Label-free Detection Of LncRNA In Cancer Cells With G-quadruplex DNA-thioflavin T Binding-induced Fluorescence

Only 2% of the human genome is translated into proteins,and the rest mostly is transcribed into noncoding RNAs(ncRNAs).Non-coding(nc)RNAs play a vital role in biological processes and they are associated with various diseases including cancer.Long non-coding RNAs(lncRNAs)belong to non-coding RNA and they are transcripts with a length of greater than 200 nt.The lncRNAs were usually considered as the transcriptional noise without biological function.However,recent evidence reveals that lncRNAs play critical roles in biological processes(e.g.,chromatin remodeling,transcriptional regulation,post-transcriptional regulation,and trafficking of proteins in cytoplasm),pathological processes(e.g.,amino acid metabolism,glucose metabolism disorders,altered immune system function,lipid metabolism disorders).Moreover,lncRNAs are engaged in diverse tumor-related biological events such as cell differentiation,apoptosis,migration,invasion and metastatic.High expression of lncRNAs(e.g.,HOX gene antisense intergenic RNA(HOTAIR))can promote tumor growth and metastasis by silencing the expression of tumor suppressor genes,while down-regulation of HOTAIR expression may reduce tumor growth and metastasis.Moreover,lncRNAs may function as the biomarkers for the diagnosis and treatment of cancers.Consequently,sensitive detection of lncRNAs is essential to lncRNAs-related biomedical research,clinical diagnosis and therapy.Conventional methods for lncRNA assay include microarray,RNA sequencing,digital PCR(dPCR),but they are limited by expensive instrumentation,poor specificity,complicated procedures,long analysis time,poor sensitivity,complex probe design and false positivity resulting from incomplete fluorescence quenching.Therefore,detection of low-abundance lncRNAs remains a great challenge.In this thesis,we develop a new label-free method to detect lncRNAs in cancer cells based on DSN-assisted target recycling,TdT-mediated amplification strategy,and human telomere G-quadruplex DNA-Thioflavin T binding-induced fluorescence.The 5′-terminal biotinylated DNA capture probe can specifically hybridize with lncRNAs to obtain a DNA-RNA heteroduplex.Subsequently,duplex-specific nuclease(DSN)selectively hydrolyzes the DNA capture probe in the heteroduplex,resulting in the release of both lncRNAs and the cleaved ssDNA fragments with 3′-OH termini.The hybridization of the released lncRNAs with new capture probes initiates new rounds of DSN cleavage reaction,generating abundant ssDNA fragments with 3′-OH termini.Because capture probe is biotinylated at the 5′end,the resultant ssDNA fragments can assemble onto the surface of magnetic beads(MBs)via biotin-streptavidin interaction.Then terminal deoxynucleotidyl transferase(TdT)catalyzes the incorporation of dATPs at 3′-OH termini of ssDNA fragments to obtain a long poly-adenine(p A)strand.The resultant long poly(A)-strand on the surface of magnetic beads can hybridize with numerous HTG signal probes to construct the MB-poly(A)-strand-HTG signal probe complex.After the magnetic separation,the captured signal probes are released through deionized water and heating treatment.Finally,thioflavin T(ThT)specifically binds to the G-quadruplex DNA domain of the released HTG signal probe to generate an enhanced fluorescence signal at 485 nm.This method displays high sensitivity with a limit detection of 1.7 fM.This method can be further employed to detect lncRNAs in different kinds of cancer cells and even distinguish cancer cells from normal cells.Moreover,this method can be used to quantify the siRNA-mediated silencing of the lncRNA HOTAIR gene in cancer cells,offering a new approach for lncRNA-based biomedical research,clinical diagnosis,and therapeutics.