iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP

janelia7_blocks-janelia7_biblio_header | block

bioRxiv. 2023 Aug 24;. doi: 10.1101/2023.08.24.554624

Marvin Jonathan, Kokotos Alexandros, Kumar Mukesh, Pulido Camila, Tkachuk Ariana, Yao Jocelyn, Brown Timothy, Ryan Timothy

janelia7_blocks-janelia7_biblio_abstract | block

Abstract

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ∼ 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 μM to 500 μM. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

node:body | entity_field

bioRxiv PrePrint https://doi.org/10.1101/2023.08.24.554624

janelia7_blocks-janelia7_biblio_authors | block

janelia7_blocks-janelia7_biblio_tools | block

Main Menu (Mobile)- Block

Main Menu - Block

iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP

Abstract

Janelia Authors