New generation of bright fluorogenic RNA probes for sensitive gene expression monitoring in live-cell imaging experiments – BrightRiboProbes

The homeostasis of the cell and its adaptation to environmental changes rely on tight and coordinated gene expression regulation occurring especially through pathways, which involve not only proteins but also non-coding RNAs. Identifying the different actors of a regulatory pathway but also its dynamics are key factors to the proper understanding of these mechanisms, their integration as well as the consequences of their dysfunctioning. Because each cell of a population adapts differently its gene expression profile, such studies must be performed at a single cell resolution. Therefore, characterizing the dynamics of the regulatory pathways requires the use of non-disruptive and non-invasive technologies compatible with cell life. Imaging fulfills these criteria provided that bright enough fluorescent probes are available. Whereas the study of protein-coding genes has been greatly facilitated by the development of fluorescent proteins, visualization of non-coding RNAs is technically more challenging.
BightRiboProbes project is motivated by a strong technological gap existing in live-cell imaging of RNA. Indeed, imaging of low-abundant RNAs is currently limited to hybridization-based technologies starting by fixing and permeabilizing cells. Consequently, despite their high sensitivity (made possible by the elevated brightness of the probes), these technologies do not allow for monitoring expression dynamics of target genes at the RNA level in living cells. Such information could be accessed using fluorogenic RNA aptamers able to bind and activate the fluorescence of fluorogenic organic dyes. Nevertheless, the systems currently available present either a low brightness or a poor affinity to RNA. Therefore, the use of current fluorogenic aptamers is essentially restricted to abundant RNAs and does not allow monitoring low abundant RNAs such as messenger RNAs and regulatory RNAs.
This project ambitions the development of a breakthrough technology to monitor low-abundant RNAs in live-cell imaging experiments using any type of cells such as bacterial and human cells. We will develop a new generation of very bright fluorogenic dyes, non-fluorescent in their free state and cell membrane-permeable. In parallel, we will select RNA aptamers interacting specifically and with high affinity with these dyes to efficiently turn their fluorescence on. We will develop dye/RNA couples fluorescing in green, red and near infrared; and we will validate them (efficient RNA expression and cell entry of the dyes) in cellulo in bacteria and eukaryotic cells. Among the numerous possible biological applications, we will apply this new generation of probes in combination with conventional fluorescence microscopy as well as super resolution microscopy to characterize the dynamics of a network regulating the expression of virulence genes in Staphylococcus aureus. This data will not only lead to accurate kinetic characterization of the control and the expression of virulence genes but also will allow discriminating those cells in the population that activate a given regulatory pathway and will analyze the cell responses in response to stress. Overall, we hope to gain a better appreciation of the collective behavior of the whole bacterial population. In addition and as pointed above, even though we will use a bacterial biological model, the whole technology will developed to be compatible to any cell type.
These ambitious objectives will be reached by associating three teams with necessary and complementary skills in chemistry, RNA evolution, genetics, microbiology, microfluidics and super resolution microscopy.