Christopher J Kuffner, Alexander M Marzilli, John T Ngo
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引用次数: 0
Abstract
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 subcompartments 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-containing and PP7-containing RNAs.
期刊介绍：
Nature Methods is a monthly journal that focuses on publishing innovative methods and substantial enhancements to fundamental life sciences research techniques. Geared towards a diverse, interdisciplinary readership of researchers in academia and industry engaged in laboratory work, the journal offers new tools for research and emphasizes the immediate practical significance of the featured work. It publishes primary research papers and reviews recent technical and methodological advancements, with a particular interest in primary methods papers relevant to the biological and biomedical sciences. This includes methods rooted in chemistry with practical applications for studying biological problems.
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