DNA Repair Enzymes Activity Detection Methods Based On Fluorescence Enhancement Function Of ?-cyclodextrin Polymer

DNA repair enzymes can participate in the DNA damage repair process,protecting the genome from DNA lesions caused by internal factors or external environment and maintaining the integrity of the genetic information.Abnormal activities of the DNA repair enzymes would interrupt the repair process of DNA lesions and directly relate to various symptoms,including aging,death,tumors and et al.Therefore,the detection of DNA repair enzymes activity is important for genetic information protection,clinical diagnosis and treatment.Cyclodextrin polymer has many advantages such as excellent stability,good water-solubility,special molecular recognition capability and multivalent binding effect.It was reported that p-cyclodextrin polymer had super fluorescence enhance capacity to pyrene.In this thesis,fluorescent methods to detect apurinic/apyrimidinic endonuclease 1(APE1)and uracil-DNA Glycocasylase(UDG)were constructed based on nucleic acid probes and fluorescence enhancement of ?-cyclodextrin polymer to pyrene.The details are summarized as follows:1.A simple and sensitive fluorescent method for APE1 activity detection was developed based on the super fluorescence enhance capacity of ?-cyclodextrin polymer to pyrene.In this method,functional pyrene-labeled dsDNA probes(AP-S1S2)and ?-CDP functioned as fluorescent signal producer and enhancer,respectively.When APE1 was absent,pyrene could not enter the cavity of ?-CDP because of steric hindrance,leading to a weak fluorescence intensity/anisotropy;when APE1 was present,it could recognize and cleave apurinic/apyrimidinic(AP)site in AP-S1S2,thus producing a dissociated pyrene-labeled oligonucleotide segment.Pyrene labeled at the 3' end of oligonucleotide segment could be easily trapped into the cavity of ?-CDP via.host-guest interaction,leading to a significant enhancement of fluorescence intensity/anisotropy.Under the optimized conditions,the proposed assay allows sensitive and selective detection of APE1 activity with a detection limit of 0.05 U/mL.Furthermore,assessment of APE1 activity in HeLa cell extracts was successfully implemented,showing great potential to be a universal method for DNA repair enzymes detection.2.A simple method for UDG activity detection was developed based on the previous work and APE1 could recognize and incise apurinic/apyrimidinic(AP)site.In this method,U-P1P2(consisted of P1 and P2)were employed in the system.Both terminals of Pl were prolonged,P2 had a uracil as the UDG cleavage site and labeled pyrene at the 5' end.Pyrene and ?-CDP were employed as the fluorescent signal producer and enhancer,respectively.In the absence of UDG,U-PIP2 could form a stable double-stranded structure,pyrene was difficult to enter the cavity of p-cyclodextrin polymer due to the steric hindrance,leading to a weak fluorescent signal/anisotropy.In the presence of UDG,it would recognize and cleave uracil.The resulting AP site was then cleaved by APEI.And the pyrene-labeled oligonucleotide segment would fall away from the U-P1P2.Then,pyrene was easily trapped into the hydrophobic cavity of ?-cyclodextrin polymer,leading to significant fluorescence enhancement/anisotropy.This assay offered an estimated detection limit of 0.01 U/mL due to the high enhancement factor of ?-CDP.And the proposed method was successfully applied to detect UDG activity in cell extracts.3.A sensitive assay for APEl activity detection has been developed based on the super fluorescence enhance capacity of ?-cyclodextrin polymer to pyrene and the separation ability of magnetic beads.AP-P1P2(consisted of P1 and P2)were employed in the system.3' end of P1 was prolonged by ten bases and 5' end of P1 was labeled with biotin.P2 had a AP site at the 31 end and labeled with pyrene at the 5' end.In the absence of APE1,AP-P1P2 could form a stable double-stranded structure and were fixed on the surface of magnetic beads.There was no pyrene in supernatant and the fluorescence signal/anisotropy was low.In the presence of APE1,it could incise AP site of AP-P1P2 on the magnetic beads.The short fragment would dissociate from AP-P1P2 on the magnetic beads,and then pyrene on the segment would enter the cavity of ?-CDP in detection system,leading to a remarkable increase of fluorescence signal/anisotropy.This assay reduced background signal by enrichment and separation ability of magnetic beads.This assay offered a detection limit of 0.01 U/mL.Furthermore,the proposed strategy was successfully applied to detect APE1 activity in cell extracts.