Novel Biomolecules Sensing Methods In Living Cells Based On Nucleic Acids Probes

Living organisms is highly compartmentalized.The multicellularity enables the growth of complex life forms as it allows for the specialization of cell types,differentiation,large-scale spatial organization and also the cellular communication.To achieve these goals,various biomolecular machines inside the cell work together to orchestrate multiple intracellular and extracellular cues rapidly and simultaneously.If something went wrong during the process,it may lead to the dysfunction,diseases and even cancer.Thus,to study the character,the differences and the behavior of these biomolecular at a single molecule and a single cell level has significant importance for us to understand life and utilize it for a better life.In the context,the exquisite advantages of nucleic acids have motivated extensive efforts.Using the specific Watson-Crick interactions,it could be manipulated in a predictable manner.With combination of versatile modifications of reactive and reporting groups,or functional DNA modules such as DNA probes,DNA triplex,aptamers,and DNAzyme,it could further be engineered to constructing nanostructures and programmable circuits.So far,many biosensors based on nucleic acids has been successfully constructed and have good performance.Here,we have developed several novel biosensors for sensing specific disease-related biochemical species in living cells with high sensitivity and high selectivity.The details are summarized as follows:In Chapter 2,we have developed a novel branched hybridization chain reaction(b HCR)circuit for efficient signal-amplified imaging of m RNA in living cells.The b HCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit.The b HCR circuit enables to generate a hyperbranched assembly seeded from a single target initiator,affording the potential for localizing single target molecules in live cells.In vitro assays show that b HCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 f M.Live cell studies reveal that b HCR displays intense fluorescence spots indicating m RNA localization in living cells with improved contrast.The b HCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics.In Chapter 3,We have developed a p H-responsive,fully-reversible hybridization chain reaction(HCR)assembly that allows sensitive sensing and imaging of p H in living cells.Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly.The HCR assembly has shown dynamic responses in physiological p H ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of p H.Moreover,this method affords HCR assembles with highly localized fluorescence responses,offering advantages of improving sensitivity and better selectivity.This proton-fueled,reversible HCR assembly may provide a useful approach for p H-related cell biology study and disease diagnostics.In Chapter 4,We have developed a dynamic DNA nanomachine that is anchored on cells surface and undergoes p H-responsive triplex-duplex conformation switching,allowing tunable sensing and imaging of extracellular p H.The results reveal that the DNA nanomachine can be stably anchored on cell surface via multiple anchors,and the adjustment of C~+·G-C content in the switch element confers tunability of p H response windows.The anchored DNA nanomachine also demonstrates desirable sensitivity,excellent reversibility and quantitative ability for extracellular p H detection and imaging.In Chapter 5,we have further reported the development of gold nanoflares with logic computing function used as diagnostic automata,sensitively handling with multi-parameter nucleic acid cues in living cells.The results show that the diagnostic automata entered cells with high efficiency and has good biocompatibility.They enabled accurate diagnosis on multi-mi RNAs pattern in different cell lines and also the fluctuations in the same cell line.Moreover,the diagnostic automata innovate on the diagnostic mode,instead of exporting exact data for each parameter,they output the diagnostic result(“YES”or“NO”)directly according to the built-in computation code.