290 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 these reviews. For example, a complete overview of enzymatic synthesis is the usual method of prepara J-couplings in the nucleic acid bases has been pub- tion; although chemical synthesis is possible it is still lished [49] and proton structural chemical shifts have prohibitively expensive when large quantities are been calculated and compared with experimental data required. Chemical synthesis is the usual approach [50]. We will incorporate these aspects into this for the preparation of DNAs of defined sequence review, together with a detailed description and criti- Zimmer and Crother [39]have shown how large quan- cal evaluation of the present state of the art NMr tities of DNA can be made via enzymatic synthesis, methodology for determining the structure of labeled thus demonstrating the feasibility of C and N DNA and RNa molecules. This review is divided into labeling of DNA via this method. However, C and eleven sections. In Section 2, C andN labeling, as N labeled DNA phosphoramidites have also recently well as other labeling methods, are described, albeit become commercially available, so that labeled briefly, in view of the quite detailed descriptions that DNAs can conveniently be prepared via chemical have recently appeared. The IUPAC nomenclature is synthesis [40]. We refer the reader to the original introduced in Section 3. In Section 4, we present an papers or reviews for the detailed protocols and for overview of the distances found in dna and rna discussions of the relative merits of the various molecules and discuss their relevance for NMR approaches [36,45,. 47, 48, 51-59]. Here we will structural studies. Section 5 gives an overview of all concentrate on some general and qualitative aspects homonuclear and heteronuclear J-couplings and A certain amount of confusing terminology has describes their structural dependencies. We also give crept into the literature with regard to labeling. We an overview of the NMr methods that are or can be will use the following terms: uniform labeling, when used to determine these J-couplings In Section 6, we every atom of a certain type in the molecule describe the chemical shifts and discuss their use both enriched: re esidue-type-specific labeling, if all residues for assignment purposes and as structural parameters. of a certain type(e.g. all Adenines) in the molecule Section 7 forms the heart of this review, and describes are enriched; site-specific labeling, if a particular resi- nd discusses in detail the currently available methods due or a number of particular residues are enriched for assignment both in unlabeled and C and N e.g. Al0 partial labeling, if the labeling of a certain labeled compounds. Section 8 concentrates on a residue is on, say, CI'only. In order to indicate that description of relaxation. Isotope enrichment has labeling is not 100%, we add the percentage after the opened up the way for detailed relaxation studies in word labelin the field of proteins. Such relaxation studies are still For the enzymatic synthesis of RNA, a DNA tem- scarce in the field of nucleic acids. We place relaxa- plate is required from which the RNA is transcribed tion studies on nucleic acids in the context of parallel by T7-polymerase using NTPs as building blocks tudies on proteins, and give an overview of the The C and/or N and/or H labeled NTPs are theoretical background. In Section 9 we briefly usually obtained from E coli cells, which are grown describe the actual structure determination from on either C enriched glucose, and/or 5N enriched NMr data. In Section 10, we discuss the prospects ammonium chloride. The RNA isolated from the cells for extension of NMR studies to larger systems and is broken down to C and/or N labeled NMPs we attempt to draw some conclusions in Section 11 which are subsequently converted into NTPs. Th method thus allows uniformly labeled RNAs to be made, or residue-type-specific labeled RNA when 2. RNA and DNA synthesis and purification the in vitro transcription occurs on a mixture of labeled and unlabeled NTPs. The method can in prin- Two strategies are available for preparing large ciple easily be extended to achieve deuteration or par quantities of DNA and RNa of defined sequence tial labeling. For example, Michnicka et al. [60]have nd high purity for NMR studies: (1)chemical suggested partial C labeling using acetate as a car synthesis by the phosphoramidite method, and (2) bon source, most recently Nikonowicz et al. [57]have enzymatic synthesis of RNAs via T7-polymerase demonstrated uniform HN labeling via the nd of dNAs via DNA-polymerase. For RNA enzymatic more complicated tothese reviews. For example, a complete