Quantification of peptides uptake by fluorescence techniques

Novel approaches to deliver either biologics (genes, RNAi...), or traditional therapeutics include cell penetrating peptides (CPPs) named due to an apparent endocytosis independent ability to enter cells. Interestingly, CPPs' concentration is key to uptak e and trafficking understanding with a threshold in the low micromolar: above, CPPs' uptake may involve non-endocytotic mechanisms while endocytosis is thought to be the predominant below. Most studies used micromolar concentrations thus potentially induc ing bias when the potential therapeutic range is likely to be in the nanomolar region. To understand delivery in this region, the project will focus on Tat peptide, the most studied CPP originating from HIV 'transactivator' domain. To assess delivery kine tics, a precise determination of the amount, the localisation and binding of Tat peptide is needed. Thus we hypothesise that a careful combination of fluorescence microscopy, Raster Image Correlation Spectroscopy (RICS) and Total Internal Reflection Fluor escence (TIRF) should help to unravel the delivery of Tat peptide at potentially therapeutic concentrations. TIRF, based on the evanescent field generated at the surface when laser light hitting the surface will be used to quantify Tat peptide binding to specific areas of the plasma membrane and whether this is concentration dependent. RICS will be used to quantify amount of peptides delivered cells by RICS and the associated kinetics on a two photon microscope. These two approaches will also consider the influence of ATP-driven mechanisms. Due to the paucity of data on the distribution of cell penetrating peptides in cells and their associated kinetics at relevant concentrations, this work is likely to have a significant impact in this area. This excitin g project will allow a bidirectional transfer of knowledge a novel approach to quantify fluorescence signal, RICS, will be provided to NTNU lab and, the host lab will offer training on 2-photon microscopy and TIRFM.