Construction And Sensing Of Proximity Bioorthogonal Reactions Driven By DNA Circuits

It is important to achieve sensitive and accurate detection and imaging of biomolecules at the cellular level,as well as understanding and elaborating their biological functions at the molecular level to provide more biological information for early diagnosis and treatment of diseases.Nucleic acid molecular probes are widely applied to the construction and design of biosensors due to their distinctive programmability,specificity and sensitivity.However,nucleic acid molecular probes are prone to degradation by nucleases in complex environments such as blood and cells,resulting in high background signals and false positive results,which limits further applications of nucleic acid molecular probes.Bioorthogonal reactions are widely used in the regulation of intracellular biological reaction processes and the detection of biomolecules in virtue of their high chemical specificity,tunable reaction kinetics,and excellent biocompatibility.Therefore,researchers’ interests were greatly attracted by the nucleic acid template-driven biorthogonal reaction,which became an important technology for RNA detection and imaging.In the traditional template reaction of nucleic acid,a nucleic acid chain is used as a template to hybridize with two functional DNA probes to realize the spatial proximity of orthogonal reaction chemicals and efficiently drive the reaction to produce signal output.However,such methods have disadvantages such as insufficient signal amplification efficiency and limitation of only detecting nucleic acid targets,which precludes the further applications of nucleic acid template reactions.In response to these problems,this paper develops a highly accurate and specific bioorthogonal sensor for the detection of tumor markers in living cells,as detailed in the following:In Chapter 2,we designed and synthesized a pair of bioorthogonal partners Tz(tetrazine molecules)and PF(prefluorescent small molecules blocked by vinyl ether).Fluorescence of small molecules can be quenched by the introduction of vinyl ether at the hydroxyl site of the fluorophore active phenyl group.Tz and PF are modified on two DNA strands by SPAAC click reactions,respectively.After adding the template strand,due to the specific hybridization between DNA,the spatial proximity between Tz and PF,so that the PF decage reaction occurred successfully and fluorescence was activated.It is indicated that the hybridization of nucleic acid templates can accelerate the orthogonal reaction rate between small molecules,allowing the Tz and PF reactions to occur smoothly under physiological conditions,which provides a new idea for the detection of tumor markers in cells.In Chapter 3,we have developed a DNA-mediated proximity-driven bioorthogonal reaction(PBRC)for accurate and amplification detection of non-nucleic acid targets Intracellular and extracellular.By combining bioorthogonal reaction with DNA-mediated aptamer biotechnology,bioorthogonal fluorescent probes(HA-Tz and HA-PF)were constructed using template amplification reactions(HCR).In vitro and in vivo studies have shown that the improved efficiency of Tz-mediated PF decage reaction is due to DNA circuit-mediated proximity effects rather than chemical specificity.The unique "one-to-one orthogonality" of bioorthogonality allows PBRC probes to effectively avoid false positive signals.PBRC probes have the ability not only to specifically detect ATP in vitro and in living cells,but also distinguish different levels of ATP expression in cells.To verify the universal applicability of the approach,we redesigned another PBRC probe for the detection of thrombin protein.This approach is robust and extends the application of template bioorthogonal reaction in multifunctional aptamers and provides a promising tool for biomarker detection and early diagnosis of diseases.In Chapter 4,we constructed a catalytic hairpin assembly(CHA)mediated bioorthogonal reaction of DNA-template for precise imaging of mRNA in live cells.Two DNA hairpin probes(H1-Tz and H2-PF)containing tetrazine molecule(Tz)or vinyl ether caged fluorophore(PF)were designed and synthesized.When the target mRNA was added,CHA was triggered,and the bioorthogonal reaction partners was spatially brought closer,the local effective concentration increased,and the reaction rate increased,so that PF fluorescence was activated.These findings demonstrate that the fluorescence sensor with a high signal-to-background ratio of ～30,and can detect sensitively detect the target mRNA with a detection limit of 4.6 p M.In addition,the bioorthogonality of the fluorescence sensor reduces interference from complex samples and avoids false positive signals caused by non-specific degradation.DNA templatemediated bioorthogonal fluorescence sensors can not only accurately image mRNA in cells,but also distinguish between relative mRNA expression levels in tumor cells and normal cells.Based on the above features,this method provides a useful tool for basic research and early clinical diagnosis of bioorthogonal chemistry.