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80 RNA labeling and dynamics in live bacterial cells

Implementation of the fluorescent proteins has made labeling proteins and DNA in live cells a routine procedure. At the same time labeling RNA in vivo is still a challenge. Most methods for labeling RNA make use of RNA aptamers attached to RNA. These aptamers are then labeled with a cognate RNA-binding protein that is fused with the fluorescent protein. Several years ago we modified this aptamer-based approach by introducing protein complementation (PC). In this approach the fluorescent protein is split and non-fluorescent until it is bound to RNA. Application of PC allowed to reduce the fluorescent background and thus to increase sensitivity of RNA detection (Valencia-Burton et al., 2007, 2009). To overcome another important limitation of the aptamer-based RNA labelingthe necessity to modify RNA by aptamer taggingwe combined protein complementation with the split aptamer approach (Toran et al., 2014, see Fig). In this method the cells are programmed to express two RNA probes that contain split aptamer sequences flanked with the anti-sense arms. Both RNA probes are expressed within one transcript. In the presence of the target RNA the RNA probes bind to the target via the anti-sense sequences which brings together the fragments of the split aptamer. The aptamer re-assembles and binds two split proteins which trigger protein complementation. In the course of this study we uncovered a mechanism of RNP complex formation that resulted in very high sensitivity of RNA detection. Our experiments revealed specific localization of an endogenous bacterial mRNA expressed on a physiological level. This work is the first example of the RNA-templated reassembly of split aptamers in vivo, which expands the set of available tools for the RNA-dependent circuit engineering. This research has been supported by the NSF Award 1050609.