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

用于 mRNA 成像和活细胞识别的亮度和对比度增强 RNA 杂交探针

• 批准号: 429038820
• 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: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/429038820?language=en
• 关键词: Brightness; contrast; enhanced; RNA; hybridization

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.

荧光寡核苷酸杂交探针允许在不进行基因细胞工程的情况下检测和定位活细胞中的特定RNA分子。目前可用的探针提供的检测限不足以检测/成像在培养基中表达的RNA分子，更不用说低拷贝数了。该项目的目标是缩小技术差距。我们将开发RNA杂交探针（RNA-FIT探针），其将i）通过改进的荧光增强来报告从未结合状态到靶结合状态的转变，ii）提供高亮度的荧光信号，以及iii）允许在独立的检测通道中监测探针的局部浓度。RNA FIT探针含有碱基替代物，其在探针杂交时经历荧光发射的增强。我们将开发新的荧光碱基替代物，并引入增加荧光量子产率的选择性氨基修饰。多荧光体FIT探针将提供亮度和对探针杂交的响应的增强。将homo-FRET最小化将是决定性的。通过在碱基替代物处实现点击缀合，我们将引入通过能量转移增加亮度的光捕获辅助染料。一种不能参与能量转移的非响应性染料，作为浓度报告者。如果有一种技术可以根据特定转录本的表达来分选和扩增活的、非遗传修饰的细胞，那么细胞生物学的许多领域都将受益。我们将应用并评估对比度和亮度增强的RNA-FIT探针在编码T细胞受体（TCR）互补决定区3（CDR 3）的mRNA成像中的应用。探针将通过微孔或通过两亲性嵌段共聚物递送到充分表征的T细胞系中。荧光显微镜和基于荧光的细胞计数将揭示探针区分不同细胞系的能力。T细胞是否识别癌组织由TCR CDR 3决定。长期目标是评估已经通过测序方法鉴定的癌症反应性T细胞是否适合通过FACS进行分选。