S.S. Wijmenga, B N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 overlap in NMR spectra of these compound this is more reliable resonance assignments. In addi- Advances in crystallization techniques have in recent tion, more extensive constraints lists could be years resulted in the structure determination of the obtained for subsequent structure determination. In RNA hammerhead enzyme [2, 3], one of the two fold- the past two years a number of RNA structures with ing domains of the group I intron self-splicing RNa a size up to 30 to 40 nucleotides have been published [4, 5], and a few RNA-protein complexes [6, 7].In [17-301, together with RNA-peptide complexes addition, the structures of several DNA duplexes, as [31-36] and an RNA-protein complex of total mol well as of a DNa quadruplex, have been determined ecular weight 22 kDa 37, 38]. These studies have also by means of X-ray crystallography [8]. However, made it clear that the upper size limit for RNAs which despite the two X-ray structures of the hammerhead, can be studied by NMr lies around 30 nucleotides catalytic mechanism of this ribozyme has not yet when uniform labeling is employed, a size limit clarified. Crystal packing forces sometimes considerably below that for proteins affect RNA or DNA structures. For example, there Only quite recently has it become possible to enrich is still no crystal structure available of a DNA or DNA withC> N isotopes. Zimmer and Crothers RNA hairpin, since these tend to crystallize in bio- 39]demonstrated that DNA can be enriched via an duplex structures enzymatic approach, while even more recentlyC [9, 10- Solution structures, which can be determined and N labeled dNa phosphoramidites have become via NMR, are therefore particularly important in dna available [401, so that C andN enriched DNAs can and RNA structural biology as a complement to now also be obtained via chemical synthesis. It is to be crystallography. In addition, nucleic acids often con- expected that these possibilities will also have an tain regions of higher conformational flexibility. effect on NMR structural studies of DNA of larger NMR is particularly suited for identifying such size. Larger DNA systems, such as those forming regions three- and four-way junctions, have already been In the field of NMR of nucleic acids, advances were studied [41], but these have not yet produced detailed made in the 1980s with the introduction of synthetic solution structures, again due to the extensive signal methods for preparing well defined DNA sequences. overlap(see, for example, Refs. [42-44D) It is note- This development also made it possible to produce worthy that Altona and co-workers used an extremely well defined RNA sequences from DNA templates interesting approach to achieve the assignments in by enzymatic synthesis via T7-polymerase. These their studies of four-way junctions [43, 44]. They developments led to the determination of several solu- used well-determined hairpins as building blocks for tion DNA and RNA hairpin structures, from which the the larger four-way and three-way junctions they main folding principles of hairpin loops could be studied. This made it possible to obtain resonance determined [11, 12]. In addition, these developments assignment in very crowded spectra. The future will led to the determination of the solution structure of a reveal whether combining this approach with labeling DNA quadruplex [13, 14] and solution structures of will allow an extension to larger systems, both for triple helix molecules [151, as well as to the determi- RNAs and dNAs nation of a new DNA multi-stranded fold the C-motif Naturally, as isotope enriched nucleic acid mol [16. Still, the overlap encountered in NMR spectra ecules are now used in NMR studies, we will pay limited the size of the molecules that could be studied particular attention in this review to the related and the detail by which the structures could be deter- NMR methods. Various other reviews [36, 45-48] mined. In the early 1990s, methods were developed to have recently appeared, but they have focused gener- produce C or N enriched RNAS, via enzymatic ally on specific aspects of the NMR of isotope synthesis, in quantities large enough for NMR studies. enriched RNA. We try here to provide a broad over This possibility enabled more detailed studies of bio- view, covering as much as possible of the various logically relevant RNA sequences and folds. Initial aspects that come into play when performing NMR NMR studies have been performed and methods structural studies of both DNA and RNa molecules have been developed for assignment of resonances Furthermore, the field is developing rapidly and new of C and N labeled RNAS. The direct result of aspects have been published since the appearance ofoverlap in NMR spectra of