Titolo: More than meets the I-motif and G-quadruplex: light-up probes for sensing non-canonical DNA structures in cells

Codice Progetto: 2022R994M2

Responsabile scientifico per il DMM: Prof. Enrico Lavezzo- Università degli Studi di Padova
Coordinatore: Università degli Studi di BRESCIA - Prof.ssa Alessandra GIANONCELLI

Partner-Unità di ricerca: Università degli Studi di Padova

Bando: PRIN 2022 - Decreto Direttoriale n. 104 del 02-02-2022
Durata: 07/02/2025 - 06/02/2027 (24 mesi)

Finanziamento progetto: € 211.401,00 – CUP D53C24003200006

 

Abstract del progetto

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are flexible biomolecules that can fold into peculiar three-dimensional arrangements. Besides the canonical double strand, sequences showing repeats of guanines (Gs) or cytosines (Cs) can form secondary structures known as G-quadruplex (GQ) or I-motif (iM), respectively, for which multiple roles in gene regulation are well supported. While the investigation of the GQs and iMs dynamic behavior in living organisms is crucial to fully address their biological effects, their visualization and quantification in physiological environments remain challenging. In the past decade, detection methods relied on antibodies, which come with unavoidable limitations, while some examples of low molecular weight fluorescent probes appeared recently.

 With this project, we aim at implementing the available tools for GQs and iMs sensing, by developing new small molecule probes that possess drug-like features. Our approach is based on the synthesis of compounds interacting with biologically relevant GQs and iMs and discriminating among them on a shape-selective base. Our ligands will be designed to undergo conformational rearrangements upon binding, thus leading to the “light-up” effect. This task will be approached adopting different synthetic strategies, leading to three sets of probes based on different sensing mechanisms: compounds synthesized through click chemistry reactions bearing fluorescent cores in which the response will be modulated by the orientation of side chains, sensors based on the photoinduced electron transfer (PET) effect and molecules formed by two chromophores connected through a flexible linker. The design will take advantage of molecular modeling tools, and synthesized compounds will be prepared in adherence with drug-likeness and atom economy criteria. Then, the spectroscopical properties of ligands will be studied in detail via UV-Vis and fluorescence analyses.

 A combination of screening techniques (mass spectrometry, fluorescence melting and competition) will be employed to evaluate the binding towards two model non-canonical nucleic acid structures. On the selected hits, nuclear magnetic resonance (NMR), spectroscopic (fluorescence, circular dichroism) and calorimetry (ITC, DSC) analyses will be applied to fully describe the recognition process. Moreover, the selectivity of the best candidates will be tested considering already characterizes biologically relevant sequences as well as additional key nucleic acid elements selected by in house refined bioinformatics screenings. The combined results, together with in cell imaging studies, will provide insights on the effectiveness of the probes for different sensing applications via structural recognition, paving the way for their use to follow the evolution in terms of number and folding topologies of these nucleic acid secondary structures in the physiological environment.