Quinolones are gyrase inhibitors that are widely used as antibiotics in the clinic. When covalently attached to oligonucleotides as 5'-acylamido substituents, quinolones were found to stabilize duplexes of oligonucleotides against thermal denaturation. For short duplexes, such as qu-T*GCGCA, where qu is a quinolone residue and T is a 5'-amino-5'-deoxythymidine residue, an increase in the UV melting point of up to 27.8 degrees C was measured. The stabilizing effect was demonstrated for all quinolones tested, namely nalidixic acid, oxolinic acid, pipemidic acid, cinoxacin, norfloxacin, and ofloxacin. The three-dimensional structure of (oa-T*GCGCA)2, where oa is an oxolinic acid residue, was solved by two-dimensional NMR spectroscopy and restrained molecular dynamics. In this complex, the oxolinic acid residues disrupt the terminal T1:A6 base pairs and stack on the G2:C5 base pairs. The displaced adenosine residues bind in the minor groove of the core duplex, while the thymidine residues pack against the oxolinic acid residues. The "molecular cap" thus formed fits tightly on the G:C base pairs, resulting in increased base-pairing fidelity, as demonstrated in UV melting experiments with the sequence oa-T*GGTTGAC and target strands containing a mismatched nucleobase. The structure of the "molecular cap" with its disrupted terminal base pair may also be helpful for modeling how quinolones block re-ligation of DNA strands in the active site of gyrases.

-Aminopurine (2AP) is a structural isomer of adenine (A), in which the amino group is at C2 instead of C6, and can form stable Watson-Crick (WC) type base pairs with thymine (T) (Figure 1). [1,2] While natural nucleobases do not emit at all, 2AP shows appreciable fluorescence. More importantly, its fluorescence quantum yield decreases 100-fold upon duplex formation. [3] Numerous studies exploit 2AP fluorescence to investigate problems in structural biology and biophysics: methyltransferase-induced base flipping, [4][5][6] conformational changes and enzymatic cleavage of the hammerhead ribozyme, [7,8] promoter binding and clearance of T7 RNA polymerase, [9,10] binding and strand separation of primertemplate DNA by T4 DNA polymerase, [11][12][13] and chargetransfer mechanisms in DNA coupled to polar solvation. [14][15][16] Alternatively, structural changes can be monitored by a lowenergy circular dichroism band observed with 2AP, as was demonstrated with RNA and DNA hairpin loops. [17,18] The high number of publications relating to 2AP reflects its importance in studies of biological macromolecules.When structural transitions in biological systems are examined with a molecular probe, the assumption is that the modified system behaves like the natural one. Consequently the introduction of the probe must leave the structure and dynamics unchanged. Fluorophore-induced perturbations have been analyzed by solution NMR spectroscopy in many cases. [19][20][21][22][23] With 2AP, however, the changes are so small that the results in the original studies were inconclusive. [24,25] With high-field spectrometers and an expanded set of NMR parameters at hand we can now investigate 2AP-induced changes in detail.We present herein the NMR solution structure and basepair dynamics of two 13-mer DNA duplexes (Figure 1) with X = A in the reference sequence and X = 2AP in the modified sample (in the following called 13merRef and 13mer2AP, respectively). The only change introduced into the helix is the position of the amino group in A and 2AP. To what extent are structure and dynamics affected by this change? To answer this question we employed 2D NMR spectroscopy and measurements of residual dipolar couplings (RDC) in conjunction with simulated annealing calculations to determine the solution structure, selective NMR T 1 experiments to evaluate base-pair dynamics, and temperature-dependent absorption and fluorescence spectroscopy to characterize local melting. By combining information from these different approaches the effect of a single substitution A!2AP can be evaluated.All NMR resonances except for the severely overlapped H5'/H5'' signals could be assigned by intra-and internucleotide NOEs.[26] WC base-pairing of 2AP is evidenced by the imino proton signal of T20 which is observed-though broadened-for 13mer2AP at 298 K. [26] In contrast to an earlier report, [24] all cross peaks expected for regular B-DNA are present in the NOESY spectra of both samples. However, for the diagonal imino proton signal for T20, fast decay with increa...

