In-Gel Imaging of RNA Processing Using Broccoli Reveals Optimal Aptamer Expression Strategies

• F30 is a highly effective RNA scaffold for expressing aptamers in cells
• Commonly used scaffolds for expressing aptamers are prone to intracellular cleavage
• Broccoli and Spinach-tagging enables RNA processing to be easily imaged in gels
• RNAs can be tagged with cassettes containing multiple Broccoli aptamers

SummaryRNA aptamers can be expressed in cells to influence and image cellular processes. Aptamer folding is maintained by inserting the aptamers into highly structured RNA scaffolds. Here, we show that commonly used RNA scaffolds exhibit unexpected instability and cleavage in bacterial and mammalian cells. Using an in-gel staining approach for rapid and simple detection of Spinach- or Broccoli-tagged RNAs in cells, we monitored the processing of RNAs tagged with scaffolded aptamers, revealing endonucleolytic cleavage, RNA instability, and poor expression. We reengineered a natural three-way junction structure to generate an alternative scaffold that enables stable aptamer expression in cells. This scaffold was used to create cassettes containing up to four Broccoli units, markedly enhancing the brightness of mammalian cells expressing cassette-tagged RNAs. These experiments describe methods for screening RNA cleavage events in cells and identify cell-compatible scaffolds that enable efficient tagging of RNAs with aptamers for cellular expression.

Graphical AbstractFigure optionsDownload high-quality image (161 K)Download as PowerPoint slide