Assessing the cellular uptake pathway for poly-Lysine analogues using triplet lifetime imaging

Research on synthetic delivery vectors is of major interest for cell imaging and manipulation, as they allow an efficient transfer of nucleic acids, therapeutic proteins or small drugs into the cells. We have developed a library of L-lysine analogues that allow for highly efficient gene delivery with low cytotoxicity. However little is known on the exact mechanism of uptake and the final intracellular destination of the synthetic carriers. Therefore we have developed a novel optical technique based on a modulated excitation allowing for intracellular imaging of the triplet-lifetime and -yield of fluorophores attached to the delivery vector. Both these parameters are highly dependant on the intracellular environment thus provide insight into the subcellular localization of the labelled carrier. The method combines high temporal and spatial resolution and is compatible with a multiplicity of fluorophores. We performed series of model experiments to compare the triplet lifetime and triplet yield behaviour during the natural uptake mechanism to a series of controlled conditions. The latter include microinjection of fluorescently labelled carriers directly into the cytoplasm and cell nucleus as well as in vitro measurements under conditions mimicking physiological, acidic, or DNA rich environments. To validate our technique the results from the triplet imaging were compared with two complementary methods: carrier localization by subcellular fractionation and confocal laser scanning microscopy. --- Reference --- Geissbuehler et al. Triplet imaging of Oxygen consumption during the contraction of a single smooth muscle cell (A7r5). Biophysical Journal (2010) vol. 98 (2) pp. 339-349

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful and informative methods to probe molecular dynamics. Specifically, chemical exchange is an important phenomenon and one of the earliest and most vigorously investigated in NMR spectroscopy. Its effects can be observed in many NMR spectra; lines in a spectrum are broadened or averaged because the nuclei are exchanging magnetic environments. A wide variety of experimental methods exists to determine exchange rates of protons that hop between amino acids and water in the slow or intermediate exchange regime. However, so far there is a lack of methods to investigate hydrogen exchange in the fast exchange regime. In this thesis, we focus on the determination of very fast chemical exchange rates from the effects of scalar relaxation on a coupled nucleus. We quantify the effects of scalar relaxation caused by exchanging hydrogens, by detecting the decay of a 15N coherence under a multiple-refocusing Carr-Purcell-Meiboom-Gill (CPMG) pulse train in the presence or absence of hydrogen decoupling. In particular, we have adapted to the case of deuterium a method that was originally designed to study the exchange rate of the indole proton of tryptophan in water as a function of pH and temperature. We will present the exchange rates of the indole deuterium in tryptophan with solvent heavy water. After that, the proton exchanges rates have been compared with the deuterium exchange rates in order to describe the kinetic isotope effect. In a similar manner, we have quantified the proton exchange rates in pure water by using multiple refocusing of transverse 17O magnetization as a function of pH and temperature. Such measurements are widely used to obtain valuable information about molecular dynamics and structure of proteins, protein complexes and nucleic acids. The knowledge of the kinetic isotope effect may contribute to the characterization of reaction mechanisms and other dynamic parameters that can give insight into the stability of hydrogen bonded secondary structures in biomolecules. This information could form a foundation for a more thorough theoretical or modeling development of a number of long and outstanding fundamental questions. In the second part of the thesis, we combine fast exchange with dissolution dynamic nuclear polarization (D-DNP) to study intrinsically disordered proteins (IDPs). IDPs constitute a very large and functionally important class of proteins that lack stable secondary and tertiary structures. As a consequence of the broad dynamics and flexibility of IDPs, and specially under physiological conditions, the NMR spectra of IDPs typically show a strong signal overlap and broadening due to proton exchange. We present a method to overcome these limitations by means of hyperpolarized HDO produced by D-DNP. We could study the effects of conformational adaptations in Osteopontin (OPN), a metastasis- associated intrinsically disordered protein (IDP) by chemical exchange. The magnetization from hyperpolarized water is transferred to the rapidly exchanging protons of the solvent-exposed residues of the protein. The exchange with hyperpolarized water boosts the signals of fast exchanging residues above the detection limits. This allows one to follow individual resonances and draw conclusions regarding conformational adaptations due to ligand binding under physiological conditions. Thus, with our approach, exchange with the solvent is turned into an advantage

EPFL2017

Study of fast exchanging processes by NMR Spectroscopy

Estel Canet Martinez

EPFL2017