# Riboglow: a robust multi-color riboswitch-based platform for imaging RNA in living cells

> **NIH NIH R01** · UNIVERSITY OF COLORADO · 2020 · $303,043

## Abstract

SUMMARY.
The complex spatiotemporal dynamics of messenger RNAs and non-coding RNAs affect virtually all aspects of
cellular function. In addition to serving as the central intermediary between DNA and proteins, RNAs regulate
gene expression at multiple levels, play roles in epigenetic regulation and genome organization, and serve as
physical scaffolds to assemble and integrate macromolecular complexes, with important implications for
normal development, as well as disease etiology. Yet, despite the importance of RNA in biology and growing
evidence of complex and dynamic localization patterns, robust tools for visualizing RNA molecules in live cells
are highly limited. The most widely used RNA tagging system involves addition of 24 MS2 stem loops and
binding of 48 molecules of the MS2 binding protein fused to GFP, adding > 1300 nucleotides and > 2.6 MDa to
an RNA of interest. While this system has revealed tantalizing glimpses at the individual steps of gene
expression regulation, perhaps not surprisingly, it has also been shown to perturb mRNA processing, splicing,
localization, and decay. Thus, there is a pressing need for robust, complementary, and minimally perturbing
tools to visualize individual RNA molecules in living cells to map the complex and evolving landscape of RNA
biology. In this work, we will meet this need by generating a suite of diverse riboswitch-based RNA tags and
corresponding fluorescent probes for simultaneous, multi-color imaging of individual RNA molecules in live
mammalian cells. Our approach builds on preliminary work from our labs that exploits one of nature’s
aptamers, the cobalamin (Cbl)-binding riboswitch as an RNA tag that binds a series of Cbl-linked fluorophores
to induce fluorescence turn-on, thus lighting up the RNA of interest. We called this new RNA tagging platform
Riboglow and demonstrated its ability to visualize mRNA and small U1 snRNA in live mammalian cells. While
the performance of Riboglow was impressive compared to other dye binding aptamers and the gold standard
24xMS2 system, there is significant room for improvement. In this proposal, we will create Riboglow 2.0, with
dramatically improved properties by systematically optimizing modules of the RNA/probe platform
(Aim 1). In three independent subaims, we will exploit the modular nature of riboswitch structural motifs, the
diversity of riboswitch sequences and power of in vitro selection to engineer optimized aptamer-linker pairs,
RNA/probe combinations with enhanced fluorescence turn-on, and orthogonal aptamer/probe pairs to enable
simultaneous detection of multiple RNAs with spectrally distinct probes. In our second aim, we will create a
robust and systematic pipeline for characterizing, validating and benchmarking Riboglow 2.0 (Aim 2).
We will define in vitro biochemical and biophysical properties, cellular contrast and single molecule sensitivity,
demonstrate functionality for tagging different RNAs in diverse cellular assays, and ensure minima...

## Key facts

- **NIH application ID:** 9904726
- **Project number:** 5R01GM133184-02
- **Recipient organization:** UNIVERSITY OF COLORADO
- **Principal Investigator:** Robert T Batey
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $303,043
- **Award type:** 5
- **Project period:** 2019-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9904726

## Citation

> US National Institutes of Health, RePORTER application 9904726, Riboglow: a robust multi-color riboswitch-based platform for imaging RNA in living cells (5R01GM133184-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9904726. Licensed CC0.

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