Fuel Molecule Driven Nucleic Acid Amplification For Intracellular MicroRNA Fluorescence Imaging

The expression information of miRNAs in cells is of great value for resolving the complexity and heterogeneity of miRNA-related diseases,as well as the early diagnosis and treatment of diseases.However,to achieve this,the following challenges must be confronted: First,how to overcome the barrier of cell membrane and effectively deliver the DNA probes into living cells.Sencond,how to improve the anti-interference ability,to ensure that the probes can work normally in living cells and the interference of complex environment is effectively reduced or avoided.Third,how to improve the sensitivity.Conventional methods are usually not sensitive enough,and can’t directly be applied for in-situ detection.In recent years,enzyme-free nucleic acid amplification technology has the advantages of mild reaction conditions,protease-free and flexible sequence design,which make it a powerful tool in miRNA detection.Herein,we take intracellular miRNA as the research object,gold nanoparticles and DNA nanostructures as the nanocarriers,then construct new nucleic acid probes driven by fuels based on enzyme-free nucleic acid amplification technology,nanotechnology and fluorescence resonance energy transfer(FRET)to realize sensitive and accurate imaging of miRNA in living cells or in vivo.Contents in this dissertation include five parts as following:1.The traditional nanobeacons based on one target to one signal readout mode are not suitable for imaging low-abundance miRNA in living cells owing to the relatively low sensitivity and insufficient readout signal.To solve this problem,we designed a hairpin-fuelled catalytic nanobeacon for amplified micro RNA imaging in living cells.The intracellular specific miRNA could be used as a catalyst to trigger the hybridization reaction between a large number of nanobeacons and fuels,then significantly enhanced fluorescence signals were generated upon adding the DNA fuels.In this process,the target miRNAs were recycled,achieving the purpose of activating multiple signal probes by one target,thus the sensitivity was improved.Compared with traditional nanobeacons,the catalytic nanobeacons greatly improve the sensitivity,and can be used for highly sensitive fluorescence imaging of specific miRNAs in tumor cells.2.In view of the complex design and the slow kinetics of cascaded circuits,herein,we proposed a newly designed paradigm called catalyst accelerated circular cascaded circuits,where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration.This new design is so simple that only requires three hairpin probes.The reaction system started with the HCR circuits where target miRNA initiated the hybridization chain reaction to form a linear polymer with hairpins structure.Then,the catalyst strand catalyzed CHA circuits to generate a long linear DNA duplex polymer.Meanwhile,a large number of re-assembled targets analogs were formed,which acted as new initiator to trigger the cross-active reaction of HCR and CHA,thus the structure of the product will change from linear to dendritic DNA nanostructure and enhanced ratiometric fluorescent signals were obtained.Compared with the traditional HCR,CHA and linear circuit(HCR-CHA),the circular circuit has higher signal and two orders of magnitude lower detection limit than linear circuit.More importantly,the circuit can achieve sensitive imaging of low abundance miRNA in living cells by employing endogenous m RNA as catalyst without adding additional catalyst.3.In the first two parts,we employed gold nanoparticles and liposomes as carriers to improve the internalization efficiency of DNA probes.However,the synthesis and functional modification of these nano carriers are time-consuming,and have high toxicity and low biosafety in high concentrations,which greatly limits their application in vivo.In order to solve these problems,we prepared DNA nanospheres of different sized based on DNA hybridization,that is,a single chain was extended at the end of the construction unit,and the assembly of DNA nanospheres was realized through mutual hybridization among single chains of the construction units.In addition,we also examined the effects of the number of branches,the proportion,and the concentration of the construction units on the size of nanostructures.The results show that the DNA nanosphere have high cell internalization efficiency and excellent stability.The DNA nanospheres are an ideal nanocarrier to effectively transport DNA probes into cells.4.We constructed endogenous ATP-powered DNA nanospheres for amplified imaging of miRNA in living cells and in vivo based on the part three.The DNA nanospheres were self-assembled from Y-shaped DNA(Y-DNA)monomers through predesigned base pair hybridization,which contained the ATP aptamer and the recognition region of the target miRNA.Once the nanospheres were internalized into cells,the intracellular specific target miRNA would trigger the cyclic dissociation of the DNA nanospheres driven by ATP,resulting in amplified FRET signals.The DNA nanospheres employed endogenous ATP as the fuel without any auxiliary additives,which simplified the operation procedures,improved the sensitivity,and successfully realized the sensitive imaging of miRNA in live cells and mice.The design of the DNA nanospheres provide a simple,flexible and universal direction for chem/ biosensing in living cells.5.Aiming at the logical relationship between tumor cells and various miRNAs,we developed “AND” and “OR” logic DNA nanospheres for the simultaneous analysis of two miRNAs in living cells.The “AND” and “OR” logic DNA nanospheres were assembled from Y-DNA and I-DNA through predesigned base pair hybridization.When the DNA nanospheres were internalized into cells,for the “AND” logic DNA nanosphere,the cyclic dissociation of the DNA nanosphere driven by ATP could be triggered only when miR-21 and miR-155 were both at present,resulting in amplified Cy3 fluorescence signals.For the“OR” logic DNA nanosphere,the disassembly of “OR” logic DNA nanospheres powered by ATP was triggered at the presence of miR-21 or miR-155,leading to enhanced fluorescence signal readout.The logic DNA nanospheres integrate different functional units,which can be used to distinguish the target tumor cells from others and normal cells through the recognition of multiple specific miRNAs in cells.In this dissertation,we take intracellular miRNA as the research object,aming at improving sensitivity,internalization efficiency and anti-interference ability of DNA probes,a series of nucleic acid amplification strategies driven by fuel are constructed by combining nucleic acid signal amplification technology,nanotechnology and fluorescence resonance energy transfer technology,which realize the sensitive and specific imaging of miRNA in living cells and in vivo.It will provide an effective way to research the heterogeneity and complexity of miRNA-related diseases in living cells and in vivo.