inhibit DNase I activity in MCF-7 cells about 57%and 61%,respectively.On the contrary,20 ?M of Schisanlactone E can activate DNase I activity in BEL-7404 and SMMC-7721 cell lines about 28%and 33%,respectively.The above results indicate that small molecule drugs can differentiate DNase I activity at different levels.(3)We have developed a novel fluorescent probe based on GO-quenched single-stranded DNA fluorescence probe for detecting S1 nuclease levels and screening inhibitors.The method was further applied for S1 activity analysis and kinetics study of S1 nuclease.Results indicated that the detection limit is 0.5 U/mL,The Km and kcat at 45?,are 1.4 ± 0.12 ?M and 0.6 min-1,respectively.Moreover,by monitoring the effect of chemical drugs on S1 activity,we found that 2 mM of erythromycin,sodium penicillin,carbenicillin disodium and ampicillin can inhibit S1 activity about 8%,60%,61%and 66%,respectively.Above all,the assay platform based on graphene oxide quenched fluorescence probe is successfully constructed to study the enzymatic activity of S1 and used for screening antibiotics.