Within the last few decades, structure-based drug design (SBDD) has evolved into a powerful tool for the optimization of many low-molecular-weight lead compounds into highly potent drugs.[1] The principle of SBDD lies in the combination of different chemical moieties with the aim of obtaining a molecule that, while possessing the pharmacological properties necessary for a drug, is complementary in shape to the receptor binding pocket. This process requires knowledge of the exact structure of the protein/ligand complex. At present, structural genomics initiatives provide protein structures of biomedically relevant targets at an increasing rate [2] and recent structures of ion channels [3] and G-protein-coupled receptors (GPCRs) [4][5][6] bring these protein classes within reach for SBDD. Despite these successes, the daily work of pharmaceutical discovery is often limited by the ability to obtain high-resolution crystal structures of the target proteins in complex with the lower affinity ligands (lead structures) that are commonly identified by high-throughput screening or by fragment-based lead discovery.[1] In view of this limitation, SBDD would benefit from methods providing the relative orientations of different chemical fragments binding competitively to a receptor site. Such an approach would provide protein/ligand structures of novel ligands or fragments in relation to the known cocrystal structure of a reference ligand.Recently, we reported the observation by NMR spectroscopy of interligand NOE peaks occurring between two small ligands binding weakly and competitively to the same binding pocket of a common macromolecular receptor (Figure 1). [7,8] The measured mixture in solution contained two ligands (L A and L B ) in a 10-to 50-fold excess relative to the target receptor (T). As the ligands were competitive binders, these NOEs did not originate from a direct transfer of magnetization between the two ligands, [9] but rather from a spindiffusion process mediated by the protons of the receptor binding pocket. We proposed that such interligand NOEs can be used to define the relative orientation of the two ligands in the receptor binding pocket (the relative binding mode) and we termed the novel effect INPHARMA (internuclear NOEs for pharmacophore mapping; [8] Figure 1). INPHARMA can be observed for complexes with a dissociation constant (K d ) in the low micromolar to millimolar range.The scope of this work is to demonstrate for the first time that the INPHARMA method allows the determination of the relative, and in favorable cases even the absolute, binding mode of two low-affinity ligands binding competitively to a common receptor site and that it can thus be applied in the context of SBDD. In accordance with existing SBDD workflows, the experimental information derived from INPHARMA was used to select the correct binding mode

Abstract:The comparison of Fö rster resonance energy transfer (FRET) efficiencies between two fluorophores covalently attached to a single protein or DNA molecule is an elegant approach for deducing information about their structural and dynamical heterogeneity. For a more detailed structural interpretation of single-molecule FRET assays, information about the positions as well as the dynamics of the dye labels attached to the biomolecule is important. In this work, Rhodamine 6G (2-[3′-(ethylamino)-6′-(ethylimino)-2′,7′-dimethyl-6′H-xanthen-9′-yl]-benzoic acid) bound to the 5′-end of a 20 base pair long DNA duplex is investigated by both single-molecule multiparameter fluorescence detection (MFD) experiments and NMR spectroscopy. Rhodamine 6G is commonly employed in nucleic acid research as a FRET dye. MFD experiments directly reveal the equilibrium of the dye bound to DNA between three heterogeneous environments, which are characterized by distinct fluorescence lifetime and intensity distributions as a result of different guanine-dye excited-state electron transfer interactions. Sub-ensemble fluorescence autocorrelation analysis shows the highly dynamic character of the dye-DNA interactions ranging from nano-to milliseconds and species-specific triplet relaxation times. Two-dimensional NMR spectroscopy corroborates this information by the determination of the detailed geometric structures of the dye-nucleobase complex and their assignment to each population observed in the single-molecule fluorescence experiments. From both methods, a consistent and detailed molecular description of the structural and dynamical heterogeneity is obtained.

The solution structure of a synthetic DNA mini-hairpin possessing a stilbenediether linker and three G:C base pairs has been obtained using (1)H NMR spectral data and constrained torsion angle molecular dynamics. Notable features of this structure include a compact hairpin loop having a short stilbene-guanine plane-to-plane distance and approximate B-DNA geometry for the three base pairs. Comparison of the electronic spectra of mini-hairpins having one-to-four G:C base pairs and stilbenediether or hexamethyleneglycol linkers reveals the presence of features in the UV and CD spectra of the stilbene-linked hairpins that are not observed for the ethyleneglycol-linked hairpins. Investigation of the electronic structure of a stilbene-linked hairpin having a single G:C base pair by means of time-dependent density functional theory shows that the highest occupied molecular orbital, but not the lowest unoccupied molecular orbital, is delocalized over the stilbene and adjacent guanine. The calculated UV and CD spectra are highly dependent upon hairpin conformation, but reproduce the major features of the experimental spectra. These results illustrate the utility of an integrated experimental and theoretical approach to understanding the complex electronic spectra of pi-stacked chromophores.