Live-cell Imaging Of RNA Based On Fluorescent Light-up Aptamers

RNA is an important genetic material of all life forms.The genetic information carried by DNA translates into functional protein with m RNA as an intermediate.In addition,some non-coding RNAs also play an important role in regulation of gene expression.Mutation or abnormal expression of RNAs often lead to diseases,such as innate immune diseases,neurodegenerative diseases,vascular diseases,and cancers.Understanding the distribution,transport,and molecular interactions of RNAs at the sub-cellular level is of great significance to the study of how RNA participates in gene regulation.Such information can be directly obtained by live-cell imaging of RNA.There are many methods for fluorescent labeling of RNAs.Among them,fluorescence light-up RNA aptamers hold great potential due to their small size,low background,and high biocompatibility.In this thesis,fluorescence light-up RNA aptamers-based cell imaging approaches were constructed to observe the abundance and subcellular distribution of circular RNA and m RNA in real time,which helps to monitor the transcription and translation process of m RNA.The main research contents and results are summarized as follows:1.Construction of split-aptamer-based fluorescent probe for live cell imaging of circular RNACircular RNA is a type of newly found non-coding RNA,which plays a vital role in both physiological and pathological processes.Although hundreds of thousands of circular RNAs have been identified through bioinformatics,their cellular processes and regulatory mechanisms remain unexplored.Imaging of circular RNA can provide direct information about its abundance,distribution,and translocation,which helps to understand its function and regulation.The current methods for visualizing circular RNA are mainly limited to fixed samples,and live-cell imaging methods for circular RNA are in demand.The low abundance and the presence of precursor linear RNA make the visualization of circular RNA difficult.In this part,we constructed a genetically encoded split-aptamer-based fluorescent probe(SAFP),and applied it in live-cell imaging of CDR1as,which plays an important role in gene regulation.SAFP can successfully image CDR1as in real time with high selectivity.Imaging-based analysis and comparison of the expression levels of CDR1as in a variety of cell lines were performed.The results prove that CDR1as is mainly expressed in neurons and embryonic tissues.In addition,SAFP realizes real-time monitoring of the expression levels of CDR1as in the long-term process(9 days)of neuronal differentiation of P19cells.The above results indicate that SAFP probe has the potential to function as a universal method for circ RNA imaging in living cells.2.Long-term tracking of transcription and translation by fluorescence light-up RNA aptamersThe transcription and translation processes are strictly controlled by spatiotemporal regulation to ensure that the proteins are synthesized in the correct developmental stage and tissue.Therefore,the establishment of methods for real-time imaging of transcription and translation in living cell is essential for understanding the dynamic processes of gene transcription,m RNA transport,and translation regulation.The commonly used m RNA fluorescent labeling system MS2-GFP system has limitations such as high background,extra burden of expression,and possible interference with the ransport and physiological functions of target m RNAs.It is necessary to establish a new m RNA fluorescent labeling method.In this part,we developed a RNA labeling module based on fluorescence light-up RNA aptamers,and used this module to label the m RNA encoding red fluorescent protein to construct RFP-3WJ-4Corn.The plasmid can be used for long-term imaging and tracing of m RNA and its protein products in living cells.Through this method,we tracked the transcription,transport,and translation of m RNA in He La cells in real time for up to 20 hours.m RNA was observed to be transported from the nucleus to the cytoplasm,and was partially localized in the endoplasmic reticulum.The signal of the protein initially co-localized with the m RNA signal,and then diffused into the cytoplasm.In addition,the definition of I_RFP/I_Corn as the translation expression level coefficient can be used to evaluate the translation level of a single cell.The above results indicate that the RFP-3WJ-4Corn system is suitable for long-term detection and tracing of the translation process of m RNA at single cell level.