Development of single molecule-sensitive, imaging probes targeting native RNA
Lifland, Aaron William
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The localization, trafficking and regulation of messenger ribonucleic acids (RNA) and viral RNA play crucial roles in cellular homeostasis and disease pathogenesis. In recent years biochemical and molecular biology methods used to study RNA function have made several important advances in the areas of RNA interference, expression of transgenes, and the sequencing of transcriptomes. In contrast, current technologies for imaging RNA in live cells remain in limited use. Previous studies of RNA localization and dynamics have relied primarily on the expression of a reporter RNA and a fluorescent protein fusion that binds to aptamer sequences in the expressed RNA. While these plasmid based systems offer methodological flexibility, there remains a need to develop methods to image native, non-engineered RNA as plasmid derived RNAs may not have the same regulatory elements (3'UTR and introns) or copy number as the native RNA. Additionally, viral pathogenesis is often sensitive to the size and sequence of their genomic RNA and may not be suitable for study using engineered systems. We sought to develop and validate a new method for imaging native, non-engineered RNA with single molecule-sensitivity. These probes have four important properties. They are modular, compatible with fixation and immunostaining, bind quickly and specifically to targets, and do not interfere with RNA function. We built upon the technique of delivering exogenous, linear probes that bind to their target by Watson-Crick base pairing. The probes are multiply labeled and tetramerized to increase their brightness. To validate the probes, targeting and utility was demonstrated in two model systems: beta-actin mRNA to show targeting of an endogenous target and the genomic RNA of human respiratory syncytial virus to show targeting of a viral RNA target. All video files are in QuickTime format.