Application Of Intelligent DNA Strand Displacement Reaction In MicroRNA In Situ Detection

MicroRNA(miRNA)is a class of endogenous,non-coding single-stranded small molecule.As key regulator of gene expression,it plays an important role in various physiological and pathological processes.The abnormal expression of miRNA is related to the occurrence and progression of many diseases,such as cancer,cardiovascular disease and diabetes et al,which has been regarded as a potential biomarker and gene therapy target.However,the design and construction detection system for intracellular miRNA detection with high sensitivity and good selectivity is still urgently needed since its short sequence,low expression abundance,high family homology,complex intracellular and extracellular environment and heterogeneity of cell expression.DNA strand displacement amplification reactions show great potentials for intracellular miRNA detection.Herein,we constructed three types of intelligent probes for miRNA detection through the design of strand displacement location,reaction method and space,which realize intracellular tumor-related miRNA detection with the highly specificity and sensitivity.The works are presented as following:1.Sensitively distinguishing intracellular precursor and mature microRNA abundanceWe develop a novel cascade reaction to sensitively distinguish miRNAs versus pre-miRNAs in living cells based on two pairs of programmable hairpin oligonucleotide probes with a simple sequence design.The programmable hairpin probes can metastably coexist until the introduction of miRNAs or pre-miRNAs,which can trigger a specific hybridization chain reaction(HCR),respectively,leading to the self-assembly of nicked DNA duplex structures and a remarkable specific fluorescence intensity increase.Compared with the conventional molecular beacon(MB),the detection system can sensitively assess the miRNA and pre-miRNA abundance in a homogeneous solution.More importantly,it avoid the false positive signal due to the presence of mature miRNA sequences in their pre-miRNA form.Thus,direct in situ imaging intracellular miRNA and pre-miRNA with high sensitivity and selectivity in diferent living cells is realized.2.DNA "nano wheel" Probe for visualizing intracellular microRNA via localized DNA cascade reactionWe designed a DNA "nano wheel"(DNW)probe for high sensitivity miRNA imaging in living cells.In this system,six modified DNA hairpin hybrid with a specially programmed DNA nanowire,miRNA trigger DNA hairpins sequential hybridization through accelerated localized DNA cascade reaction(LDCR)along the DNA nanowire and open of the self-quenched hairpins to assemble six-arm branched junction "nano wheel" structure lighted with three fluorescence dye.Notably,the recycled miRNA target further acts as a catalyst to generate multiple lighted DNW structures,thus one target miRNA enable to trigger strong and amplified fluorescence signal for intracellular sensitive imaging.It outperforms the "one-to-one" conventional molecular beacon(MB)and intermolecular hybridization-based six-arm branched junction(SFHM).It main because the LDCR endows the system with fast reaction kinetics owing to the high local concentration.The programmed DNW provides a useful and valuable tool for rapid and highly efficient visualization of low-abundance miRNA in living cells,which is beneficial to understand the miRNA function and exploration of its biomedical application.3.Bioinspired framework nucleic acid capture sensitively and rapidly resolving microRNAs in living cellsInspired by the "tentacles" of an octopus,herein,we present a framework nucleic acid(FNA)capture for sensitive,rapid,and multiplexed imaging of miRNAs cancer biomarkers in living cells.The programmable FNA is designed using three DNA triangular prism(DTP)nanostructures carrying two pairs of metastable catalytic hairpin assembled(CHA)probes,AS1411 aptamer,and pendent biotinylated DNA strand in different vertexes and is further assembled via streptavidin to form the multivalent DTP(SA-DTP).The SA-DTP system acts as an octopus that captures the target cancer miRNAs quickly and delivers them preferentially among DTPs' "tentacles" in the SA-DTP system to produce strong,amplified fluorescence for detection.Precise multiplexed imaging of miRNA-155 and miRNA-21 cancer biomarkers' aberrant expression and dynamic change in different cells demonstrates the feasibility of both monitoring disease progression and evaluating therapeutic efficacy.