# An Orthogonal Bioluminescent Platform For Multiplexed Imaging And Control

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $228,427

## Abstract

PROJECT SUMMARY
In this project, we will engineer bright, small luciferases with bioavailable substrates orthogonal to
NanoLuc/furimazine—the brightest and most widely employed bioluminescent system currently available to
researchers. Bioluminescence is rapidly gaining traction as a general means to observe and control biochemical
and electrophysiological signals in cells and is particularly advantageous for tracking and manipulating cells non-
invasively in intact animal models of spinal cord injury, neurodevelopment, and neurodegenerative diseases
such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS). The
addition of one or more small, bright, independently controllable luciferin-luciferase systems will enable far more
informative studies of the complex dynamics of the brain and other tissues. Here, we will take inspiration from
the naturally orthogonal luciferin, vargulin (derived from marine ostracods), which produces very bright
bioluminescence in its native context. We will take advantage of the small and bright coelenterazine-consuming
luciferase variant SSLuc (derived from Oplophorus shrimp), an enzyme engineered by the project team. By
making stepwise changes to the side groups of the luciferin coupled with directed evolution of the luciferase to
increase specific activity, we will evolve SSLuc into an enzyme that efficiently generates light from vargulin rather
than coelenterazine. In parallel, we will generate membrane-permeant caged analogs of vargulin that enable it
to be delivered efficiently into cells in the central nervous system, along with caged analogs tailored for delivery
to cells expressing specific enzymes, extending the utility of the vargulin platform. Our work is highly innovative,
as it combines a unique blend of chemical synthesis and protein engineering to fill a long-standing void in
bioluminescence-based imaging and cell manipulation. The proposed research is significant, as the
bioluminescent tools will enable the direct and independent interrogation of multiple cells and pathways using
bioluminescence, which is not currently possible with existing toolsets. The luciferin substrates generated in this
work will be highly bioavailable, stable, and genetically targetable. Additionally, like other imaging technologies,
the bioluminescent probes developed in this project will likely catalyze new discoveries in a broad spectrum of
fields.

## Key facts

- **NIH application ID:** 10992538
- **Project number:** 1R21EY036659-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Jennifer Prescher
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $228,427
- **Award type:** 1
- **Project period:** 2024-09-30 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10992538

## Citation

> US National Institutes of Health, RePORTER application 10992538, An Orthogonal Bioluminescent Platform For Multiplexed Imaging And Control (1R21EY036659-01). Retrieved via MORVS 2026-10-27 from https://api.morvs.ai/grant/nih/10992538. Licensed CC0.

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