# Two novel red-FRET ERK Biosensors in the 670-720 nm range

## TL;DR

Researchers developed two new red-FRET ERK biosensors that allow better multiplexed imaging of cell signaling.

## Contribution

Two novel red-FRET ERK biosensors (REKAR67 and REKAR76) were developed for improved single-cell signaling studies.

## Key findings

- REKAR67 and REKAR76 biosensors operate in the 670–720 nm range with minimal spectral overlap.
- REKAR67 has a higher dynamic range but greater signal variance compared to REKAR76.
- Both biosensors are spectrally compatible with existing CFP/YFP biosensors for multiplexed imaging.

## Abstract

Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central node in the control of cell proliferation and differentiation. ERK typically cooperates with a network of other regulators, making it necessary to study multiple signaling pathways simultaneously at the single-cell level. Many existing fluorescent biosensors for ERK and other pathways have significant spectral overlap, limiting their utility for multiplexing. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670–720 nm range using the fluorescent proteins miRFP670nano3 and miRFP720. REKAR67 and REKAR76 differ in fluorophore position, which impacts biosensor characteristics; REKAR67 displayed a higher dynamic range but greater signal variance than REKAR76. In both polyclonal and clonal populations, REKAR67- or REKAR76-expressing cells displayed similar Signal-to-Noise ratios (SNR). Overall, the red-FRET ERK biosensors were highly consistent with existing CFP/YFP biosensors in reporting ERK activity. Both REKAR biosensors expand the available tools for measuring single-cell ERK activity by being spectrally compatible with other CFP/YFP FRET and cpGFP -based biosensors, allowing for multiplexed imaging.

The online version contains supplementary material available at 10.1186/s13036-025-00541-9.