CONTEXT

Calcium is known to act as a modulator of many important physiological responses and pathophysiological conditions, participating as a second messenger in the control of a myriad of cellular processes. Among all Ca2+ sensors available, aequorin is a reporter of choice in numerous applications owing to its remarkable flexibility. It is a Ca2+-sensitive photoprotein, a complex of the protein apoaequorin, the coelenterazine (CLZ) and molecular oxygen. However, the light emission from wild type-aequorin, as well as from other photoproteins, is characterized by intrinsically low quantum yield that is proportional to its relatively low sensitivity to calcium. In order to circumvent this issue, incorporation of different CLZ analogs and genetic mutagenesis have been used to alter calcium sensitivity, decay kinetics, spectral emission and thermostability. However, so far no apoaequorin mutant with increased affinity for calcium has been described. Hence, the need for a new generation of apoeaequorin-based calcium biosensors, displaying high affinity to calcium that will significantly enhance the signal-to-noise ratio. Such property is highly desired and would be an essential complementary asset to practically measure minute changes in cellular calcium, which concentration is around several hundreds of nanomolars.

TECHNICAL DESCRIPTION

This invention provides novel apoaequorin-based calcium biosensors (named aequorinXS, 465 nm peak), which have increased affinity for calcium compared to known photoproteins, and exhibit enhanced bioluminescence relative to calcium concentration.

Furthermore, novel red light-emitting aequorinXS versions (RedquorinXS, 581 nm peak) have been enhanced more specifically for multicolor and multiplexing assays, as well as for in vivo imaging.

BENEFITS

AequorinXS and RedquorinXS biosensors have been developed to circumvent the limited calcium sensitivity of the existing photoproteins, hence allowing the following unique properties:

Improved sensitivity to calcium, and therefore increased signal-to-noise-ratio and bioluminescence ouput, while maintaining a low-buffering capacity on cytosolic calcium

Exhibit the necessary characteristics of bioluminescence kinetics and thermostability for detection of changes in intracellular calcium levels, therefore adapted to in vitro, in cellulo and in vivo assays

The red light-emitting variant, RedquorinXS will specifically allow for highly advantageous applications in multicolor modes:

Multiplexed detection of calcium from different cells and/or cellular organelles,

Multiplexed  detection of different analytes, calcium and other molecules (e.g. cAMP),

in vivo imaging, giving the low absorbance and scattering of red wavelengths in living tissues (deep tissues and blood cells).

INDUSTRIAL APPLICATIONS

This innovation could be used for:

Quantitative cellular calcium imaging, while allowing multiplexing of blue, green and red emission signals,

High-throughput drug screening based on cell lines expressing GPCRs,

Engineering of stable cell lines for applications in toxicology, genotoxicity and environmental pollution tests,

Engineering of self-illuminating quantum dot conjugates for adapting more colors hues,

Engineering kits with viral particles/AequorinXS/RedquorinXS for delivery into specific or hard-to-transfect cell lines, including ex vivo tissue samples,

Engineering kits with self-penetrating peptide/AequorinXS/RedquorinXS for delivery into hard-to-transfect cell lines, including ex vivo tissue samples,

Immunoassays based on photoprotein bioluminescence, where AequorinXS/RedquorinXS can be modified to target specific functional groups/residues. Aequorin-based labelling is known to reach beyond attomole sensitivity of detection,

Development of bioluminescence imaging systems for dual to three-color (blue, green and red) measurement and screening

DEVELOPMENT STAGE

Accomplished tests:

In vitro characterization of AequorinXS/RedquorinXS (determination of spectral emission, calcium affinity, bioluminescence intensity, bioluminescence kinetics, thermostability)

Assays on live cells (CHO; HEK293T) expressing RedquorinXS using various types of GPCR related agonists/drugs (ATP, Carbachol, Isoproterenol) and other chemical compounds like Forskolin

Engineering of stable cell lines (CHO/RedquorinXS) and characterization in terms of detection limit of ATP molecules, ATP sensitivity and Z-factor determination.

Engineering of viral particles (Sindbis/RedquorinXS), infecting pyramidal neurons from neocortical slices of mice and performing ex vivo imaging of calcium dynamics

Current developments:

Multiplexing synchronous signal detection from mobilization of calcium and cAMP in HEK293T cell line (expressing RedquorinXS and GloSensor cAMP biosensors) after external stimulation

Self-penetrating peptide/RedquorinXS for delivery into blood cells, a hard-to-transfect tissue samples, followed by calcium measurement

In vivo dual-color (green and red) imaging of calcium dynamics in developing zebrafish embryos, by injecting the biosensors in form of DNA, RNA or protein

In vivo imaging of intracranial calcium dynamics in young mice after viral infection of neurons and subsequent expression of RedquorinXS