Quantitative Model for Reversibly Photoswitchable Sensors

Abstract

Composed of a reversibly photoswitchable unit allosterically linked to a sensing module, reversibly photoswitchable sensors (rs-sensors) represent a new and attractive strategy to quantitatively read-out analyte concentrations. However, their kinetic response to illumination is complex, and much attention is required from the design to the application steps. Here, we exploit a generic kinetic model of rs-sensors which enables us to point to key thermokinetic parameters, such as dissociation constants and kinetic rates for exchange toward the analyte, and cross-sections for photoswitching. The application of the model allows to evaluate the robustness of the analyzed parameters and to introduce a methodology for their reliable use. Model and methodology have been experimentally tested on a newly reported calcium sensor based on a reversibly photoswitchable green fluorescent protein allosterically linked to a calcium-sensing module integrating calmodulin and an RS20 peptide.

Absolute measurement of cellular activities using photochromic single-fluorophore biosensors

“…A more detailed investigation of the dynamics of photochromic sensors was recently published in Ref. [59].…”

OptoAssay – Light-controlled Dynamic Bioassay Using Optogenetic Switches

“…Through a photoreceptor that can light-dependently interact with a binding partner, a competitor molecule can be released and later removed from the detection area by applying far-red and red light. Although other photoswitchable assays already exist 16,17 , they only allow for detection of specific molecules, whereas the OptoAssay can be universally applied by fusing the molecule of interest to the phytochrome B interaction partner PIF6. For example, the integrating of a z domain 18 , an IgG-binding domain, antibodies can be attached to the PIF6 construct and act as detection antibodies to add even more versatility.…”

Correlation of fluorescence evolution for quantitative analysis of labels and sensors

“…[5,6,11,22] Using a different scaling, we harness the reaction of calcium with a reversibly photoswitchable fluorescent sensor and employ the correlation method to titrate calcium. [20,21] We conclude in section 5. In a first step, light intensities are calibrated using Dronpa-2 for which the photoisomerization cross-section σ 480 = 196 m 2 .mol −1 under illumination at 480 nm is known.…”

“…Using different scalings of the correlation output, we apply the correlation method to either the direct quantification of the concentration of a targeted reversibly photoswitchable fluorescent protein (RSFP) or the titration of calcium in interaction with a one-color ratiometric reversibly photoswitchable fluorescent sensor. [20,21] The manuscript is organized as follows. Section 2 provides Material and Methods.…”