Tran Jeremy C, Kuffner Christopher J, Marzilli Alexander M, Miller Ryan Emily, Silfen Zachary E, McMahan Jeffrey B, Sloas D Christopher, Chen Christopher S, Ngo John T
Department of Biomedical Engineering, Boston University, Boston, MA, USA.
Biological Design Center, Boston University, Boston, MA, USA.
Nat Commun. 2025 Jan 20;16(1):852. doi: 10.1038/s41467-025-56148-7.
Synthetic Notch (SynNotch) receptors function like natural Notch proteins and can be used to install customized sense-and-respond capabilities into mammalian cells. Here, we introduce an adaptor-based strategy for regulating SynNotch activity via fluorescein isomers and analogs. Using an optimized fluorescein-binding SynNotch receptor, we describe ways to chemically control SynNotch signaling, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o-nitrobenzyl-caged fluorescein conjugate. We further show that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can be used to regulate SynNotch activity depending on the folding state of collagen-based ECM networks. To demonstrate the utility of these tools, we apply them to activate dose-dependent gene expression responses and to induce myogenic-like phenotypes in multipotent fibroblasts with spatiotemporal and microenvironmental control. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to ligands based on the clinically-approved fluorescein dye.
合成Notch（SynNotch）受体的功能类似于天然Notch蛋白，可用于将定制的感知和响应能力植入哺乳动物细胞。在此，我们介绍一种基于衔接子的策略，用于通过荧光素异构体和类似物调节SynNotch活性。利用优化的结合荧光素的SynNotch受体，我们描述了化学控制SynNotch信号传导的方法，包括基于生物正交化学连接的方法和通过邻硝基苄基笼蔽荧光素缀合物的光图案化解笼蔽实现的空间可控策略。我们进一步表明，荧光素缀合的细胞外基质（ECM）结合肽可用于根据基于胶原蛋白的ECM网络的折叠状态调节SynNotch活性。为了证明这些工具的实用性，我们将它们应用于激活剂量依赖性基因表达反应，并在多能成纤维细胞中通过时空和微环境控制诱导肌源性样表型。总体而言，我们引入了一种优化的结合荧光素的SynNotch，作为一种基于临床批准的荧光素染料调节对配体转录反应的通用工具。
