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

荧光寡核苷酸杂交探针允许在没有遗传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极限不足以检测/成像在介质中表达的RNA分子，更不用说低拷贝数了。缩小技术差距是这个项目的目标。我们将开发RNA杂交探针(RNA-Fit探针)，它将1)通过改进的荧光增强来报告从非结合状态到靶结合状态的转变，2)提供高亮度的荧光信号，3)允许在独立的检测通道中监测探针的局部浓度。RNA Fit探针包含一个碱基替代品，它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品，并引入选择性的氨基修饰来提高荧光量子产率。多荧光配对探针将提供对探针杂交的亮度和响应的增强。将同性恋情绪降至最低将是决定性的。通过在基础代理上启用点击共轭，我们将引入捕光辅助染料，通过能量转移增加亮度。一种不能参与能量转移的非响应性染料充当浓度报告器。如果有一种可用的技术，允许根据特定转录本的表达来分类和扩大活的、转基因的非转基因细胞，细胞生物学的许多领域都将受益。我们将应用和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(CDR3)的mRNA成像中的应用。这些探针将通过微孔化或通过两亲性嵌段共聚物传递到特征良好的T细胞系。荧光显微镜和基于荧光的细胞细胞术将揭示探针区分不同细胞系的能力。T细胞能否识别癌症组织由TCR CDR3决定。长期的目标是评估通过测序方法鉴定的癌症反应性T细胞是否可以通过流式细胞仪进行分选。

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.