Efficient Detection And Imaging Of Intracellular RNA Based On Functional Nucleic Acid Nanoprobes

RNA is a class of biomolecules that play an important role in biological activities such as gene coding,decoding,regulation and expression.MicroRNA?miRNA?is a class of endogenously expressed non-protein-encoded single-stranded RNA of approximately 19-23 nucleotides that regulates in viral and animal and plant gene expression.Messenger RNA?mRNA?transfers genetic information from DNA to ribosomes and specif ies the amino acid sequence of the gene expression product protein.MiRNA or mRNA plays important regulatory roles in a range of life activities in organisms,and abnormal expression of miRNA or mRNA is associated with the development of cancer.Therefore,miRNA or mRNA is considered as a potential tumor-related biomarker.The development of reliable,practical,highly sensitive,highly specific,rapid and effective RNA in vivo detection technology is of great significance for cancer detection,prevention and treatment.The specific research contents of th e thesis are as follows:In Chapter 2,we developed a functional nucleic acid nanoprobe based on the hybridization chain reaction?HCR?to achieve nucleic acid signal amplification for ultrasensitive fluorescence activated imaging of miRNA in living cells.The functional nucleic acid nanoprobe was assembled by electrostatic interaction and?-?interaction of the g-C₃N₄ nanolayer and molecular beacons H1 and H2?labelled with BHQ2 and Cy5?.The g-C₃N₄ nanolayer had excellent biocompatibility,and could efficiently load DNA hairpin probes and had high efficiency in cell delivery,and could be used as a tracer to avoid false negative signals.HCR provided efficient signal amplification with a detection limit of 0.13nM for miRNA-21.The strategy developed in this chapter might provide a valuable platform for the detection of low-abundance biomarkers in living cells.In Chapter 3,we constructed a multicolor nanoprobe for simultaneous detection and imaging of three tumor-related mRNAs in living cells.The multicolor nanoprobe consist ed of three parts:the core was a gold nanoparticle,the middle layer was a cationic peptide bonded by an Au-thiol bond,and the outer layer was an electrostatically assembled three molecular beacons labelled with different fluorophores for targeting three mRNAs in cells,respectively.The functional nucleic acid nanoprobe was simple to prepare and exhibit ed high specificity,high sensitivity and good biocompatibility.Cell imaging experiments showed that the nanoprobe could effectively distinguish cancer cells from normal cells and could identify changes in the expression levels of tumor-related mRNA in living cells.Compared to the traditional detection of single tumor-related mRNA,this strategy could provide valuable information for early cancer diagnosis and could effectively avoid false positive results.