Protein-PNA conjugates as conditional BRET platforms for life cell mRNA imaging and quantification

在这项提议的框架内，将开发一种新的高灵敏度成像方法，以实现实时可视化和空间量化细胞中低丰度的mRNA异构体。尽管现有的RNA成像方法多种多样，但由于目前的策略缺乏敏感性，实时研究活细胞中的mRNA异构体仍然是一个主要障碍。重要的是，这些主要通过选择性剪接形成的低丰度转录本对蛋白质多样性有重要贡献，并在发育、神经元可塑性和神经系统疾病(如阿尔茨海默氏症、脊髓性肌萎缩症(SMA)等)中发挥核心作用。基于SMA相关的已有研究的mRNA亚型，我们的目标是开发一个PNA-NanoBRET生物成像平台，使后续基因在活细胞中实时表达。这一新方法最终将在转基因细胞和患者成纤维细胞中进行测试。

Within the framework of this proposal, a new highly sensitive imaging method will be developed to enable visualization and spatial quantification of low abundance mRNA isoforms in cells in real time. Despite the variety of existing RNA imaging methods, it is major hurdle to study mRNA isoforms in real time in living cells due to the lack of sensitivity of current strategies. Importantly, these low abundance transcripts formed mainly by alternative splicing contribute significantly to protein diversity and play a central role in development, neuronal plasticity, and nervous system disorders such as Alzheimer's, spinal muscular atrophy (SMA), etc. Based on the well-characterized mRNA isoforms responsible of SMA, we aim to develop a PNA-NanoBRET bioimaging platform, which allows follow gene expression in real time in living cells. This novel method will ultimately be tested in genetically modified cells and patient fibroblasts.