DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Investigating Biomolecular Interactions by PICOsecond Fluorescence in PICOliters – PICO2

PCIO2

Biomolecular interactions revealed by picosecond fluorescence in picoliters.

Sensing and unraveling the mechanisms of biomolecular interactions

Biomolecular interactions are characterized by structural heterogeneity, i.e. coexistence of different conformers, and structural dynamics over time scales from picoseconds to over minutes. The detailed investigation of these interactions is of essential biological relevance for the design of new therapeutic strategies targeting specifically biomolecular interactions. Here we gather the complementary expertise of 3 partners in order to develop the use of time-resolved fluorescence (TRF) of biomolecules labeled with fluorescent, environment-sensitive probes in order to investigate/assay biomolecular interactions.<br />Throughout the project, innovative implementations of TRF for biomolecular interactions studies/assays will improve our fundamental knowledge of the underlying molecular mechanism and our technological capabilities for TRF-based biosensing. Expected outcomes of the present research program comprise emerging research directions as well as innovative applications to biotechnologies possibly involving industrial partners.

A first target (Task 2) will exploit an original experimental approach combining TRF and droplet microfluidics (DmF) to investigate interactions within out-of-equilibrium biomolecular complexes. Here TRF is used to reveal distributions of fluorescence lifetimes, which are representative of distributions of conformers (i.e. structural heterogeneity). Simultaneously, DmF is used to produce out-of-equilibrium biomolecular complexes (fast mixing). We exploit this innovative experimental approach to investigate the molecular mechanism of the chaperone activity (i.e. promoting DNA structural rearrangements) of the NCp7 protein of HIV (expertise of Partner 3, LBP, Illkirch), used here as a model system for dynamic protein-nucleic acid interactions.

A second research line (Task 3) will develop the use of TRF, measured by time-correlated single photon counting (TCSPC), for high-throughput (HT) biomolecular interactions assays. Unlike fluorescence intensity detection, which is abundantly used in HT biosensing, TRF is an intrinsic, hence more reliable, measure of biomolecular interactions with fluorescently-labeled biomolecules. Here the aim is to develop the technology (CMOS microelectronics) enabling the detection and real-time data processing of single photon events to perform HT flux cytometry and screening based on TCSPC, a technology patented by Partner 1 and 2. A prototype will be built and validated by demonstrating a proof-of-principle screening of known inhibitors of NCp7.
A final, purely technological objective (Task 4) will enable a 10-fold increase in the data acquisition and processing rates. This will in turn enable a 10-fold increase of the fluorescence lifetime acquisition rate for HTS, or the conception and operation of a 1D linear array of sensors for TCSPC, i.e. a CMOS integrated streak camera.

• Combinaison TRF/Microfluidique pour l’étude de dynamique structurale :
o Approche expérimentale TRF+droplet microfluidique développée et appliquée à des experiences de « saut de pH » pour l’étude de la relation structure ?photophysique de protéines fluorescentes (IPCMS, Coll. H. Pasquier initiée avant début ANR) => un article en cours de rédaction
o Hybridation (+)/(-) PBS : expériences préliminaires en cours dans gouttes microfluidiques (IPCMS)
• Etude/application nouvelles sondes fluorescentes de structure :
o Etude photophysique d’un dérivé d’hydroxychromone (absorption transitoire femtoseconde) article en préparation (IPCMS/LBP)
o Caractérisation de la cinétique (+) / (-) PBS, à l'aide de la sonde 3HCnt
o Perturbation de la structure d'un duplex par l'introduction de la sonde 3HCnt
• Application TRF aux biotechnologies :
o démonstration de bonnes performances pour un test d’activité enzymatique par TRF dans des microgouttelettes à haut débit (IPCMS, ICUBE, coll Novartis) : article publié.
o projet prématuration CNRS (2016-17) : conception d’un prototype pour contact avec partenaires industriels (IPCMS, ICUBE, Coll. A. Griffiths)
o Implémentation routine de fit rapide en FPGA (real-time analysis) => proceeding de conference (IPCMS,ICUBE, coll. D. Fey)
• Développement microélectronique :
o Générateur d’impulsion laser picoseconde à haut taux de répétition à base de diode laser finalisé. Puissance moyenne de l’ordre de 1mW à 80MHz, largeur d’impulsion de l’ordre de 100 ps FWHM (ICube).
o Capteur CMOS monovoie développé et retournée de fonderie. Convertisseur temps vers numérique déjà testé et offrant une résolution temporelle de 10 ps. Test du système de détection optique en cours (ICube).

o Developpement de nouvelles sondes de fluorescence pour la comprehension interaction porteine/ADN
o Recherche partenariats industriels pour l'application de TCSPS en haut débit

Revues à comité de lecture:
“Towards Sensitive, High-Throughput, Biomolecular Assays Based on Fluorescence Lifetime”, Anastasia Ioanna Skilits et al., Methods and Applications of Fluorescence (submitted) 2017
M. SHOLOKH, et al. «Annealing mechanism of HIV-1 (-)/(+)PBS DNA sequences and its chaperoning by the nucleocapsid protein as revealed by site-selective fluorescence labeling.« Submitted.
L. ZARGARIAN, et al. «Structural and dynamical impact of a universal fluorescent nucleoside analogue based on 3-hydroxychromone inserted into a DNA duplex.« Submitted.

