Studies On The Fluorescent Assay Of Protein And DNA Modification Enzyme Based On Nicking Enzyme Signal Amplilfcation Technique

Amplification of specific nucleic acid sequences is one of the basictechniques in molecular biology. With the development of molecular biology aswell as the widespread applications of interdisciplinary subjects like biochemistry,biophysics in the field of molecular biology, there have emerged many newnucleic acid amplification techniques, such as polymerase chain reaction (PCR),rolling circle amplification (RCA), strand displacement amplification (SDA) andso on.Nicking enzyme signal amplification (NESA) is a novel strand displacementamplification technique, which has attracted much interest among researchers.This method is based on the fact that nicking endonuclease is an enzyme that cutsone strand of a double-stranded DNA at a specific recognition nucleotidesequences known as a restriction site. The principle of the amplification method isas follows, the polymerase extension can be initiated at the3’-hydroxyl termini ofthe nicked double-stranded DNA by polymerase in the presence ofdeoxyribonucleoside triphosphates (dNTPs), leading to the replication of thesingle-stranded DNA (ssDNA). Replication of the ssDNA yields the duplex thatincludes the nicking site for nicking endonuclease. Scission of the replicatedstrand results in a new replication site for polymerase and the concomitantdisplacement of the nicked ssDNA, leading to a “polymerization-nicking” cycleand displaced plenty of ssDNA after many cycles. Finally, the signal detection wasachieved through other signal conversion methods. The NESA method can obtain asignificant signal enhancement through the “polymerization-nicking” cycle.Combining the above methods and previous reports, we have developedseveral fluorescent biosensors for immunoassay and the detection of severalimportant DNA modification enzymes based on the nicking enzyme signalamplification method. The main contents are as follows:(1) We developed a fluorescent immunoassay strategy based on the nickingenzyme signal amplification. In this work, human IgG was chosen as a detectionmodel. Firstly, sheep against human IgG antibody, human IgG and biotin-labeledantibody were sequentially fixed on the polystyrene microplate to form theimmuno-sandwich structure by means of the traditional ELISA method. Then, the biotin-labeled antibody was linked with biotin-labeled primer probe bystreptavidin through biotin–SA specific interaction. Next, the primer probehybridized with the template and initiated nicking enzyme signal amplification inthe cooperation of polymerase and nicking endonuclease. Finally, the displacedstrand after the NESA was hybridized with molecular beacon, the opening ofmolecular beacon results in great fluorescence enhancement. The determination oftarget antigen could be realized by assaying the fluorescence generated. Bycombining the traditional ELISA method with the novel NESA technique, theestablished strategy holds advantages of high efficiency, high sensitivity with thedetection limit as low as0.1ng/mL.(2) We developed a novel fluorescence assay for DNA phosphatase based onthe nicking enzyme signal amplification. The method aims at the detection of DNA3’-phosphatase, using T4polynucleotide kinase phosphatase (PNKP) as a detectionmodel. The designed3’-phosphoryl hairpin probe can be dephosphorylated into3’-hydroxyl end by T4PNKP, leading to the initiation of polymerase extension andtriggering the nicking enzyme signal amplification. The displaced plenty ofssDNA after many “polymerization-nicking” cycles hybridized with molecularbeacons, yielding significant fluorescence signal after the opening of themolecular beacons. The detection of T4PNKP can be accomplished by monitoringthe fluorescence signal change. The proposed strategy is convenient, highlysensitive with a low detection limit of0.167U/mL.(3) We developed a label-free fluorescent biosensor for assay of uracil–DNAglycosylase (UDG) based on the nicking enzyme signal amplification. The methodutilized UDG as the detection model. Firstly, a single-stranded DNA was partiallyhybridized with a hairpin substrate probe which contains uracil-rich sequences andthe nicking site for nicking endonuclease to form the double-stranded substrate forUDG. After the treatment of UDG, uracil bases were removed from thedeoxyribose phosphate backbone, leading to the decrease of the meltingtemperature of the double-stranded substrate, causing the dissociation the dsDNAunder the experimental conditions. The released substrate probe can selfhybridized to form a hairpin structure at its3’ end, then the nicking enzyme signalamplification can be triggered in the cooperation of polymerase, nickingendonuclease and dNTPs. The displaced plenty of ssDNA after the amplificationprocess can form the G-quadruplex in the presence of K~+, which can result insignificant fluorescence signal after binding with N-methyl mesoporphyrin IX (NMM). By combining the recognition process of UDG and the nicking enzymesignal amplification, the method shows an obvious improved sensitivity throughthe specific binding affinity between the G-quadruplex and NMM. The establishedstrategy is label-free and convenient which can be applied to effectively andsensitively assay of UDG with a low detection limit of2U/L.