Construction Of DNA Logic Gate Enzyme-free Signal Sensing Analysis Method And Application Of MiRNA Detection

Small molecule ribonucleic acid(miRNA)is an evolutionarily conserved family of endogenous 19-22 yuan long noncoding RNA,involved in post-transcriptional regulation and cell differentiation,proliferation,apoptosis,metabolism ? The mechanism of miRNA in a variety of physiological and pathological processes is to achieve precise early diagnosis of major diseases and gene therapy for prognosis.In this work,the microfluidic chip integrated platform and DNA enzyme-free signal amplification method are introduced into DNA logic gates,then the surface-enhanced Raman scattering(SERS)technology is used to construct a new type of functionalized DNA enzyme-free signal amplification logic system.The logic system can realize a series of logic operations,and apply the logic operations to highly sensitive and selective mi RNA detection.On this basis,we further introduced the target-initiated cascade reaction DNA machine into the logic gate operation,and designed a new cascade reaction DNA nanomachine logic gate platform.Combining SERS and fluorescence technology,the dual-mode signal imaging analysis of miRNA in tumor cells and the output and treatment of silenced genes are realized.This paper includes the following two parts:(1)We adopt SERS technology and DNA enzyme-free signal amplification technology,and use microfluidic chip as an integrated platform to construct a new type of functional DNA enzyme-free signal amplification logic system.In the logic gate conversion unit,a kind of displacement reaction zone with folding fan structure as the main body was constructed.On this basis,for the first time,the combination of microfluidic technology and biological logic gates,overcome the problem of slow DNA hybridization using its rapid enrichment function and further improve the reaction efficiency.We have designed three logic gates of AND,XOR and OR to realize the signal response of different logic gates.We have constructed a series of general logic operations based on fan-shaped structure,and simultaneously realized accurate quantitative detection in vitro.The success of this method has demonstrated the potential of biological logic system with DNA without enzymatic signal amplification in the detection of trace amounts of tumor-related characteristic molecules.(2)On the basis of DNA functionalized logic operations,we constructed a DNA nanomechanical(DNM)logic system based on miRNA driven cascade reaction to realize self-assembly reaction induced by miRNA and release SiRNA in living cells.The DNM logic system was constructed by combining two pairs of DNA/RNA hybridization(DR and D 'R ')with gold nanoparticle scaffolds and swinging arm hairpins.The dual inputs of miR-21 and mi R-155 used as logic inputs triggers the DNA cascade strand displacement reaction in DNM,outputs SiRNA,and releases the SERS signal.The released SiRNA can inhibit the expression of the mRNA and protein in the cell,and silence the highly efficient genes that promote tumor growth in the body.This work realized the real-time monitoring and detection of miRNA,delivered silent genes for the diagnosis and treatment of tumor,constructed a cascade reaction DNA nanomachine logic gate platform based on in living cells,and provided new ideas and ways for early diagnosis and treatment of diseases.