Abstract: The Genetically Encoded Small Illuminant (GESI) technology proposed here consists of using an one-bead-one-compound (OBOC) combinatorial library screen to identify short peptide(s) that activate fluorophores/dyes,which can be coupled to alterations of their chemical environment including conformational change upon ligandbinding and phosphorylation of the peptide. These peptides can then be genetically fused to a target protein toenable functional cellular imaging in vitro and in vivo. As proof of concept, we will fuse the identified peptide tointegrin and use the exogenously added GESI-peptide-activating dye to demonstrate the utility of the proposedgenetically encoded functional imaging system in living cells. We will test their ability to measurespatiotemporal dynamics, interactions with ligands and phosphorylation of intracellular domain. Integration ofthese distinct GESIs into one integrin protein will allow concurrent probing of multiple integrin functions in realtime in living cell. Impact: GESI peptides can specifically bind to and activate the fluorescence of selected organic dyes.Some GESI peptides will do so only after binding to Ca2+, conformational changes or post-translationallymodified, such as phosphorylation. Therefore, when transfected into a living cell, they can illuminate thespatiotemporal regulation and modification of proteins of interest. Like GFP, GESI can also be expressed intransgenic animal, or in tumor cells implanted into nude mice as xenograft. If the dye can be delivered to thetissue of interest, GESI reporting can occur in living animals as well. The genetic illuminants are small (1200-1900 daltons), thus can be readily inserted along the sequence of the native proteins without interfering withtheir physiological functions. We can create functional peptide-dye pairs without overlapping spectrum tosimultaneously monitor a number of PTMs or other cellular functions concurrently, to reveal system levelregulation in living cells. This research will expand the catalogue of fluorescence imaging tools; their versatilitywill be broadly applicable to many fields of biology, including cancer.Specific aims of this proposed project are:Aim 1. Design and synthesis of a series of organic dyes suitable for GESI development.Aim 2. Multiplex tracking of protein dynamics and ligand-receptor interactions with GESIs using integrin as a model system.Aim 3. GESIs for post-translational modification of intracellular domain of integrin.
|Effective start/end date||8/15/16 → 7/31/19|
- National Institutes of Health: $362,749.00