Grant: Research Tools and Instruments

It has been reported by the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of Mouse (FANTOM) consortiums that coding transcripts represent less than 3% of the total genome. These findings surprisingly suggest that the vast majority of transcription products have no coding potential. Importantly, recent work suggests that a significant proportion of non-coding transcripts are functionally active RNA molecules, which are expected to primarily exert their biological function through their structure and interaction with cellular partners. Since RNA folds sequentially during transcription (i.e., cotranscriptional folding), the functional RNA conformation can be strongly influenced by kinetic parameters directing the RNA polymerase during transcription. The understanding of RNA cotranscriptional folding remains in its infancy and still constitutes one of the long-standing challenges in biology. Currently, single-molecule Förster Resonance Energy Transfer (smFRET) provides the highest level of structural dynamics information that can be obtained from RNA structures without interference from ensemble averaging. This is in part achieved by the very high sensitivity and speed of the electron-multiplying charge-coupled device (EMCCD) camera that allows to record in real time the fluorescence emission of single molecules, which is crucial to study the structural dynamics of nascent RNA molecules. We are requesting funds to replace our 16 years old EMCCD camera that is showing signs of imminent failure. If this critical piece of equipment were to break without replacement available, the majority of our funded projects would be stopped, thus preventing the progression of one research assistant, three Ph.D. and four M.Sc. students. Additionally, to our knowledge, our smFRET imaging system is one of the first built in Canada and is now lagging behind in term of technical capacities and in ease of use for new users. Thus, to remain competitive and to stay at the forefront of the field, we want to take this opportunity to upgrade the possibilities of our system by adding a laser, a new set of filters, a beam splitter and by updating the imaging software. Overall, the requested EMCCD camera will ensure that our research activities are not jeopardized due to a hardware failure of the 16 years old camera. The additional requested components are also highly important as they will expand the possible measurements that can be made on a single sample and will facilitate the maintenance of the equipment as well as the introduction of the technique to new users.