Kuffner Christopher J, Marzilli Alexander M, Ngo John T
bioRxiv. 2024 Nov 21:2024.11.21.624393. doi: 10.1101/2024.11.21.624393.
RNA localization and regulation are critical for cellular function, yet many live RNA imaging tools suffer from limited sensitivity due to background emissions from unbound probes. Here, we introduce conditionally stable variants of MS2 and PP7 coat proteins (which we name dMCP and dPCP) designed to decrease background in live-cell RNA imaging. Using a protein engineering approach that combines circular permutation and degron masking, we generated dMCP and dPCP variants that rapidly degrade except when bound to cognate RNA ligands. These enhancements enabled the sensitive visualization of single mRNA molecules undergoing differential regulation within various sub-compartments of live cells. We further demonstrate dual-color imaging with orthogonal MS2 and PP7 motifs, allowing simultaneous low-background visualization of distinct RNA species within the same cell. Overall, this work provides versatile, low-background probes for RNA imaging, which should have broad utility in the imaging and biotechnological utilization of MS2- and PP7-containing RNAs.
RNA定位和调控对细胞功能至关重要，但许多活细胞RNA成像工具由于未结合探针的背景发射而灵敏度有限。在这里，我们引入了MS2和PP7外壳蛋白的条件稳定变体（我们将其命名为dMCP和dPCP），旨在降低活细胞RNA成像中的背景。使用结合了环形排列和降解子屏蔽的蛋白质工程方法，我们生成了dMCP和dPCP变体，这些变体在未与同源RNA配体结合时会迅速降解。这些改进使得在活细胞的各个亚区室中经历差异调控的单个mRNA分子能够被灵敏地可视化。我们进一步展示了使用正交MS2和PP7基序的双色成像，允许在同一细胞内同时对不同的RNA种类进行低背景可视化。总体而言，这项工作为RNA成像提供了通用的、低背景的探针，这在含MS2和PP7的RNA的成像和生物技术应用中应该具有广泛的用途。
