Brightness- and contrast-enhanced RNA hybridization probes for mRNA imaging and recognition of living cells

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Fluorogenic oligonucleotide hybridization probes allow the detection and localization of specific RNA molecules in live cells without genetic cell engineering. The detection limit provided by the currently available probes is not sufficient to detect/image RNA molecules expressed in medium let alone low copy numbers. It is the objective of this project to close the technology gap. We will develop RNA hybridization probes (RNA-FIT probes), which will i) report the transition from the unbound to the target bound state by improved enhancements of fluorescence, ii) afford high brightness of fluorescence signaling and iii) allow monitoring of the probe’s local concentration in an independent detection channel. RNA FIT probes contain a base surrogate, which experiences enhancements of fluorescence emission upon probe hybridization. We will develop new fluorescent base surrogates and introduce select amino modifications that increase the fluorescence quantum yields. Multifluorophor FIT probes will provide enhancements of both brightness and the response to probe hybridization. It will be decisive to minimize homo-FRET. By enabling click conjugation at the base surrogate we will introduce light harvesting auxiliary dyes that increase brightness via energy transfer. A non-responsive dye, which cannot participate in energy transfer, serves as a concentration reporter.Many areas in cell biology would profit if there were a technology available that allowed to sort and expand living, genetically non-modified cells based on the expression of specific transcripts. We will apply and evaluate the contrast- and brightness-enhanced RNA-FIT probes in the imaging of mRNA coding for the complementarity determining region 3 (CDR3) of the T cell receptor (TCR). The probes will be delivered into well-characterized T cell lines by microporation or via amphiphilic block copolymers. Fluorescence microscopy and fluorescence-based cell cytometry will reveal the probe’s ability to distinguish between different cell lines. Whether or not T cells recognize cancer tissue is determined by the TCR CDR3. It is the long-term objective to assess whether cancer reactive T cells, which have been identified by means of sequencing methods, are amenable to sorting via FACS.