Development Of Fluorescence Method For Sensitive Detection Of Histone Deacetylase And Polynucleotide Kinase

Epigenetic inheritance is a heritable change in gene function independent of alterations in nucleotide sequence.Epigenetics regulates the normal cellular activities of the organisms by affecting gene expression and transcription,and maintains a stable environment in the body.Therefore,the detection of epigenetic biomarkers plays important roles in epigenetics-related biochemical research and clinic diagnosis.In this thesis,we develop two fluorescence methods for sensitive detection of epigenetic biomarkers.We use histone deacetylase and polynucleotide kinases as model targets to demonstrate the proof of concept.The detailed contents are as follows:1.Deacetylation-activated construction of single quantum dot-based nanosensor for sirtuin1 assay.We develop a single quantum dot（QD）-based-nanosensor for sensitive sirtuin 1（SIRT1）assay.SIRT1 is an important histone deacetylase that regulates biological functions ranging from DNA repair to metabolism.The alteration of SIRT1 is associated with a variety of diseases including diabetes,inflammation and cancers.Consequently,the detection of SIRT1 activity is of great therapeutic importance.This single QD-based nanosensor is composed of a Cy5-labeled peptide substrate and a streptavidin-coated QD.The peptide with one lysine acetyl group acts as both the Cy5 fluorophore carrier and the substrate for sensing SIRT1.In the presence of SIRT1,it removes the acetyl group in the acetylated peptide,and the resultant deacetylated peptide can react with the NHS-activated biotin reagent to form the biotinylated peptide.The multiple biotinylated peptides can assemble on single QD surface via biotin-streptavidin interaction,inducing efficient fluorescence resonance energy transfer（FRET）from the QD to Cy5,generating distinct Cy5 signal which can be simply quantified by total internal reflection fluorescence-based single-molecule detection.This single QD-based nanosensor can sensitively detect SIRT1 with a detection limit of as low as 3.91×10^-12 M,and it can be applied for the measurement of enzyme kinetic parameters and the screening of SIRT1 inhibitors.Moreover,this nanosensor can be used to detect the SIRT1activity in cancer cells,providing a powerful platform for epigenetic research and SIRT1-targeted drug discovery.2.Sensitive detection of polynucleotide kinase with hairpin template-based circular exponential amplification assay.We develop a label-free fluorescent method for sensitive detection of polynucleotide kinase（PNK）on the basis of circular exponential amplification in combination with hairpin templates.PNK plays a critical role through the regulation of the 5’-phosphate terminal in many cellular events including DNA recombination,DNA/RNA repair,and m RNA degradation.Abnormal PNK activity is closely associated with various human diseases such as spinocerebellar ataxia and neurodegenerative diseases.Consequently,the development of sensitive methods for PNK activity assay is highly desirable.In this work,the 5’end of probe-A substrate is phosphorylated through the catalysis of PNK.Then the phosphorylated probe-A and probe-B are ligated to produce template-AB with the assistance of DNA ligase.The hairpin template-AB can initiate two consecutive strand displacement amplification（SDA）reactions,constituting a circular exponential amplification which enables the conversion of a small amount of target PNK signal to large numbers of DNA trigger signals.This assay exhibits high sensitivity with a detection limit of as low as 9.5×10^-8 U/μL,and it enables the monitoring cellular PNK activity at the single-cell level,holding great potential in clinical diagnosis and biomedical research.