Novel Living Cell Imaging Methods Of Functional DNA Nanoprobes

As the carrier of genetic material,DNA is the most important material in the organism and plays vital roles in the organism.The discovery of DNA nanotechnology has broken the inherent cognition that DNA is just genetic material.DNA itself has unique properties that make it an excellent nanomaterial for constructing DNA nanostructures.At the same time,it also endows DNA nanostructures with unique properties such as editability,addressability,and specificity.Based on these advantages,DNA nanotechnology can be used to construct nanostructures not only with different shapes and sizes,but also with different functions.Therefore,DNA nanostructures have been widely utilized in the field of biosensing.The construction of functionalized DNA nanoprobes is an important development direction of DNA nanotechnology.In this thesis,we have constructed some new biosensors using DNA nanoprobes and applied them to live cell imaging and gene therapy.The isothermal nucleic acid amplification technology can amplify signals by several,dozens,or even hundreds of times under mild conditions,providing a powerful tool for the detection of trace biomarkers.Enzyme-free isothermal nucleic acid amplification enables the detection of intracellular biomarkers.DNAzyme’s specific cleavage ability and changeable double-arm sequences enable it to cleave any substrate RNA.In Chapter 2,we designed a Target induced reconstruction of DNAzymatic amplifier nanomachines in living cells for concurrent imaging and gene silencing.In this design,we combined catalytic hairpin assembly with DNAzyme,and inserted DNAzyme into the hairpin probe through the reasonable design of the DNA hairpin.In the absence of target,no signal was generated,and DNAzyme was inactivated.However,in the presence of the target,the amplification reaction was triggered and the DNA hairpin probes were opened,followed by the release of DNAzyme.DNAzyme was activated and cut the substrate RNA,thereby achieving gene silencing.This method provided a multifunctional platform for simultaneous intracellular imaging and gene silencing.With the development of DNA nanotechnology,the assembly of nanostructures with different morphologies has been realized.In order to make the assembly of DNA nanotubes simpler and more convenient,a modular assembly method is proposed.In Chapter 3,we proposed a "one-pot" assembly method that further simplified the synthesis step of DNA nanotubes,which can be assembled into DNA nano tube in a single step of annealing.Human apurinic apyrimidinic endonuclease/redox effector factor 1(APE1/Ref-1)is an important enzyme in the process of base excision and repair,which is involved in many physiological processes in vivo.APE1 cleaves the abasic sites in the double strand but does not show much activity in the single strand containing abasic sites.In addition,APE1 is sequence independent and can cleave debasing sites in any sequence.The structure of DNA nanotubes consisted of two parts:a "rung" building unit and a linking chain.Therefore,we used the double strands containing abasic sites as the connecting strands and assemble d them into DNA nanotube structures.The DNA nanostructure serve d as a delivery vector to transport the double-stranded DNA probes in response to APE1 into cells and performed specific and sensitive intracellular imaging of APE1.Hybridized DNA nanostructure is an important kind of DNA nanostructure,which is assembled by combining DNA with other nanomaterials.I-motif,cytosine-rich DNA sequence,is a p H-specific sequence that folds into a four-stranded structure under acidic conditions and a free single-stranded structure under alkaline conditions.Therefore,the i-motif configuration can be changed by adjusting p H.In Chapter 4,we assembled a p H-responsive nanostructure based on the specific binding of streptavidin and biotin and the i-motif sequence.DNA nanostructures were assembled by DNA complementary hybridization using streptavidin as scaffold and i-motif sequence as network,which maintained stable nanostructures in alkaline environment.When the p H of the environment became acidic,the i-motif sequence was induced to fold into a four-strand structure,followed by the disintegration of DNA double chain and DNA nanostructure.The nanostructure could specifically respond to p H and realize intracellular p H imaging.Meanwhile this DNA nanotube is a promising drug carrier.DNA nanostructures constructed based on protein and DNA provide a new strategy for the assembly of nanostructures.One molecule of streptavidin can bind four biotin molecules with high specificity and affinity.Avidin is a protein that has similar binding properties to streptavidin.In Chapter 5,we assembled a nanostructure that specifically responded to APE1 based on the specific strong binding of avidin and biotin.First,two avidin-based DNA tetrads were constructed,and then the two tetrads were linked by complementary base pairing using an DNA strand to form DNA nanostructures.Two DNA chains modified with biotin con tain abasic sites Therefore,in the presence of APE1,the abasic sites in the nanostructure were sheared,and the hybrid double-strand became unstable and melted,leading to the disintegration of the nanostructure.The unique interaction between avidin and APE1 enabled DNA nanostructures to specifically respond to APE1 while providing protection for DNA probes.The nanostructure provided a detection method with excellent performance,and the released single chain could be designed as a chain with specific functions,giving the nanostructure the potential for biomedical applications.