| DNA glycosylase is an enzyme that can recognize and remove damaged bases on DNA strands,and participates in DNA damaged base repair by initiating base-excision repair(BER)pathway.Therefore,DNA glycosylase can protect the genome from the damage of damaged bases and maintain the integrity of the genome.Abnormal DNA glycosylase activity can lead to neurodegenerative diseases,developmental disorders and tumors.Therefore,the detection of DNA glycosylase activity is of great significance for the diagnosis of diseases,the clinical evaluation of DNA damage repair process and the functional study of DNA glycosylase.DNA molecular machine is an artificial molecular machine constructed from DNA nanomaterials,which can perform similar mechanized movement under specific stimuli,and has high predictability,programmability,and good biocompatibility.DNA molecular machines,which include DNA loops,DNA walkers,DNA tweezers,and DNA robots,have been used for the detection of nucleic acids,proteins,small molecules and metal ions,and other targets.In this paper,we construct a DNA molecular machine driven by Nt.Bbv CI and use it to detect DNA glycosylase activity without the label.Using uracil-DNA glycosylase(UDG)as an analysis model,this paper is divided into four chapters:The first chapter is the introduction,which mainly summarizes the concept,classification,function,research significance,detection methods,existing problems,and the main research content of this paper.In chapter two,we construct a DNA loop driven by Nt.Bbv CI for label-free detection of UDG activity.Firstly,we designed a double-strand recognition probe and a hairpin signal probe,in which the double-strand recognition probe contains the uracil base(U),1/2Nt.Bbv CI recognition sequence and trigger sequence,which can recognize and signal transduction of UDG.The hairpin signal probe contains 1/2 Nt.Bbv CI recognition sequence and G-quadruplex sequence,which can be used for signal amplification.Under the action of UDG,the U base in the double-strand recognition probe is removed,resulting in an apurinic/apyrimidinic site,the Tm value of the double-strand is reduced,and the trigger sequence is released.The trigger sequence is then hybridized with a hairpin type signal probe,exposing the complete Nt.Bbv CI recognition sequence and be cleaved,releasing the G-quadruplex sequence.The free trigger sequence can be hybridized with the next signaling probe to form an Nt.Bbv CI-assisted DNA loop,which eventually produces a large number of G-quadruplex sequences,which fluoresce after insertion of fluorescent dye N-methyl-mesoporphyrin IX(NMM).This method achieved the marker-free detection of UDG activity,the detection limit was 0.0006 U/m L,and the detection of UDG activity in human cervical cancer(Hela)cell lysate and UDG inhibitor.In Chapter three,we construct a DNA walker driven by Nt.Bbv CI and use it for label-free detection of UDG activity.Based on the work in Chapter two,the double-chain identification probe and hairpin signal probe are modified on Au NP to form the running track of the walker.Under the action of UDG,the trigger sequence in the double-strand recognition probe is released.As a walking chain,the trigger sequence can hybridize with the adjacent hairpin signal probe on the track,exposing the complete Nt.Bbv CI recognition site,so that the signal probe is cut and the G-quadruplex sequence is released.The free walking chain can be hybridized with the next adjacent signal probe,and the Au NP track can be walked with the aid of Nt.Bbv CI.This strategy increased the local concentration of the hairpin signal probe,improved the reaction rate and amplification efficiency,and the detection limit of UDG activity was 0.00004 U/m L.Meanwhile,it also realized the labeling free detection of UDG activity in Hela cell lysate.The fourth chapter is the conclusion and prospect part,which mainly summarizes the research results of this paper and prospects for the application prospect of the work. |
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