Energy transfer and enforced intercalation: Responsive as well as bright DNA-based high performance probes for RNA imaging in live cells

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Fluorogenic hybridization probes enable real-time measurements of RNA transport within living wild-type cells. Powerful probes furnish significant enhancements of fluorescence emission upon binding of the RNA target. However, the brightness of fluorescence is another important parameter which critically affects the signal-to-noise ratio. Previous work fell short in meeting the challenge to simultaneously improve responsiveness and brightness of probes. By contrast, most newly developed probes have been fashioned to include additional options for fluorescence quenching. To enable the imaging of less abundant RNA, we will develop fluorogenic oligonucleotides, which combine high responsiveness with high brightness. Another important aim concerns the extension of the repertoire of colors, which is required for simultaneous detection of multiple RNA targets. In addition, we want to develop a tool for the quantitative expression analysis (RNA counting) beyond quantitative imaging.To achieve these aims, we will develop novel DNA-based FIT-probes. These probes contain cyanine dyes of the thiazole orange (TO) family of dyes. The TO and TO-like dyes serve as fluorogenic nucleobase surrogates which are forced to intercalate at selected sites of the probe-target complex. The introduction of Locked Nucleic Acid (LNA) rigidifies the backbone in the vicinity of the dye. The accompanying improvement of base stacking interaction will impede torsional twisting around a cyanine methin bridge. As a result, quantum yields of cyanine fluorescence will be increased. Additional, slightly red-shifted cyanine dyes will interact with TO in a synergistic fashion. The spectral overlap will contribute to the increased brightness of the probes. Without using additional fluorophores or modifications, we will further the brightness as well as the color palette. A tag repeat system will involve the adjacent hybridization of multiple differently colored FIT-probes which interact via FRET. To achieve quantitative RNA imaging we will equip FIT-DNA with additional NIR dyes, the fluorescence of which will remain insensitive to collisions or contact with the TO-nucleotide. As a result, emission of the NIR dye will provide information about the concentration of probes, while TO emission will report the hybridization status of the probe. The powerful hybridization probes will enable the imaging of less abundant mRNA molecules at the early stage of an infection by Influenza. Up to three different mRNA molecules will be localized simultaneously. The study aims at deciphering the temporal orchestration of the mRNA localization within the nucleoli during infection. In further collaborative work we will characterize the transport of oskar mRNA within developing oocytes from Drosophila. To examine the influence of the probe backbone (ionic vs. non-ionic) on the outcome of localization experiments both DNA-based and PNA-based FIT-probes will be introduced by microinjection.