In our project, we showed that G-quadruplex-specific ligands PhenDC3 and 360A bind more strongly to hybrid forms of the polymorphic quadruplex-duplex hybrid sequences of the PIM1 oncogene than to the antiparallel form. We explored the atomic interactions between two PIM1-derived quadruplex-duplex hybrid sequences and the ligands using advanced NMR and well-tempered metadynamics. PhenDC3 and 360A bind at the same pocket in the hybrid QDH, characterized by a 3' G-quartet and coaxially stacked Watson-Crick base pairs. No significant binding-induced refolding was observed for the antiparallel sequence. PhenDC3 favored a single binding pose, with its middle phenanthroline moiety intercalated between G4 and the duplex.
In contrast, 360A occupied two mirror-image poses, each with a terminal quinolinium group, while pyridine B and the other quinolinium ring remained solvent-exposed. Notably, we found ligand-induced conformational preferences for the hybrid forms of the PIM1 sequence, which exists as a mix of hybrid and antiparallel forms in vitro. Optimizing ligand aromatic scaffolds is crucial for effective binding, as seen with PhenDC3 and 360A, and their ability to differentiate quadruplex-duplex junctions can aid in developing molecular switches for supramolecular recognition processes.
In the next segment, we utilized 19F NMR to investigate nucleic acid conformational polymorphism and ligand binding. We selected various polymorphic G4 families from different genomes, each with at least two conformations, and incorporated a 19F probe (FANA, 2′-Fluoro-arabinonucleic Acid) into the sequences without disrupting their native equilibrium. The 1D 19F NMR effectively distinguished these conformational populations by chemical shifts and intensity differences. Additionally, 19F labeling allowed for precisely identifying ligand-bound G4 sub-states, a feat often unattainable with 1H NMR and other low-resolution techniques. Thus, we reported the application of FANA labeling and 19F NMR to reveal the conformational polymorphism in different nucleic acid forms. The cellular environment influences conformational polymorphism, folding topology, and nucleic acid stability, which may contradict in vitro observations. The Xenopus laevis (African clawed frog) system is genetically similar to humans and shares approximately 79% of the identified human disease genes. Notably, under in-cell NMR conditions (in X. laevis), the 1H spectra were poorly resolved and broad because of an unfavorable transverse relaxation pathway and inherent sample inhomogeneity in the cellular context, making further analysis impossible without labeling. Therefore, to evaluate the role of an intracellular viscoelastic microenvironment in modulating interactions between polymorphic quadruplex-duplex hybrid topologies and projected ligands, we investigated the conformations of prefolded complexes in Xenopus laevis oocytes using 19F in-cell NMR. The observation of 19F peaks for hybrid quadruplex-duplex hybrid-ligand complexes in the cellular environment of oocytes matched well with the in vitro ligand recognition patterns of polymorphic sequences. Therefore, the in-cell NMR results indicated two distinct orthogonal conformations when injected under prefolded conditions with and without the ligand(s). The complexes retain their native folding inside the cellular environment with reduced off-target and non-specific binding to proteins or other nucleic acids and refolding to unbound conformations. For the 1st time, 19F NMR spectroscopy using FANA-modified oligos demonstrated the stability, folding, and ligand interactions of non-canonical nucleic acids in living cells (i.e. Xenopus laevis oocytes).
In the final segment, we tried several single nucleotide mutations in the quadruplex-duplex junction by natural DNA bases (Adenine, guanine, cytosine, and thymine). It is known that duplex stem-loop interactions drive folding and ligand binding, which requires the formation of an intercalation cavity by demolishing the interfacial quartet and duplex-stacking interactions. Our preliminary mutational study demonstrated that the duplex stem-loop is crucial for folding and ligand binding at the Q-D junction. One immediate Watson-Crick base pairing at the lateral loop can stabilize the unimolecular conformation. Duplex stem loops without base pairs or immediate bulges can provide polymorphism and non-specific ligand-bound states. A detailed structural and thermodynamic characterization of the effect of mutations is in progress.
