BLANC - Blanc

Labelling of peptides and oligonucleotides by two-color fluorophores for investigating biomolecular interactions. – Fluo-aa-nt

Submission summary

The sequencing of the human genome and the progresses in proteomics raised a strong demand for the characterization and quantification of protein/protein and protein/nucleic acid interactions in order to understand the molecular basis of various diseases and establish assays for high throughput screenings and molecular diagnostics. Through its capability to decrease the measurements down to the single molecule level, fluorescence spectroscopy is the technique of choice to reach these objectives. However, this technique is limited by the availability of optimized fluorophores. Limitations are especially important for nucleic acids since natural nucleosides are almost non fluorescent and fluorescent nucleoside analogues suffer from either low fluorescence or poor sensitivity to their environment. In proteins, Trp residues have numerous applications but exhibit limited spectroscopic properties. Fluorescently labelled aminoacids have been developed but most of them exhibit single band emission that provides only limited information on their environment. In this context, the objective of this project is to develop two-colour fluorescently labelled nucleosides and aminoacids, which being introduced in peptides and oligonucleotides (ODNs) by solid-phase synthesis will site-specifically report on their interactions with biomolecular targets. This objective will be reached by using fluorophores of the 3-hydroxychromone (3HC) and 3-hydroxyquinolone (3HQ) family that exhibit a dual fluorescence highly sensitive to the environment. By labelling the peptides or ODNs at selected positions, the two-colour signal from these fluorophores will provide site-selective information on the environmental changes induced by the interactions. Moreover, 3HC and 3HQ derivatives are small and likely disturb less the structure of the labelled peptides and ODNs than conventional dyes. This project will be based on three interconnected work packages: i) Design, synthesis and spectroscopic characterization of novel two-color fluorophores based on 3HC and 3HQ dyes for aminoacid and nucleoside labelling; ii) Solid phase synthesis of fluorescently labelled peptides and characterization of their interactions with biomolecules and; iii) Development of fluorescently labelled nucleosides, incorporation into ODNs by solid phase synthesis and characterization of the ODN/biomolecule interactions. Preliminary data have shown that the selective labelling of a peptide and a protein with a first generation of our dyes does not significantly alter their structure and functions and, enables to sensitively monitor interactions with target biomolecules. Moreover, a panel of nucleoside analogues have already been synthesized based on a 3HC dye that remains highly fluorescent when stacked with bases. Our labelling technology will be further validated on peptides and ODNs selected in fields where the participants are expert. In this respect, the labelling strategy will be applied to the 55 amino-acid NCp7 protein of HIV-1 as well as its target ODNs, including the cTAR sequence of HIV-1. It will also be validated with peptides interacting with antibody fragments as well as with aptamers directed against a plant viral protein. Using fluorescence spectroscopy, surface plasmon resonance and circular dichroism, the labelled sequences will be compared with non-labelled sequences to check whether the fluorophores perturb the structure, functions and binding properties of the labelled sequences. Moreover, by labelling the ODNs and peptides at several positions, the potency of the 3HC- and 3HQ-labelled nucleosides and aminoacids to site-specifically characterize and quantify the interactions with proteins and nucleic acids will be evaluated. The project is highly multidisciplinary (at the interface between chemistry, biophysics and biology) and will require the complementary expertises (in organic, ODN and peptide chemistry, fluorescence spectroscopy, Biacore and biomolecular interactions) of the participating teams. This project is expected to show the general applicability of these two-colour fluorescently labelled aminoacids and nucleosides for solid-phase synthesis of peptides and oligonucleotides to detect, characterize and quantify biomolecular interactions in one-step.

Project coordination

Yves MELY (Université)

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

Help of the ANR 350,000 euros
Beginning and duration of the scientific project: - 36 Months

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