Background

The use of fluorescence-based assays has significantly improved our understanding of various biologic functions and facilitated novel developments across the fields of clinical chemistry, engineering, molecular biology, medical diagnostics, and life sciences. There is therefore a need to improve current fluorescence-based assays in order to provide continued support to high value research initiatives.

The development of novel fluorescent labelling proteins may improve upon current limitations present in fluorescence systems. Most existing fluorescence-based assays have a similar flaw, specifically quenching of the fluorescence signal. This limitation prevents a number of cellular receptors under investigation from being accurately measured, as it does not provide enough time for extended imaging of excitation and emission cycles that would represent the receptors activity. The average number of excitation and emission cycles that occur for a particular fluorophore before photo bleaching occurs is dependent upon the molecular structure and the local environment. Yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired.

Technology Overview

The inventors have developed a suite of novel fluorescent labelling proteins derived from mVenus, a basic (constitutively fluorescent) yellow fluorescent protein with high brightness and low acid sensitivity. Variants of mVenus were developed through modulation of amino acid sequences. They have introduced several variants with results demonstrating up to a 4-fold improvement in photostability with equivalent brightness compared to precursor variants. This increased photostability slows the process of photo bleaching and extends imaging time. 

Stage of Development

The inventors have successfully demonstrated the functional performance of their novel yellow fluorescent proteins. To support performance of the novel fluorescent proteins, inventors monitored photostability and brightness. They further performed extensive characterization studies using both in cellulo and in vitro models. The inventors further presented optimized methods for making and applying the proteins that allow it to be used within the same context as similar fluorescent proteins. This invention further supports advancement in high value research initiatives by mitigating current limitations present in current commercialized products, making it an attractive technology for the market. 

Further Detail

Versatile phenotype-activated cell sorting Lee J, Liu Z, Suzuki Ph, Ahrens Jf, Lai S, Lu X, Guan S, St-Pierre F (2020). Science Advances, 6(43) , eabb7438. doi: 10.1126/sciadv.abb7438 Fpbase.org

Benefits

• Increased Photostability - improved structural integrity of the proteins resists photo bleaching which extends imaging time
• Retains Spectral Properties - emitted brightness was maintained despite modulations to improve stability
• Increased Viscosity - provides easier application to the tissue and maintained contact throughout the imaging process