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<p>RNA is one of the key molecules responsible for controlling gene expression regulation, and visualizing individual RNA molecules in living cells offers unique insights into the dynamics of this process. Recently, the RNA-regulated destabilization domain was developed for live-cell imaging of single RNA. However, this method is limited to single-color RNA imaging and its long RNA tag induces destabilization of the tagged RNA. Here we describe two orthogonal RNA-regulated destabilization domains (mDeg and pDeg) that enable three-color messenger RNA (mRNA) imaging in living cells. We show that these destabilization domains can image mRNA tethered to the endoplasmic reticulum membrane, the inner surface of the plasma membrane and in the cytosol. In addition, we show that mDeg can detect mRNA more effectively than the previously reported tDeg system. Moreover, mDeg can be combined with a short RNA tag (9XMS2) for single-molecule RNA imaging without perturbation of RNA stability.</p>

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Orthogonal RNA-regulated destabilization domains for three-color RNA imaging with minimal RNA perturbation

  • 期刊论文

  • 发表日期：

    2025年12月17日

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  • Tien G. Pham,
  • Omoyemi Ajayi,
  • Jiaze He,
  • Irina Sagarbarria,
  • Jeanne A. Hardy,
  • Jiahui Wu

摘要

RNA is one of the key molecules responsible for controlling gene expression regulation, and visualizing individual RNA molecules in living cells offers unique insights into the dynamics of this process. Recently, the RNA-regulated destabilization domain was developed for live-cell imaging of single RNA. However, this method is limited to single-color RNA imaging and its long RNA tag induces destabilization of the tagged RNA. Here we describe two orthogonal RNA-regulated destabilization domains (mDeg and pDeg) that enable three-color messenger RNA (mRNA) imaging in living cells. We show that these destabilization domains can image mRNA tethered to the endoplasmic reticulum membrane, the inner surface of the plasma membrane and in the cytosol. In addition, we show that mDeg can detect mRNA more effectively than the previously reported tDeg system. Moreover, mDeg can be combined with a short RNA tag (9XMS2) for single-molecule RNA imaging without perturbation of RNA stability.