Electrochemical Study Based On DNAzyme Of Biomolecular Interactions

The nature of life activity is the process of biomolecular interactions.Exploration of life phenomena such as gene expression,signal transduction,and immune response can be ascribed to the study of the biomolecular interactions.DNA and protein are two most important biological macromolecules in life.Among them,DNA is the genetic material of life and protein is the function carrier of life.Therefore,the study of DNA-protein interactions and protein-protein interactions will further reveal the esoteric nature of life from multiple dimensions such as molecules,cells,and organisms.At present,there are many limitations such as complex steps,low sensitivity,and high cost for existing analysis methods.In recent years,biosensing technologies,especially electrochemical biosensors based on enzymatic catalysis for signal amplification,have gained widespread attention due to their advantages of simple operation,high sensitivity and low cost.Among them,the deoxyribozyme,essentially a single strand of DNA with good ability for structure recognition and high catalytic activity,can provide new auxiliary means while a new biosensor is constructed.This dissertation introduces a stratage based on biomolecular interactions-induced specific migration of deoxyribozymes on the surface of the electrode to obtain differential electrical signals.The dynamic binding process of protein complexes,the screening of DNA nucleic acid base sequences,specific detection of proteins and other multi-purpose targets were studied.The details are as follows:1.Electrochemical Study Based on DNAzyme of DNA-proteininteraction analysis.In this study,we have demonstrated that the attachment of protein molecule onto the DNAzyme strand may result in a reduced enzymatic activity.Based on this finding,we have then introduced an electrochemical assay method for specific and sensitive analysis of interaction between DNA and protein in nucleic extracts,based on the protein-induced distinctive motion behavior of DNA deoxyribozyme(DNAzyme)on an electrode surface.In order to further verify the experimental principle,we have also presented assays for the rapid,sensitive,and selective detection of aptamer(Apt29 as an example)and transcription factors(NF-κB,SP6 RNA polymerase and HNF-4α as examples)for analysis and verification.And the specific nucleic acid sequence was analyzed for binding to the target protein under partial base mutation conditions,and the nucleic acid sequence specifically binding to the HNF-4α protein was even screened in vitro.This work may not only attain quantitative information on DNA-binding protein(at nanomolar level)and may open new opportunity for in-depth profiling of the sequence specificity of DNA-binding proteins and study of nucleotide polymorphisms in known protein-binding sites.Finally,the concept proposed in this report may also be further developed for sensitive,on-chip,and simultaneous detection of multiple molecular interactions,making this method economical and easily adaptable into currently available microfluidic platforms.2.Electrochemical Study Based on DNAzyme of protein-proteininteraction analysis.In this work,we have developed a novel,versatile signal transduction and amplification strategy for the one-step detection of protein and PPI,named dSTEA based on the protein binding-induced distinctive motion behavior of DNAzyme on the electrode surface.In this method,we controlled the particle size of the target protein with the aid of the gold-nanoparticle and find that the attachment of a large protein complex can significantly reduce the activity of DNAzyme to cleave substrate DNA on the electrode surface,thus producing a distinguished signal response.The result demonstrated that the cleavage activity of DNAzyme toward the substrate DNA modified on the electrode surface is inversely correlated with the hydrodynamic diameter of the macromolecule attached to it.This work can not only detect subpicomolar protein interaction events but also analyze the assembly of kinase in the whole cell extract.Moreover,this novel signaling mechanism proposed in this work may broaden the applicability of DNAzyme-based electrochemical assays and it may also have great potential for applications in other interfacial sensor developments.