DNAyme-based Fluorescence Analysis For Protein Detection Via Terminal Protection Of Small-molecule-linked DNA

Protein,a basic component of living organisms,is the important active material which accurately regulates the organism normal physiologic function and sustains life.Protein is closely related to many physiological activities such as nutrient metabolism,material transport,start of defense mechanism,heredity and so on.And some research shows that some special proteins are abnormal expression and distribution in the development of disease.So the assay of proteins plays an important significance in the research of the physiologic functions of proteins and the diagnosis and treatment of interrelated diseases with proteins.The terminal protection based on small-molecule-linked DNA has aroused wide interest of researchers in protein assay.The terminal protection is that the DNA terminally tethered to a small molecule can be protected from the degradation by exonuclease when the small molecule moiety is bound to its target protein.The terminal protection is sequence-independent.It is beneficial to combination of several nucleic acid amplifications,detection methods and the terminal protection.Terminal protection provides a new way for the sensitive detection of protein.In this dissertation,we have established a series of fluorescence assay based on terminal protection of small-molecule-linked DNA for protein detection.The main contents are as follows:The first method: fluorescence quenching analysis strategy for protein detection based on terminal protection of small-molecule-linked DNA and DNAzyme.In the experiment,a hairpin DNA probe whose 3’ terminal is tethered to a biotin molecule is designed.In absence of target protein STV,the double stranded DNA(dsDNA)moiety of DNA probe is degraded from 3’ terminal by Exonuclease Ⅲ(Exo Ⅲ).The remaining undegradable single stranded DNA(ssDNA)moiety of DNA probe can form DNAzyme.DNAzyme combines with molecular beacon(MB)by hybridization and cleaves the MB at the RNA base in the middle of MB,generating fluorescence signal.In the meantime,the DNAzyme is released and combines with a new MB.The DNAzyme is recycled until all of the MB is completely used.The fluorescence signal gradually increases as more and more fluorescence groups are released.In the presence of STV,STV combines with biotin.The DNA probe is protected from the degradation by Exo Ⅲ and keep the hairpin structure.The MB cannot be cleaved and keeps fluorescence quenching without DNAzyme.In the proposed method,target protein is quantitatively detected via the decrease of fluorescence signal.As low as 50 pmol STV can be accurately detected.The new strategy is simple,fast and low cost,but it is necessary to improve its sensitivity.The second method: fluorescence growth analysis strategy for protein detection based on terminal protection of small-molecule-linked DNA and DNAzyme.In this experiment,the DNA probe tethered to a small molecule is designed to be DNAzyme sequence.The DNA probe is protected from the degradation by exonucleaseⅠ(ExoⅠ)when the small molecule moiety is bound to its target protein.DNA probe combines with MB and DNAzyme is activated.The MB is cleaved and its fluorescence group is released.The DNAzyme is recycled used to combine with new MB continually.The fluorescence signal gradually increases as more and more fluorescence groups are released.In the absence of target protein,DNA probe is degraded by ExoⅠand cannot form the DNAzyme.So,the MB keep fluorescence quenching.the fluorescence signal is very low.In this method,target protein is quantitatively detected via increase of fluorescence signal.As low as 0.1 pmol STV and 100 ng FR can be accurately detected in the volume of 100 μL.Compared of the first method,the sensitivity is improved 500 times.