Simultaneous Detection Of Two Long Non-coding RNAs In Cancer Cells At Single-molecule Level

Non-coding RNAs(ncRNAs)are non-protein-coding RNA molecules transcribed from genomes,and they play a vital role in epigenetics,post-transcriptional regulation and chromatin modification.The long ncRNAs(lncRNAs)are a type of non-coding RNA,which accounts for an important proportion in the transcriptome.LncRNAs participate in genomic imprinting,chromatin modeling and various biological processes,and has become a hot spot in biomedical research.Aberrant change in lncRNA is closely related to the occurrence and development of diseases.It affects the expression of oncogenes,tumor suppressor genes,metabolic enzyme genes and transcription factors,and is an important marker for the diagnosis and treatment of cancer.Therefore,it is particularly important to develop a sensitive and efficient method for the accurate quantification of lncRNA in clinical samples.However,the current detection techniques for lncRNA usually involve amplification steps,complex operations and expensive enzymes,which makes the application not ideal.Although great progress has been made in the identification of lncRNA with the improvement of technology,the simultaneous detection of lncRNAs has remained a great challenge due to their large size and extensive secondary structure.Herein,we develop an single-molecule detection method for simultaneous detection of multiple lncRNAs in cancer cells based on target-catalyzed strand displacement.We designed the magnetic bead(MB)-capture probe-multiple Cy3/Cy5-modified reporter unit complexes to isolate and identify lncRNA MALAT1 and lncRNA HOTAIR.In the presence of lncRNA MALAT1 and lncRNA HOTAIR,the target-catalyzed strand displacement reactions lead to the release of Cy3 and Cy5 fluorescent molecules from the complexes,which can be subsequently quantified by single-molecule detection.Where Cy3 indicates the presence of lncRNA MALAT1 and Cy5 indicates the presence of lncRNA HOTAIR.This method has the characteristics of dual-targetability,prominent selectivity and high sensitivity with a detection limit of 0.10 a M for lncRNA MALAT1 and 0.031 a M for lncRNA HOTAIR.This method can not only detect lncRNA MALAT1 and lncRNA HOTAIR in different cell extracts with ultrasensitive detection,but also detect lncRNA in living cells through single-molecule imaging.Importantly,single-molecule imaging can distinguish tumor cells from normal cells.This method has distinct advantages of simple sequence design and free of any enzymes,holding great potential in early clinical diagnosis.