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Single molecule FRET microscopy with upconverting nanoparticules

Research unit

UMR 7213 - Laboratoire de biophotonique et pharmacologie (LBP)
Faculté de Pharmacie, 74 route du Rhin, BP 60024, 67401 ILLKIRCH-GRAFFENSTADEN

Group

Name: Biophotonique des interactions moléculaires et cellulaires

Group leader: MéLY Yves - yves.mely@unistra.fr

Group leader's phone: 0368854263

Website: Visit website

Group organization:
- Chercheurs: 18
- ITA: 2
- Doctorants: 19
- Post-Docs: 2
- Autres: 4

Publications of the team linked to the topic (3 last years):
1) A. REISCH, P. DIDIER, L. RICHERT, S. ONCUL, Y. ARNTZ, Y. MELY, & A. KLYMCHENKO. Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nat. Comm. 2014, 5, 4089.
2) V. KILIN, H. ANTON, N. ANTON, E. STEED, J. VERMOT, T.F. VANDAMME, Y. MELY, & A.S. KLYMCHENKO. Counterion-enhanced cyanine dye loading into lipid nano-droplets for single particle tracking in zebrafish. Biomaterials, 35, 4950-7.
3) N. KEMPF, V. POSTUPALENKO, S. BORA, P. DIDIER, Y. ARNTZ, H. de ROCQUIGNY, & Y. MELY. The HIV-1 nucleocapsid protein recruits negatively charged lipids to ensure its optimal binding to lipid membranes. J Virol., 2015, 89, 1756-67.

About PhD

PhD Director: MELY Yves - yves.mely@unistra.fr

Phone: 0368854263

Junior advisor: PRZYBILLA Frédéric

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Single molecule FRET microscopy with upconverting nanoparticules

Project: Upconverting nanoparticles (UCNPs) based on lanthanide doped nanocrystals offer a key feature as compared to all other luminescent labels, as they reemit visible photons upon excitation by near infrared photons. Anti-Stokes emission is a very specific property of these nanostructures that enables their nearly zero photon background detection, even in biological samples. Additional features that make them ideal nanoscale reporters include high photostability and non-blinking emission. On the other hand, single molecule studies of protein-oligonucleotides interaction are able to bring out details hidden by conventional ensemble measurements, such as transient states and inhomogeneous population distribution. However, single molecules studies are complicated with conventional fluorophores due to the unavoidable fluorescence background, which provides poor signal to noise ratio. In this project, we propose to develop in collaboration with H. Gorris and T. Hirsch (U. Regensburg) a single molecule FRET (Forster Resonant Energy Transfer) assay for investigating biomolecular interactions taking advantage of the unique features of UCNPs. To reach this objective, we will combine sub 10 nm UCNPs synthesized by our German partner with appropriate organic dyes as acceptor. These UCNPs will be coated by a thin polymer to allow their water-solubility and conjugation with organic dyes. We will compare various dyes as well as different polymer coatings to select the most efficient system for getting high FRET. In parallel, our German partner will provide us with UCNPs of different compositions, so that we will be able to find out the most suitable one for single molecule detection. In a second step, after selection of the best UCNP/dye couple, we will graft the selected UCNPs with oligonucleotides of a given length and sequence. Then, their complementary oligonucleotide labelled with the selected FRET acceptor at different positions will be added to form a duplex. Due to their regular structure, DNA duplexes can be used as nanometric rulers to characterize the dependence of FRET on the acceptor distance. Next, to validate our approach, we will use a system that has already been investigated on a single molecule level. We will use HIV-1 reverse transcriptase (RT) in interaction with model primer template sequences. Comparative use of the DNA and RNA versions of these sequences is of strong interest since the enzyme shows opposite orientations on the two types of sequences, so that different FRET efficiencies are expected. Careful chemistry will be required to couple the oligonucleotides at a 1:1 stoichiometry to the UCNPs, while the FRET acceptor will be coupled to an exposed Cysteine introduced at various positions of the protein by mutagenesis. The data obtained with our system will be compared to the literature to ensure that the FRET couple and especially the UCNP does not perturb the system. Due to the zero photon background detection and improved signal to noise ratio offered by UCNPs, a much more accurate investigation of the interaction of HIV-1 RT with its substrates should be achieved. Moreover, due to the photostability of UCNPs, the interactions could be observed over much longer time scales, so that multiple binding/dissociation events and slow kinetic events should be perceived. Last, we will exploit the signal multiplexing capabilities of UCNPs. Indeed, their emission spectra consist of well separated (> 100 nm) sharp emission bands, so that the same UCNP could be used with two different acceptors. This unique feature over conventional organic dyes will be used to study the interaction between three partners in a reaction. We will use again the RT/oligonucleotide system together with the HIV-1 nucleocapsid NCp7 protein to determine how NCp7 affects the kinetics of association/dissociation and the orientation of the RT on its substrates.

Wished skills: Skills at the interface chemistry/biology/physics. Good knowledge in photophysics and microscopy.

Expertises which will be acquired during the training: Highly interdisciplinary project.Expertises will be acquired in advanced microscopy techniques, as well as in coating and use of nanoparticles for biological purposes.