Mi Lan, You Mingxu
Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA; Molecular and Cellular Biology Program, University of Massachusetts, Amherst, MA 01003, USA.
SLAS Discov. 2025 Jun 16;35:100243. doi: 10.1016/j.slasd.2025.100243.
Fluorescence- and bioluminescence-based probes are valuable tools for understanding cell functions in health and disease. Bioluminescence offers an ideal complementary readout to fluorescence due to its minimal background interference and self-illuminating nature. We previously introduced the first type of genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors. These RNA-based probes are highly programmable and can be modularly engineered to detect various cellular targets. While this system was successfully validated in vitro and from the entire cell population within a microplate, the BRET signals were quite dim and difficult to visualize at the single-cell level under a microscope. The ability of single-cell bioluminescence imaging is critical for studying cell-to-cell variations and spatiotemporal changes of cellular targets in different signaling pathways or upon drug treatment. In this study, we will introduce strategies that can enhance the functionality and capability of RNA-based BRET sensors for real-time cellular imaging and sensing. Using commonly used widefield microscopes, single-cell bioluminescent detection of various metabolites and other small molecules can be achieved in both bacterial and mammalian cells. This advancement represents a significant step toward the future development of genetically encoded RNA-based bioluminescent tools for studying disease mechanisms, high-throughput drug screening, and in vivo imaging.
基于荧光和生物发光的探针是了解健康和疾病状态下细胞功能的宝贵工具。由于背景干扰极小且具有自发光特性，生物发光为荧光提供了理想的互补读数。我们之前介绍了第一种基于基因编码的RNA生物发光共振能量转移（BRET）传感器。这些基于RNA的探针具有高度可编程性，并且可以通过模块化设计来检测各种细胞靶点。虽然该系统已在体外和微孔板中的整个细胞群体中成功得到验证，但BRET信号相当微弱，在显微镜下的单细胞水平很难可视化。单细胞生物发光成像能力对于研究不同信号通路中或药物处理后细胞靶点的细胞间差异和时空变化至关重要。在本研究中，我们将介绍一些策略，这些策略可以增强基于RNA的BRET传感器用于实时细胞成像和传感的功能和能力。使用常用的宽场显微镜，可以在细菌和哺乳动物细胞中实现对各种代谢物和其他小分子的单细胞生物发光检测。这一进展代表了朝着基于基因编码的RNA生物发光工具未来发展迈出的重要一步，这些工具可用于研究疾病机制、高通量药物筛选和体内成像。
