The Construction Of Multifunctional Nucleic Acid Probes And Its Application In Detection Of Bioactive Molecules

When the body is attacked by external radiation such as ionizing radiation,chemical mutagen and virus infection,the normal physiological functions of nucleic acid molecules（DNA or RNA）will be severely damaged.For example,the bases in DNA are removed to form vacancies,the hydroxyl groups at the 3’end of DNA are phosphorylated as well as the guanine（G）in DNA is oxidized to 8-oxguanine（8-oxoG）.If these DNA damages can not be repaired in time,it may cause the death of cell,the mutation of the gene and the malignant transformation into the tumor cells.In order to maintain the normal expression and physiological function of genetic information,a large number of biologically active molecules in the cell actively participate in the process of DNA repair.For example,the tumor suppressor gene p53 DNA repairs cell growth through repairing of intracellular gene defects and controlling the growth of cells as a“genome-protective shield”.T4Polynucleotide Kinase（T4 PNK）can restore the 3’-phosphateend to normal hydroxyl and effectively prevents lesions;The FPG enzyme can recognize and remove 8-oxoG in damaged DNA fragments and then restore the normal cellular activity.Therefore,developing a sensitive and selective sensing platform for the above biologically active molecules（p53 DNA,T4 PNK,FPG enzyme）and its mutation analysis is essential,which will provide powerful theoretical and technical support for the study of the tumor diseases and cancer.As we known,the above bioactive molecules are expressed in cells with low abundance.However,it is difficult to detect the above biologically active molecules with conventional molecular beacons in cell lysates due to their low detection sensitivity.Hence,it is highly desirable to develop a novel nucleic acid molecular probe with high sensitivity for the low-abundance molecules detection and the constructed probe should meet the following aspects:（1）The signal can be enriched and amplified sensitively;（2）It can reduce the background signal of the complex substrate and improve the signal-to-noise ratio.In this paper,we combined the nucleic acid probes coupled with magnetic Fe₃O₄nanoparticles to design a multifunctional nucleic acid probe which enable to integrate the target recognition,signal transduction and signal amplification into one system.On the one hand,based on the large specific surface and fast magnetic response of magnetic nano Fe₃O₄,the false positive signal caused by redundant nucleic acid probes in the system can be eliminated,and the background signal can be reduced by separating the complex sample base.On the other hand,the linear（exponential）rolling ring amplification strateges were used to realize the multiple signal amplification to improve the detection sensitivity of p53DNA and DNA repair enzymes（T4 PNK and FPG）.The main contents include:1、Construction of a spontaneous cyclic multifunction nucleic acid probe based onrolling ring amplification reaction and magnetic Fe₃O₄ nanoparticles for the detection of FPG enzyme activityHerein,we develop a simple and sensitive FPG activity assay by taking advantage of target-induced self-primed rolling circle amplification and magnetic nanoprobe.A unique nick（8-oxoguanine,8-oxoG）was positioned in duplex DNA containing P-circle and P1.The presence of FPG specifically binds and cleaves 8-oxoG containing duplex DNA resulting 5’-phosphoryl termini.Meanwhile,a phosphodiester bond between the5’-phosphoryl and 3’hydroxyl termini was formed with the addition of T4 DNA ligase,producing an unnicked circular strand.Using the unnicked strand as RCA template,the P1tail in duplex DNA as RCA primer proceeds to do initiate the RCA reaction.Subsequently,the RCA produced amount of long tandem-repeat complementary sequences of FAM modified DNA probes（FP）and bio-modified DNA probes（BP）of magnetic microbead（MB）.Finally,multiple FP was hybridized to the RCA products,resulting in greatly amplified enrichment of fluorophores on the surface of MB.Due to the RCA enhanced highly-concentrated fluorescence accumulation on MB,an ultralow detection limit of1.033 U/mL for FPG was obtained.Combined with the high tolerance capability for human blood serum owing to magnetic isolation,the FPG assays in human blood serum by fluorescence and confocal laser scanning microscopy confocal images were also achieved.These results indicate that this robust self-primed isothermal amplification combined withmagnetic isolation is an excellent candidate for quantitatively monitoring FPG activity to serve to DNA oxidative damage-related clinical diagnosis and therapy.2、Construction of multi-functional magnetic nano probe based on hyperbranched ring amplification reaction and the activity detection of T4 polynucleotide kinaseFirstly,a partial hairpin DNA（HP）probe with 3’phosphoryl terminal was immobilized on the surface of streptavidin-magnetic nanobeads（MBs）.In the presence of T4 PNK,the HP probe was dephosphorylated,subsequently initiates the extension reaction by polymerases,generating a complete duplex containing the specific nicking site of the Nb.Bsm I.Next,the Nb.Bsm I recognized and cleaved the duplex,liberating DNA fragments.Finally,the DNA fragments subsequently initiated the HRCA reaction whose products can be monitored by SYBR GreenI（SG）.Then performing the separated function,the binding of SG and substrate probes could be avoided and a low background was acquired with a detection limit of 0.008 mU/mL.Furthermore,we detected the activity of T4 PNK enzyme in the Hela cell lysate and made a screening test for the inhibitor,and all obtained satisfactory results.3、Construction of a triple-helix magnetic probe based on rolling circle amplification and fluorescent liposomes for the detection of p53 DNAThe triple-helix probe modified streptavidin was coupled with magnetic nanoparticles modified biotin inorder to forming the triple-helix magnetic probe based on Streptavidin biotin interaction.The outer hairpin sequence of the triple-helix nucleic acid probe and the effective fragment of p53 DNA satisfy the principle of base complementation,which is beneficial to the target identification of p53 DNA.The nucleic acid short chain,which is surrounded by triple-helix probe,is the primer chain that triggers the RCA reaction.In the presence of p53 DNA,the hairpin is opened and the intermediate primer chain is released,thereby triggering a subsequent RCA reaction.The RCA product can be hybridized with the liposome-DNA probe coated with SYBR Green I（SG）.The magnetic field separation can avoid the interference of the substrate probe.After adding the fragmentation agent,strong fluorescence signals can be generated,which realizes the label free sensitive detection of p53 DNA.The detection limit of the triple-helix magnetic probe for p53 DNAcan be as low as 0.07 fM,which is better than the detection limit of p53 DNA based on optical sensing principle reported in the literature.In order to investigate the detection ability of the method for real samples,we also analyzed the p53 DNA in cell lysate by adding standard recovery method,and obtained satisfactory results.Therefore,the method proposed in this paper can be used for the sensitive detection of p53 DNA in complex biological samples and is expected to be further applied in clinical practice.