# Multicomponent, single-molecule imaging of RNA in mammalian cells

> **NIH NIH F32** · UNIVERSITY OF COLORADO · 2020 · $25,918

## Abstract

PROJECT SUMMARY
RNA lies at the center of cellular function. Its most appreciated role is to carry protein blueprints to the ribosome
for manufacture. Only recently have researchers begun to appreciate its myriad of other functions, many of which
are implicated in a variety of disease states. Long noncoding RNA (lncRNA) comprise one such important class
of RNA that does not participate in the central dogma. Alarmingly, the human genome encodes for as many
lncRNA as proteins. These transcripts are typically greater than 200 bases, and are known to participate in
binding both proteins and nucleic acids, often both at the same time. However, little else is understood regarding
their function. Where and when do they interact with their targets? How long do these interactions occur, and
what other cellular machinery is present? This lack of understanding is due in part to the lack of tools available
to image this biomolecule. Localization of RNA on a single-molecule level, and multicomponent imaging of RNA
transcripts remains difficult. Existing tools utilize aptamers that are unstable in mammalian cells, or constructs
that are too large for imaging small transcripts. Multicomponent RNA imaging is also difficult due to the design of
current tools.
To address this need, I aim to develop a platform for RNA imaging that will enable facile tracking of multiple
transcripts at single cell resolution. Riboglow is an RNA imaging platform recently developed in the Palmer lab. It
utilizes a fluorescence-quenched pair formed by cobalamin (vitamin B12) and a pendant fluorophore. In solution,
this construct shows low fluorescence. When bound to the cobalamin riboswitch aptamer domain, there is an
increase in fluorescence. This tool shows promise for RNA imaging because it solves many of the problems
faced by traditional RNA probes, however several drawbacks are keeping it from widespread utility. The proposed
work addresses these drawbacks, and seeks to utilize improved Riboglow tools to study outstanding questions in
the field of noncoding RNA.
Previously developed Riboglow constructs suffered from poor signal induction and low brightness. First, I aim to
derivatize the native cobalamin structure, linker and fluorophore with the goal of maximizing fluorescence turn-
on. These new molecules will be evaluated for quenching efficiency and signal induction. Next, the molecules I
develop will be screened against libraries of riboswitch aptamers to further improve probe properties. Screening
will be carried out in mammalian cells via flow cytometry, a specialty of the Palmer lab. Candidate probes will be
verified through single-molecule imaging of mRNA in living cells. In tandem with brightness optimization, I will
develop mutually orthogonal probes to enable labeling of different RNA transcripts in the same cell. The power
of SELEX to find selective and tight binders will be used to screen for mutually exclusive aptamer-cobalamin
pairs. These pairs will be conjugated to spectrally...

## Key facts

- **NIH application ID:** 9894643
- **Project number:** 5F32GM131666-02
- **Recipient organization:** UNIVERSITY OF COLORADO
- **Principal Investigator:** Colin Rathbun
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $25,918
- **Award type:** 5
- **Project period:** 2019-02-01 → 2020-06-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9894643

## Citation

> US National Institutes of Health, RePORTER application 9894643, Multicomponent, single-molecule imaging of RNA in mammalian cells (5F32GM131666-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9894643. Licensed CC0.

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