overview of J-couplings in the nucleic acid bases has been pub￾lished [49] and proton structural chemical shifts have been calculated and compared with experimental data [50]. We will incorporate these aspects into this review, together with a detailed description and criti￾cal evaluation of the present state of the art NMR methodology for determining the structure of labeled DNA and RNA molecules. This review is divided into eleven sections. In Section 2, 13C and 15N labeling, as well as other labeling methods, are described, albeit briefly, in view of the quite detailed descriptions that have recently appeared. The IUPAC nomenclature is introduced in Section 3. In Section 4, we present an overview of the distances found in DNA and RNA molecules and discuss their relevance for NMR structural studies. Section 5 gives an overview of all homonuclear and heteronuclear J-couplings and describes their structural dependencies. We also give an overview of the NMR methods that are or can be used to determine these J-couplings. In Section 6, we describe the chemical shifts and discuss their use both for assignment purposes and as structural parameters. Section 7 forms the heart of this review, and describes and discusses in detail the currently available methods for assignment both in unlabeled and 13C and 15N labeled compounds. Section 8 concentrates on a description of relaxation. Isotope enrichment has opened up the way for detailed relaxation studies in the field of proteins. Such relaxation studies are still scarce in the field of nucleic acids. We place relaxa￾tion studies on nucleic acids in the context of parallel studies on proteins, and give an overview of the theoretical background. In Section 9 we briefly describe the actual structure determination from NMR data. In Section 10, we discuss the prospects for extension of NMR studies to larger systems and we attempt to draw some conclusions in Section 11. 2. RNA and DNA synthesis and purification Two strategies are available for preparing large quantities of DNA and RNA of defined sequence and high purity for NMR studies: (1) chemical synthesis by the phosphoramidite method, and (2) enzymatic synthesis of RNAs via T7-polymerase and of DNAs via DNA-polymerase. For RNA, enzymatic synthesis is the usual method of prepara￾tion; although chemical synthesis is possible it is still prohibitively expensive when large quantities are required. Chemical synthesis is the usual approach for the preparation of DNAs of defined sequence. Zimmer and Crother [39] have shown how large quan￾tities of DNA can be made via enzymatic synthesis, thus demonstrating the feasibility of 13C and 15N labeling of DNA via this method. However, 13C and 15N labeled DNA phosphoramidites have also recently become commercially available, so that labeled DNAs can conveniently be prepared via chemical synthesis [40]. We refer the reader to the original papers or reviews for the detailed protocols and for discussions of the relative merits of the various approaches [36,45,47,48,51–59]. Here we will concentrate on some general and qualitative aspects. A certain amount of confusing terminology has crept into the literature with regard to labeling. We will use the following terms: uniform labeling, when every atom of a certain type in the molecule is enriched; residue-type-specific labeling, if all residues of a certain type (e.g. all Adenines) in the molecule are enriched; site-specific labeling, if a particular resi￾due or a number of particular residues are enriched, e.g. A10; partial labeling, if the labeling of a certain residue is on, say, C19 only. In order to indicate that labeling is not 100%, we add the percentage after the word labeling. For the enzymatic synthesis of RNA, a DNA tem￾plate is required from which the RNA is transcribed by T7-polymerase using NTPs as building blocks. The 13C and/or 15N and/or 2 H labeled NTPs are usually obtained from E. coli cells, which are grown on either 13C enriched glucose, and/or 15N enriched ammonium chloride. The RNA isolated from the cells is broken down to 13C and/or 15N labeled NMPs, which are subsequently converted into NTPs. This method thus allows uniformly labeled RNAs to be made, or residue-type-specific labeled RNA when the in vitro transcription occurs on a mixture of labeled and unlabeled NTPs. The method can in prin￾ciple easily be extended to achieve deuteration or par￾tial labeling. For example, Michnicka et al. [60] have suggested partial 13C labeling using acetate as a car￾bon source; most recently Nikonowicz et al. [57] have demonstrated uniform 2 H/15N labeling via the enzymatic approach. It is more complicated to 290 S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387