Simultaneous readout of multiple FRET pairs using photochromism

Abstract

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.

Synchronized, Spontaneous, and Oscillatory Detachment of Eukaryotic Cells: A New Tool for Cell Characterization and Identification

“…A schematic of the experimental setup is shown in Figure S1d of the Supporting Information. The heating element was composed of an onchip meander structure, prepared, and calibrated as described by Cornelis et al [69] Fluorescence microscopy data on S. cerevisiae were acquired simultaneously using the system specified by Roebroek et al, [70] making use of green fluorescence protein excitation and detection channel. A general CRISPR/Cas9 protocol was used to generate constitutively fluorescent S288C S. cerevisiae cells for correlative measurements.…”

Breadth and Specificity in Pleiotropic Protein Kinase A Activity and Environmental Responses

“…When fused to specific AKAPs or other proteins, these reporters can inform on the responsiveness of specific PKA signalosomes ( Surdo et al, 2017 ). Other fluorescence-based reporters have also been used, including a recent GFP reporter engineered to phase-separate into distinct foci upon PKA activation ( Zhang et al, 2018 ; Roebroek et al, 2021 ; Zhang et al, 2021 ). These tools have been used recently to uncover a wealth of information, from models of cAMP binding and diffusion to oscillatory dynamics of PKA stimulation upon GPCR activation ( Zhang et al, 2018 ; Bock et al, 2020 ).…”

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“…On the contrary, both FRET and SERS enable additional multiplexing. Lifetime and intensity multiplexing are demonstrated methods to increase the number of color channels in FRET spectroscopy, , and a number of different channels up to 10 is technologically feasible. On the contrary, SERS has less limitations in terms of multiplexing.…”

“…The optimization of the EM field confinement is a key aspect related to the properties of space dependency of the signal intensity in optical spectroscopies. For example, SERS is effective within about 1 nm from the plasmonic nanostructure, and FRET is sensitive to the distance between donor and acceptor dyes with nanometer resolution. ,, Therefore, in a plasmonic nanopore, biomolecules like DNA and protein produce strong FRET emission or SERS spectra only from the segments in the hot spot. Other parts not in close proximity to the hot spot can hardly give comparable signal, which makes optical read-out highly competitive with the electrical method once high spatial resolution in EM field confinement can be achieved .…”