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活细胞中检测和定位特定的RNA分子，而不需要基因细胞工程。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。这个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），它将i)通过改进的荧光增强来报告从非结合状态到目标结合状态的转变，ii)提供高亮度的荧光信号，iii)允许在独立的检测通道中监测探针的局部浓度。RNA FIT探针含有一个碱基替代物，在探针杂交后荧光发射增强。我们将开发新的荧光基替代物，并引入选择性的氨基修饰，以提高荧光量子产率。多荧光FIT探针将提供亮度和响应探针杂交的增强。这将是决定性的，以尽量减少同质fret。通过在碱基替代物上启用点击共轭，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反应性染料充当浓度报告者。如果有一种技术可以根据特定转录物的表达对活的、基因未修饰的细胞进行分类和扩增，那么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T细胞受体（TCR）互补决定区3 （CDR3）的mRNA编码成像中的应用。探针将通过微孔或通过两亲嵌段共聚物输送到特征良好的T细胞系中。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细胞是否识别癌组织是由TCR CDR3决定的。长期目标是评估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。

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.