Development of fluorescent probes and a novel peptide tag for intracellular live cell imaging and selective detection of ROS/RNS

Abstract

Diverse of fluorescent labeling technics has been developed to visualize and help understanding biological roles, dynamics and functions at single cell level [1]. In the last decade nanoscopy or super resolution microscopy (SRM) technics such as STED and STORM has been developed. This has enabled researchers to not visualize the localization but also understanding of biological structure and function in nanoscale (10-30 nm), which previous fluorescence confocal imaging method had a limit of 200 nm [2, 3]. Therefore fluorescence probe that can be labeled by self-labeling enzyme such as Halo, SNAP, CLIP tag has received attention due to about 20 fold higher photophysical properties than fluorescent protein [4]. Various Halotag dye has been developed and among them the green florescence probe that can be used in vivo has not been reported yet. Here I report BODIPY based Halo G that are highly stable in serum for capable in vivo system.

Despite of their relatively large size of fluorescent protein and self-labeling enzyme, they occur only minimal affect in the clustering behavior [5]. However, by small protein such as viral protein could not be labeled due to their sizes [6]. Therefore, I decided to develop a peptide tag and fluorescent probe based on sulfonyl fluoride (SuFEx) click chemistry. The SFP2 dye was developed via phenotypic high-throughput screening (HTS) with BODIPY family that could penetrate and washed out rapidly with minimal background. I found candidates that specifically label specific peptide sequence KYRGQAHDx2 (KYRG2) in vitro. I am also developing in vivo screening method to find soluble and specific to peptide sequence.

Fluorescent probe labeling are not only important for understanding biological dynamics and tracking, but also for diagnosis such as reactive oxygen/nitrogen species (ROS/RNS). In low concentrations of ROS can activate the signaling pathways and trigger important cellular proliferation and differentiation but in oxidative stress, high ROS occurs signal transduction and diverse pathological conditions, including Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s disease. Among ROS, superoxide radical anion ($O2^{•−}$) is the major reactive oxygen species (ROS) in living systems. Therefore, I have developed a hemicyanine-embedded diphenylselenide (HemiSe)-based probe for the selective detection of superoxide radical anion. The probe shows excellent sensitivity and selectively towards superoxide and showed “turn-on” fluorescence upon addition of endogenous superoxide in RAW264.7 cells. This unit will be further studied in the context of versatile approaches to detection and therapeutics in neurodegenerative disease research as confirmed by confocal microscope imaging.