Dynamic DNA Nanotechnology-Based Enzyme And Glycoprotein Detection

DNA modifying enzymes are a class of enzymes that affect changes in the chemical structure or topology of DNA.DNA modifying enzymes are involved in the synthesis,degradation and mutation of DNA and are widely found in living organisms,including eukaryotes,prokaryotes and viruses.Abnormal expression of DNA modifying enzymes has been associated with a variety of diseases,including cancer,and therefore there is an urgent need for a method that can accurately detect DNA modifying enzymes.Cell surface glycoproteins play a crucial role in various cellular processes,and the expression level of glycoproteins reflects cellular function or disease status,so detection of cell surface glycoproteins is essential.Dynamic DNA nanotechnology induces changes in systems or structures composed of DNA at the nanoscale and interacts with information from the outside.The introduction of DNA-modifying enzymes into dynamic DNA nanotechnology has enabled the development of many efficient DNA biosensors and provided new ideas for the detection of DNA-modifying enzymes themselves,while enzyme-dependent biosensors enable the detection of cell surface glycoproteins.In this thesis,an ultra-specific fluorescence detection method for DNA modifying enzymes was developed using dissipation-enabled DNA networks that can absolutely distinguish between target and non-target enzymes.Target DNA modifying enzymes exhibit a unique oscillating fluorescence signal,while non-target DNA modifying enzymes exhibit an irreversible "one-way" increase in fluorescence.This assay shows good versatility for various types of DNA modifying enzymes,including DNA repair enzymes(APE1),polynucleotide kinase(T4 PNK)and methyltransferase(Dam).Meanwhile,the dissipation-enabled probes provide a novel quantification mode based on area under curve which is more robust than those intensity-based quantifications.The detection limits of APE1,T4 PNK,and Dam reach 0.025 U/mL,0.44 U/mL,and 0.113 U/mL,respectively.This thesis combines the RNA-guided endonuclease Cas12 a in the CRISPR system with click chemistry to design a strategy for detecting cell surface glycoproteins.This strategy uses Cas12a for both efficient recognition and cleavage of target DNA and non-specific cleavage of nearby DNA strands.A DNA probe labeled with DBCO(Dibenzocyclooctyne)binds cell surface azide and activates the cis cleavage activity of Cas12a,another reporter probe labeled with both fluorescent and quenched groups corresponds to the trans cleavage activity of Cas12a and enables in situ amplification of the fluorescent signal.This strategy was used in this thesis to achieve the detection of surface glycoproteins on MCF-7(human breast cancer cells).