Jiang, X.; Li, H.; Jiang, L.; Cao, C.; Jiang, Q.; Su, N.; Fang, M.; Chen, Z.; Zuo, F.; Zhao, Y.; Tian, X.; Chen, X.; Zhu, L.; Yang, Y.

Fluorescent RNAs (FRs) have emerged as powerful tools for the visualization of RNA in live cells. However, the poor folding of FRs is a major limiting factor in many advanced RNA imaging applications. Here, we describe a novel strategy to significantly increase the folding efficiency of FRs. By introducing a stabilizing stem and performing targeted mutagenesis at key junction regions, we develop a superfolder variant of the Pepper aptamer, termed sfPepper, that exhibits a striking twofold increase in folding efficiency compared with Pepper under physiological conditions. sfPepper exhibits much improved thermostability and reduced ion dependence and, most importantly, is substantially brighter than Pepper in live cells. sfPepper enables robust imaging of diverse noncoding RNAs and messenger RNAs with increased signal-to-background ratios and stimulated emission depletion (STED) superresolution imaging of CUG trinucleotide repeat-containing toxic RNAs. Remarkably, only four sfPepper repeats facilitate sensitive single-molecule mRNA tracking, revealing dynamic heterogeneity among ER-associated mRNA molecules. Together, this study establishes an efficient strategy for improving FR folding and offers a powerful tool for fluorescently labelling RNAs in live cells.