S.S. Wijmenga, B N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32(1998)287-387 291 achieve site-specific labeling via the enzymatic des(; r)p for distances between adjacent base paired method(see, for example, Ref. [36]). Site-specific labeling, on the other hand, can quite easily be nucleotides, e.g. dcs(1, 2)3 achieved via chemical synthesis. This would be the The symbols NH and NHz represent imino and amino method of choice for the preparation of labeled dNA oligonucleotide protons, respectively. The directionality in the equential cross-strand distances has to be indicated Consider two adjacent base pairs, and define the 5 3. Nomenclature and 3 -nucleotides. It can be easily seen that dcs either between two 3 -nucleotides or between two For atom numbering and torsion angle definitions in 5-nucleotides. This is indicated by the subscript nucleic acids we will follow the IUPAC/UB guide Alternatively, when two protons I and r do not fall lines [61]. Accordingly, the chemical structure and in any of the above categories the distance is indicated atom numbering of the five common bases, the by pyrimidines C, T and U, and the purines G and A are given in Fig. 1(A), and of the B-D-(deoxy)riboses d(; r) for long -range internucleotide distances in Fig. I(B), which also indicates the torsion angles in e.g. d(T2-NB3, A9-NH,6) the sugar-phosphate backbone(a, 6, 7, 8, 8 and 5) and the glycosidic torsion angle x. Their definitions Here, T2-NH3 indicates the imino proton of Thymine are:O3’-P-O5′-C5′(a),P-O5′-C5-C4’(B), number2andA9NH26 indicates the amino group of O5′-C5′-C4-C3′(),C5′-C4-C3-03′(6), Adenine number9 C4-C3-03-P(e),C3-03′-P-O5′(3),O4 CI'-NI-C2 ( x (Py), and 04'-C1'-N9-C4 (x(Pu)). Furthermore, it gives a designation of the 4. Distances chain direction and the unit numbering in a poly- nucleotide chain. Fig. I(C)shows the two most Proton to proton distances are essential parameters common conformations of the B-D(deoxy )ribose for the three-dimensional structure determination of sugar ring, the C2'-endo (E)and the C3'-endo(E) biomolecules by NMR. Since only short distances conformers, also referred to as S-type and N-type (<5-6A)can be obtained by NMR, it is difficult to conformers, respectively determine global features, such as bending of the To describe the distances we will use the shorthand helix. On the other hand, local features can be deter- notation introduced by Wijmenga et al. [62]. In this mined quite well and most NMR structural studies notation the distance between the protons I and r is have focused on these aspects. Consequently, it is of paramount importance to have a good overview of the di(; r)for intranucleotide distances, e.g. di (8, 2) short distances in the main structural elemer as the sugar ring, the bases, the base pairs, etc ds(; r) for internucleotide distances, e.g. ds(1, 6) how these distances determine the structural of those elements. Another aspect is that several of the Here, I corresponds to the proton in the 5'-nucleotide short distances do not depend on conformation nor do unit and r with the proton in the 3-nucleotide unit. For they take on well defined values for the two major methyl protons the I or r is indicated by the letter helical conformations. A- and B-helices. for this M. To indicate that the distance is between H3 in reason, it is particularly useful to have at hand an the 5'-nucleotide and H5 or the methyl protons in overview of these distances and their characteristics, the3′- nucleotide se d(3, 5/M). Cross-strand so that one can focus on the relevant data for interesting structural aspects dci(l; r) for distances within a base pair, In the next sections we therefore discuss the short distances and how they reflect structural characteris- e.g. dci(T-NH3; A-NH26 tics, by first giving a more general overview andachieve site-specific labeling via the enzymatic method (see, for example, Ref. [36]). Site-specific labeling, on the other hand, can quite easily be achieved via chemical synthesis. This would be the method of choice for the preparation of labeled DNA oligonucleotides. 3. Nomenclature For atom numbering and torsion angle definitions in nucleic acids we will follow the IUPAC/IUB guide￾lines [61]. Accordingly, the chemical structure and atom numbering of the five common bases, the pyrimidines C, T and U, and the purines G and A, are given in Fig. 1(A), and of the b-D-(deoxy) riboses in Fig. 1(B), which also indicates the torsion angles in the sugar–phosphate backbone (a, b, g, d, « and z) and the glycosidic torsion angle x. Their definitions are: O39–P–O59–C59 (a), P–O59–C59–C49 (b), O59–C59–C49–C39 (g), C59–C49–C39–O39 (d), C49–C39–O39–P («), C39–O39–P–O59 (z), O49– C19–N1–C2 (x (Py)), and O49–C19–N9–C4 (x (Pu)). Furthermore, it gives a designation of the chain direction and the unit numbering in a poly￾nucleotide chain. Fig. 1(C) shows the two most common conformations of the b-D-(deoxy)ribose sugar ring, the C29-endo (2 E) and the C39-endo (3 E) conformers, also referred to as S-type and N-type conformers, respectively. To describe the distances we will use the shorthand notation introduced by Wijmenga et al. [62]. In this notation the distance between the protons l and r is given by: di(l; r) for intranucleotide distances, e:g: di(8; 29) ds(l; r) for internucleotide distances, e:g: ds(19; 6) Here, l corresponds to the proton in the 59-nucleotide unit and r with the proton in the 39-nucleotide unit. For methyl protons the l or r is indicated by the letter M. To indicate that the distance is between H39 in the 59-nucleotide and H5 or the methyl protons in the 39-nucleotide we use ds(39;5/M). Cross-strand distances are defined as: dci(l; r) for distances within a base pair, e:g: dci(T ¹ NH3; A ¹ NH26) dcs(l; r)p for distances between adjacent base paired nucleotides, e:g: dcs(19; 2)39 The symbols NH and NH2 represent imino and amino protons, respectively. The directionality in the sequential cross-strand distances has to be indicated. Consider two adjacent base pairs, and define the 59- and 39-nucleotides. It can be easily seen that dcs is either between two 39-nucleotides or between two 59-nucleotides. This is indicated by the subscript p. Alternatively, when two protons l and r do not fall in any of the above categories the distance is indicated by: d(l; r) for long ¹ range internucleotide distances, e:g: d(T2 ¹ NH3; A9 ¹ NH26) Here, T2-NH3 indicates the imino proton of Thymine number 2 and A9-NH26 indicates the amino group of Adenine number 9. 4. Distances Proton to proton distances are essential parameters for the three-dimensional structure determination of biomolecules by NMR. Since only short distances ( , 5–6 A˚ ) can be obtained by NMR, it is difficult to determine global features, such as bending of the helix. On the other hand, local features can be deter￾mined quite well and most NMR structural studies have focused on these aspects. Consequently, it is of paramount importance to have a good overview of the short distances in the main structural elements, such as the sugar ring, the bases, the base pairs, etc. and of how these distances determine the structural features of those elements. Another aspect is that several of the short distances do not depend on conformation nor do they take on well defined values for the two major helical conformations, A- and B-helices. For this reason, it is particularly useful to have at hand an overview of these distances and their characteristics, so that one can focus on the relevant data for the more interesting structural aspects. In the next sections we therefore discuss the short distances and how they reflect structural characteris￾tics, by first giving a more general overview and S.S. Wijmenga, B.N.M. van Buuren/Progress in Nuclear Magnetic Resonance Spectroscopy 32 (1998) 287–387 291