6533b81ffe1ef96bd1278c38

RESEARCH PRODUCT

Des outils moléculaires toujours plus multifonctionnels pour l’étude de structures alternatives d’acides nucléiques.

subject

description

G-Quadruplexes (G4) are non-canonical secondary structure found throughout the genome and transcriptome in guanine rich sequences. Their involvement in biologically relevant processes (telomeres maintenance, transcription and translation) has been demonstrated, yet their role is still not fully elucidated. To investigate their role, we focus on the development of biomimetic molecular tools that could be used either to isolate G4 by new generation sequencing techniques or visualize them directly in cell. This class of molecular tools (TASQ: Template Assembled Synthetic Quartet) is composed by four guanine moieties linked to a central template thanks to spacers of different chemical nature. In presence of the target the four Gs assemble in a synthetic tetrad that, by stacking on top of the native G4, stabilize its structure. The unique nature of the interaction (like-likes-like) ensure an exquisite level of selectivity. Moreover, the central template can be easily functionalized (i.e., by addiction of an appendix) or exhibit interesting properties (i.e., fluorescence) introducing a very desirable level of versatility. Starting from these premises our goal was to improve the chemical accebility and the molecular tools portfolio, developing new TASQ designs. We first focused on the development of TASQ bearing a biotin appendix specifically designed for sequencing purposes. We improved the chemical accessibility of BioTASQ – already implemented in RNA sequencing (G4RP-seq) – and explored the influence of the spacers developing two new designs, BioCyTASQ and BioTriazoTASQ bearing alkyl and triazole linker respectively.With excellent in vitro features (FRET melting, FAM and qPCR pull-down) BioCyTASQ became the gold standard for biotinylated TASQs; while the bulkier BioTriazoTASQ showed a certain level of preference towards quadruplex RNAs isolation.To further improve the TASQ potential we replaced the biotin moiety with fully orthogonal appendages. MultiTASQ and AzMultiTASQ, bearing an alkyne and azide respectively, were used to isolate and visualize G4s in cells, using click chemistry (CuAAC and SPAAC); photoMultiTASQ, functionalized with a triple bond and a diazirine, was successfully synthesized with the premises of covalently trapping the G4 surroundings (proteins and nucleic acids) by photoactivation.We then shifted our focus on fluorescent TASQs expressly designed for cell imaging. We focus on twice-as-smart naphthalene-based probes, whose emission is dictated by G4 interaction. We revisited N-TASQ synthesis and developed TzN-TASQ comprised of triazole spacers, the latter highlighting an improved fluorescent enhancement with quadruplex RNAs. We finally focused on near infrared emitting probes based on a squaraine template. Preliminary results show how Square-TASQ v.2 exhibited strong G4 binding feature, with an emission centered around 640 nm increased upon interaction with G4.By implementing the TASQ portfolio with new design to better isolate, visualize G4s or do both with the same probe, we focus on providing molecular tools that could shed a light on the role of quadruplex.