A parallel quadruplex derived from the Myc promoter sequence was extended by a stem‐loop duplex at either its 5′‐ or 3′‐terminus to mimic a quadruplex–duplex (Q–D) junction as a potential genomic target. High‐resolution structures of the hybrids demonstrate continuous stacking of the duplex on the quadruplex core without significant perturbations. An indoloquinoline ligand carrying an aminoalkyl side chain was shown to bind the Q–D hybrids with a very high affinity in the order Ka≈107 m−1 irrespective of the duplex location at the quadruplex 3′‐ or 5′‐end. NMR chemical shift perturbations identified the tetrad face of the Q–D junction as specific binding site for the ligand. However, calorimetric analyses revealed significant differences in the thermodynamic profiles upon binding to hybrids with either a duplex extension at the quadruplex 3′‐ or 5′‐terminus. A large enthalpic gain and considerable hydrophobic effects are accompanied by the binding of one ligand to the 3′‐Q–D junction, whereas non‐hydrophobic entropic contributions favor binding with formation of a 2:1 ligand‐quadruplex complex in case of the 5′‐Q–D hybrid.

The oligomer d(GCGTG 3 TCAG 3 TG 3 TG 3 ACGC) with short complementary flanking sequences at the 5'-and 3'ends was shown to fold into three different DNAG-quadruplex species.I nc ontrast, ac orresponding oligomer that lacks base complementarity between the two overhang sequences folds into as ingle parallel G-quadruplex. The three coexisting quadruplex structures were unambiguously identified and structurally characterized through detailed spectral comparisons with well-defined G-quadruplexes formed upon the deliberate incorporation of syn-favoring 8-bromoguanosine analogues into specific positions of the G-core.T wo (3+ +1) hybrid structures coexist with the parallel fold and feature an ovel lateral-propeller-propeller loop architecture that has not yet been confirmed experimentally.B oth hybrid quadruplexes adopt the same topology and only differ in their pattern of anti!syn transitions and tetrad stackings.

G-rich nucleic acid sequences encompassing G-tracts of varying lengths can fold into different non-canonical G-quadruplexes with distinct structural features.

Canonical G-quadruplexes can adopt a variety of different topologies depending on the arrangement of propeller, lateral, or diagonal loops connecting the four Gcolumns. A novel intramolecular G-quadruplex structure is derived through inversion of the last G-tract of a threelayered parallel fold, associated with the transition of a single propeller into a lateral loop. The resulting (3 + 1) hybrid fold features three syn•anti•anti•anti G-tetrads with a 3'-terminal all-syn G-column. Although the ability of forming a duplex stem-loop between G-tracts seems beneficial for a propellerto-lateral loop rearrangement, unmodified G-rich sequences resist folding into the new (3 + 1) topology. However, refolding can be driven by the incorporation of syn-favoring guanosine analogues into positions of the fourth G-stretch. The presented hybrid-type G-quadruplex structure as determined by NMR spectroscopy may provide for an additional scaffold in quadruplex-based technologies.

Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in-situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function. M2 is a paradigm of pH-activated proton channels and controls the proton flux into the viral interior during viral infection. We use SEIRA to track the large-scale reorientation of M2’s transmembrane α-helices in-situ during pH-activated channel opening. We quantify this event as a helical tilt from 26° to 40° by correlating the experimental results with solid-state NMR-informed computational spectroscopy. This mechanical motion is impeded upon addition of the inhibitor rimantadine, giving a direct spectroscopic marker to test antiviral activity. The presented approach provides a spectroscopic tool to quantify large-scale structural changes and to track the function and inhibition of the growing number of viroporins from pathogenic viruses in future studies.