Ouvrages ou chapitres d’ouvrage 1. Dadouche F, Turko T, Malass I, Skilitsi AI, Léonard J, Uhring W. Design, implementation and characterization of Time to Digital Converter on low cost FPGA. In: Yurish S, editor. Advances in Sensors: Reviews, Vol4 “Sensors and Applications in Measuring and Automation Control Systems.” IFSA Publishing, S.L.; 2016.

Prix d'Instrumentation (SCF/SFP) 2016. «Fluorescence résolue en temps pour les biotechnologies à haut débit », Jérémie Léonard,

Biomolecular interactions are characterized by structural heterogeneity, i.e. coexistence of different conformers, and structural dynamics over time scales from picoseconds to over minutes. The detailed investigation of these interactions is of essential biological relevance for the design of new therapeutic strategies targeting specifically biomolecular interactions. Here we gather the complementary expertise of 3 partners in order to develop the use of time-resolved fluorescence (TRF) of biomolecules labeled with fluorescent, environment-sensitive probes in order to investigate/assay biomolecular interactions.
A first, fundamental target (Task 2) will exploit an original experimental approach combining TRF and droplet microfluidics (DmF) to investigate interactions within out-of-equilibrium biomolecular complexes. Here TRF is used to reveal distributions of fluorescence lifetimes, which are representative of distributions of conformers (i.e. structural heterogeneity) in ensemble measurements. Simultaneously, DmF is used to produce out-of-equilibrium biomolecular complexes (fast mixing). In a preliminary work, Partner 1 (IPCMS, Strasbourg) demonstrated the experimental concept by implementing TRF detection with a conventional streak camera along a microfluidic channel and monitoring the structural relaxation of a biomolecular complex in microdroplets circulating over hundreds of ms in the channel. Here, the main objective is to exploit this innovative experimental approach to investigate the molecular mechanism of the chaperone activity (i.e. promoting DNA structural rearrangements) of the NCp7 protein of HIV (expertise of Partner 3, LBP, Illkirch), used here as a model system for dynamic protein-nucleic acid interactions. We will in particular focus on the NCp7-promoted hybridization reactions of complementary nucleic acid sequences, (+)/(-)PBS (“Primer Binding Site”) and cTAR/TAR. The reactions are involved in two essential steps of the virus life cycle.
A second technological research line (Task 3) will develop the use of TRF, measured by time-correlated single photon counting (TCSPC), for high-throughput (HT) biomolecular interactions assays. Unlike fluorescence intensity detection, which is abundantly used in HT biosensing, TRF is an intrinsic, hence more reliable, measure of biomolecular interactions with fluorescently-labeled biomolecules. A preliminary work published by Partners 1 and 2 (ICube, Strasbourg) showed that TCSPC can be implemented in order to detect the fluorescence lifetime of microdroplets in very HT conditions, with an accuracy enabling reliable, binary assay (“positive” versus “negative” hits) of biomolecular interactions. Here the aim is to develop the technology (CMOS microelectronics) enabling the detection and real-time data processing of single photon events to perform HT flux cytometry and screening based on TCSPC, a technology patented by Partner 1 and 2. A prototype will be built and validated by demonstrating a proof-of-principle screening of known inhibitors of NCp7.
A final, purely technological objective (Task 4) will enable a 10-fold increase in the data acquisition and processing rates. This will in turn enable a 10-fold increase of the fluorescence lifetime acquisition rate for HTS, or the conception and operation of a 1D linear array of sensors for TCSPC, i.e. a CMOS integrated streak camera.
Throughout the project, innovative implementations of TRF for biomolecular interactions studies/assays will improve our fundamental knowledge of the underlying molecular mechanism and our technological capabilities for TRF-based biosensing. Expected outcomes of the present research program comprise emerging research directions as well as innovative applications to biotechnologies possibly involving industrial partners.

Project coordination

Jérémie LÉONARD (Institut de Physique et Chimie des Matériaux de Strasbourg)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LBP- UNISTRA Laboratoire de biophotonique et pharmacologie
IPCMS-CNRS Institut de Physique et Chimie des Matériaux de Strasbourg
ICUBE - UNISTRA LABORATOIRE DES SCIENCES DE L'INGÉNIEUR, DE L'INFORMATIQUE ET DE L'IMAGERIE

Help of the ANR 491,698 euros
Beginning and duration of the scientific project: September 2015 - 48 Months

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