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Variations in the hydrogen bond network of the Oxy-1.5 DNA guanine quadruplex have been monitored by trans-H-bond scalar couplings, (h2)J(N2N7), for Na(+)-, K(+)-, and NH(4)(+)-bound forms over a temperature range from 5 to 55 degrees C. The variations in (h2)J(N2N7) couplings exhibit an overall trend of Na(+) > K(+) > NH(4)(+) and correlate with the different cation positions and N2-H2...N7 H-bond lengths in the respective structures. A global weakening of the (h2)J(N2N7) couplings with increasing temperature for the three DNA quadruplex species is accompanied by a global increase of the acceptor (15)N7 chemical shifts. Above 35 degrees C, spectral heterogeneity indicates thermal denaturation for the Na(+)-bound form, whereas spectral homogeneity persists up to 55 degrees C for the K(+)- and NH(4)(+)-coordinated forms. The average relative change of the (h2)J(N2N7) couplings amounts to approximately 0.8 x 10(-3)/K and is thus considerably smaller than respective values reported for nucleic acid duplexes. The significantly higher thermal stability of H-bond geometries in the DNA quadruplexes can be rationalized by their cation coordination of the G-quartets and the extensive H-bond network between the four strands. A detailed analysis of individual (h2)J(N2N7) couplings reveals that the 5' strand end, comprising base pairs G1-G9* and G4*-G1, is the most thermolabile region of the DNA quadruplex in all three cation-bound forms.

Structure determination of protein?RNA complexes in solution provides unique insights into factors that are involved in protein/RNA recognition. Here, we review the methodology used in our laboratory to overcome the challenges of protein?RNA structure determination by nuclear magnetic resonance (NMR). We use as two examples complexes recently solved in our laboratory, the nucleolin RBD12/b2NRE and Rnt1p dsRBD/snR47h complexes. Topics covered are protein and RNA preparation, complex formation, identification of the protein/RNA interface, protein and RNA resonance assignment, intermolecular NOE assignment, and structure calculation and analysis.

The binding of CysMeTANDEM and triostin A to DNA containing a CpI step has been studied by one- and two-dimensional 1H NMR spectroscopy. CysMeTANDEM binds sequence specifically to CpI steps as well as TpA steps as a bis-intercalator with the peptide backbone in the minor groove of the DNA. Only nonspecific, nonintercalative binding is observed between triostin A and DNA containing a CpI step. Comparison of the CysMeTANDEM-[d(GGACITCC)]2 complex to the CysMeTANDEM-[d(GGA-TATCC)]2 complex indicates that the structures of both complexes are very similar. However, CysMeTANDEM binds less tightly to [d(GGACITCC)]2 than to [d(GGATATCC)]2. The NMR evidence presented provides molecular insight into the role of stacking interactions and hydrogen bonding between the drug and the DNA in the sequence-specific binding of CysMeTANDEM to TpA sites and of triostin A to CpG sites.

The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studied by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1' and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RN oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1' and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.

New 2D and 3D 1H-13C-15N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in 13C- and 15N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective 13C-refocusing and 15N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D 1H-15N and 3D 1H-13C-15N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra- and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.

The complexes formed by the homopurine and homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4 have been investigated by one- and two-dimensional 1H NMR. Under appropriate conditions [low pH, excess d(TC)4 strand] the oligonucleotides form a triplex containing one d(GA)4 and two d(TC)4 strands. The homopurine and one of the homopyrimidine strands are Watson-Crick base paired, and the second homopyrimidine strand is Hoogsteen base paired in the major groove to the d(GA)4 strand. Hoogsteen base pairing in GC base pairs requires hemiprotonation of C; we report direct observation of the C+ imino proton in these base pairs. Both homopyrimidine strands have C3'-endo sugar conformations, but the purine strand does not. The major triplex formed appears to have four TAT and three CGC+ triplets formed by binding of the second d(TC)4 strand parallel to the d(GA)4 strand with a 3' dangling end. In addition to the triplexes formed, at least one other heterocomplex is observed under some conditions.