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

能量转移和强制嵌入：用于活细胞 RNA 成像的响应且明亮的基于 DNA 的高性能探针

基本信息

批准号：
52097295
负责人：
Professor Dr. Oliver Seitz
金额：
--
依托单位：
Institut für Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2007
资助国家：
德国
起止时间：
2006-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/52097295?language=en
关键词：
Energy transfer enforced intercalation Responsive

项目摘要

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.

荧光杂交探针能够实时测量活的野生型细胞内的RNA转运。强有力的探针在结合RNA靶时提供荧光发射的显著增强。然而，荧光的亮度是另一个重要的参数，其严重影响信噪比。以前的工作未能满足同时提高探头的响应性和亮度的挑战。相比之下，大多数新开发的探针已经被设计成包括用于荧光猝灭的附加选项。为了使不太丰富的RNA成像，我们将开发荧光寡核苷酸，它结合了联合收割机的高响应性和高亮度。另一个重要的目标是扩展颜色库，这是同时检测多个RNA靶所必需的。此外，我们希望开发一种定量成像以外的定量表达分析（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子（TO）染料家族的花青染料。TO和TO样染料充当荧光核碱基替代物，其被迫嵌入探针-靶标复合物的选定位点。锁核酸（LNA）的引入使染料附近的主链刚性化。伴随的碱基堆积相互作用的改善将阻止花青甲川桥周围的扭转。结果，花菁荧光的量子产率将增加。另外，稍微红移的花青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针的亮度。不使用额外的荧光团或修改，我们将进一步的亮度以及调色板。标签重复系统将涉及通过FRET相互作用的多个不同颜色的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NIR染料，其荧光将保持对与TO-核苷酸的碰撞或接触不敏感。因此，NIR染料的发射将提供关于探针浓度的信息，而TO发射将报告探针的杂交状态。强大的杂交探针将能够在流感感染的早期阶段对丰度较低的mRNA分子进行成像。多达三种不同的mRNA分子将同时定位。这项研究的目的是破译感染过程中的mRNA定位在核仁内的时间编排。在进一步的合作工作中，我们将描述oskar mRNA在果蝇卵母细胞中的运输。为了检查探针骨架（离子型与非离子型）对定位实验结果的影响，将通过显微注射引入基于DNA和基于PNA的FITC探针。