DNAzyme-Based Fluorescent Probes For Amplified MicroRNA Detection In Living Cells

MicroRNAs?miRNAs?are a group of single-stranded,small,endogenous noncoding RNAs that can serve as the key controller of gene expression and play significant roles in a diverse range of biological processes.Increasing researches have indicated that the dysregulated expression of miRNAs was associated with a large variety of human diseases,such as cancers.Therefore,the development of ultrasensitive,highly selective,and in situ strategies for the detection of miRNAs are expected to be of great significance.Up to date,quite a few DNAzyme-based probes have been developed and applied for detection of vital metal ions and even biomolecules,and they have shown broad prospects for detection of intracellular nucleic acids owing to the moderate reaction conditions.In this thesis,two kinds of fluorescent probes based on DNAzyme were established to amplified detection miRNA inside live cells.The main contents are as follows:?1?A new class of intracellular nanoprobe,termed AuNP loaded split-DNAzyme probe,was developed to sense miRNA-21 in living cells.Briefly,it consists of an AuNP and substrates hybridized with two hal ves of split DNAzymes.In the absence of target miRNA,the split DNAzymes form an inactive DNAzyme motif with their substrate through partial paring at the end of each str and,and the fluorescence is quenched by both AuNP and quencher.Inside the cells,the target miRNA-21 binds with both the two halves of split DNAzymes,forming an active secondary structure in the catalytic cores,which can cleave the substrates,resulting in the rupture of the substrate and recovery of the fluorescence.Meanwhile,the target is released and binds to another inactive DNAzyme to drive another cycle of activation.In the process,a very small number of target miRNAs can initiate the cleavage of many fluorophore-labeled substrate strands from AuNP surface,providing an amplified fluorescent signal of miRNA-21,offering high detection sensitivity.Furthermore,results show that the probe can quickly enter living cells and achieve the intracellular miRNA-21 imaging.?2?A DNAzyme cascade amplification system was constructed for ultrasensitive detection of intracellular miRNA-141.The system includes three parts:S1 is a upstream locked-hairpin DNAzyme containing the miRNA-141 binding domain and cages initiator strand for downstream DNAzyme cycle,S2 is downstream locked-hairpin DNAzyme.The S3 is a substrate labeled with fluorophore and quencher at its ends.In the absence of target,both S1 and S2 form a hairpin structure by intramolecular hybridization,which could inhibit the catalytic activity of DNAzyme strand,and the fluorescence of S3 is quenched by the quencher.However,in the presence of miRNA-141,the hybridization of target and upstream DNAzyme would immediately yield an active DNAzyme,and then cleave self-strand with the assist of Mg²⁺,producing the cleaved fragment to trigger downstream DNAzyme.Meanwhile,the miRNA-141 is released and automatically hybridizes with another upstream DNAzyme to drive another cycle.The active downstream DNAzyme can cleave multiple substrates,producing an increased fluorescence.Results show that the system may enormously enhance the readout of the fluorescence signal and achieve imaging of miRNA-141 in living cells.