Abstract
ABSTRACT
A broader understanding of RNA biology requires methods for visualizing transcript dynamics in native environments. Common imaging approaches rely on fluorescent probes that require excitation light and often depend on large aptamer arrays, limiting sensitivity and compatibility with many non-coding RNAs, particularly highly structured folds. To address these challenges, we developed a miniaturized bioluminescent platform for RNA imaging. This system employs a compact RNA tag that assembles luciferase fragments upon binding of monomeric RNA-binding proteins (RNA lanterns). Systematic optimization of the RNA component yielded a structured 24-nucleotide tag, representing greater than 3-fold reduction in size relative to the original
C-11-B
RNA tag, placing it among the smallest RNA imaging tags reported to date. The optimized tag enabled RNA-dependent bioluminescence detection in live cells, notably as a small, single insert within highly structured non-coding RNAs, including the
MALAT1 ENE
domain and a small HDV-like ribozyme. Collectively, this work establishes a miniaturized bioluminescent platform for imaging a broad range of RNA targets in living systems.