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

荧光杂交探针能够实时测量活的野生型细胞内的RNA运输。强大的探针在结合RNA靶标时提供了显著的荧光发射增强。然而，荧光的亮度是影响信噪比的另一个重要参数。以前的工作在同时提高探测器的响应性和亮度方面存在不足。相比之下，大多数新开发的探针都包括荧光猝灭的附加选项。为了使较少丰富的RNA成像，我们将开发荧光寡核苷酸，它结合了高响应性和高亮度。另一个重要的目标是扩展颜色库，这是同时检测多个RNA目标所必需的。此外，我们希望开发一种定量成像之外的定量表达分析（RNA计数）工具。为了实现这些目标，我们将开发新的基于dna的fit探针。这些探针含有噻唑橙（TO）染料家族的菁染料。TO和TO样染料作为荧光性核碱基替代物，被迫插入探针-靶复合物的选定位点。锁定核酸（LNA）的引入使染料附近的主干硬化。随着基层相互作用的改善，将会抑制花青素桥的扭转扭转。结果，菁荧光的量子产率将提高。另外，稍微红移的花青素染料将以协同方式与TO相互作用。光谱重叠将有助于增加探测器的亮度。不使用额外的荧光团或修改，我们将进一步提高亮度以及调色板。标记重复系统将涉及多个不同颜色的通过FRET相互作用的fit探针的相邻杂交。为了实现定量RNA成像，我们将为FIT-DNA配备额外的近红外染料，其荧光对碰撞或与To -核苷酸接触不敏感。因此，近红外染料的发射将提供有关探针浓度的信息，而TO发射将报告探针的杂交状态。强大的杂交探针将使在流感感染的早期阶段较少丰富的mRNA分子成像成为可能。最多三种不同的mRNA分子将同时被定位。该研究旨在破译感染期间核仁内mRNA定位的时间编排。在进一步的合作工作中，我们将表征oskar mRNA在发育中的果蝇卵母细胞中的转运。为了研究探针主链（离子型和非离子型）对定位实验结果的影响，我们将通过显微注射引入基于dna和基于pna的fit探针。

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.