these compounds. Advances in crystallization techniques have in recent years resulted in the structure determination of the RNA hammerhead enzyme [2,3], one of the two fold￾ing domains of the group I intron self-splicing RNA [4,5], and a few RNA–protein complexes [6,7]. In addition, the structures of several DNA duplexes, as well as of a DNA quadruplex, have been determined by means of X-ray crystallography [8]. However, despite the two X-ray structures of the hammerhead, the catalytic mechanism of this ribozyme has not yet been clarified. Crystal packing forces sometimes affect RNA or DNA structures. For example, there is still no crystal structure available of a DNA or RNA hairpin, since these tend to crystallize in bio￾logically less relevant extended duplex structures [9,10]. Solution structures, which can be determined via NMR, are therefore particularly important in DNA and RNA structural biology as a complement to crystallography. In addition, nucleic acids often con￾tain regions of higher conformational flexibility. NMR is particularly suited for identifying such regions. In the field of NMR of nucleic acids, advances were made in the 1980s with the introduction of synthetic methods for preparing well defined DNA sequences. This development also made it possible to produce well defined RNA sequences from DNA templates by enzymatic synthesis via T7-polymerase. These developments led to the determination of several solu￾tion DNA and RNA hairpin structures, from which the main folding principles of hairpin loops could be determined [11,12]. In addition, these developments led to the determination of the solution structure of a DNA quadruplex [13,14] and solution structures of triple helix molecules [15], as well as to the determi￾nation of a new DNA multi-stranded fold, the C-motif [16]. Still, the overlap encountered in NMR spectra limited the size of the molecules that could be studied and the detail by which the structures could be deter￾mined. In the early 1990s, methods were developed to produce 13C or 15N enriched RNAs, via enzymatic synthesis, in quantities large enough for NMR studies. This possibility enabled more detailed studies of bio￾logically relevant RNA sequences and folds. Initial NMR studies have been performed and methods have been developed for assignment of resonances of 13C and 15N labeled RNAs. The direct result of this is more reliable resonance assignments. In addi￾tion, more extensive constraints lists could be obtained for subsequent structure determination. In the past two years a number of RNA structures with a size up to 30 to 40 nucleotides have been published [17–30], together with RNA–peptide complexes [31–36] and an RNA–protein complex of total mol￾ecular weight 22 kDa [37,38]. These studies have also made it clear that the upper size limit for RNAs which can be studied by NMR lies around 30 nucleotides when uniform labeling is employed, a size limit considerably below that for proteins. Only quite recently has it become possible to enrich DNA with 13C and 15N isotopes. Zimmer and Crothers [39] demonstrated that DNA can be enriched via an enzymatic approach, while even more recently 13C and 15N labeled DNA phosphoramidites have become available [40], so that 13C and 15N enriched DNAs can now also be obtained via chemical synthesis. It is to be expected that these possibilities will also have an effect on NMR structural studies of DNA of larger size. Larger DNA systems, such as those forming three- and four-way junctions, have already been studied [41], but these have not yet produced detailed solution structures, again due to the extensive signal overlap (see, for example, Refs. [42–44]). It is note￾worthy that Altona and co-workers used an extremely interesting approach to achieve the assignments in their studies of four-way junctions [43,44]. They used well-determined hairpins as building blocks for the larger four-way and three-way junctions they studied. This made it possible to obtain resonance assignment in very crowded spectra. The future will reveal whether combining this approach with labeling will allow an extension to larger systems, both for RNAs and DNAs. Naturally, as isotope enriched nucleic acid mol￾ecules are now used in NMR studies, we will pay particular attention in this review to the related NMR methods. Various other reviews [36,45–48] have recently appeared, but they have focused gener￾ally on specific aspects of the NMR of isotope enriched RNA. We try here to provide a broad over￾view, covering as much as possible of the various aspects that come into play when performing NMR structural studies of both DNA and RNA molecules. Furthermore, the field is developing rapidly and new aspects have been published since the appearance of S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 289