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.
G-rich nucleic acid sequences encompassing G-tracts of varying lengths can fold into different non-canonical G-quadruplexes with distinct structural features.
Quadruplex-duplex (Q-D) junctions are increasingly considered promising targets for medicinal and technological applications. Here, a Q-D hybrid with a hairpin-type snapback loop coaxially stacked onto the quadruplex 3'-outer tetrad was designed and employed as a target structure for the indoloquinoline ligand SYUIQ-5. NMR spectral analysis demonstrated high-affinity binding of the ligand at the quadruplex-duplex interface with association constants determined by isothermal titration calorimetry of about 10 7 M À 1 and large exothermicities ΔH°of À 14 kcal/mol in a 120 mM K + buffer at 40 °C. Determination of the ligand-bound hybrid structure revealed intercalation of SYUIQ-5 between 3'-outer tetrad and the neighboring CG base pair, maximizing π-π stacking as well as electrostatic interactions with guanine carbonyl groups in close vicinity to the positively charged protonated quinoline nitrogen of the tetracyclic indoloquinoline. Exhibiting considerable flexibility, the SYUIQ-5 sidechain resides in the duplex minor groove. Based on comparative binding studies with the non-substituted N5-methylated indoloquinoline cryptolepine, the sidechain is suggested to confer additional affinity and to fix the alignment of the intercalated indoloquinoline aromatic core. However, selectivity for the Q-D junction mostly relies on the geometry and charge distribution of the indoloquinoline ring system. The presented results are expected to provide valuable guidelines for the design of ligands specifically targeting Q-D interfaces.
A G-rich sequence was designed to allow folding into either a stable parallel or hybrid-type topology. With the parent sequence featuring coexisting species, various related sequences with single and double mutations and with a shortened central propeller loop affected the topological equilibrium. Two simple modifications, likewise introduced separately to all sequences, were employed to lock folds into one of the topologies without noticeable structural alterations. The unique combination of sequence mutations, high-resolution NMR structural information, and the thermodynamic stability for both topological competitors identified critical loop residue interactions. In contrast to first loop residues, which are mostly disordered and exposed to solvent in both propeller and lateral loops bridging a narrow groove, the last loop residue in a lateral three-nucleotide loop is engaged in stabilizing stacking interactions. The propensity of single-nucleotide loops to favor all-parallel topologies by enforcing a propeller-like conformation of an additional longer loop is shown to result from their preference in linking two outer tetrads of the same tetrad polarity. Taken together, the present studies contribute to a better structural and thermodynamic understanding of delicate loop interactions in genomic and artificially designed quadruplexes, e.g. when employed as therapeutics or in other biotechnological applications.
Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.
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