Other 2D experiments permit accurate coupling constants to be measured and from these the conformation
may be determined, for example of the ribose or dexoyribose sugar pucker. In a section of a PE-COSY(COSY
stands for correlated spectroscopy) spectrum (Figure 11.7), note that the cross-peaks have a fine structure from
which coupling constants (J) may be measured. The coupling between protons on adjacent carbon atoms (or
any nuclear spin separated by three bonds,^3 J) is related to the torsional angle ( ) between their bonds as
described by the Karplus Equation(11.4):^9
(11.4)With appropriately labelled samples, it is possible to measure^3 J for^1 H–^1 H,^1 H–^13 C,^1 H–^31 P and^13 C–^13 C
nuclei and thereby describe not only the sugar pucker but also the geometry of the phosphate backbone. If
the heterocyclic base is both^15 N and^13 C enriched, then the glycosylic torsion angle (see Section 2.1.4)
may also be determined via measurement of the H1 to C6 (or C8) coupling constant. This also connects
a base to its sugar. Measurement of these heteronuclear couplings requires more complicated experiments that
have acronyms such as HMQC(heteronuclear multiple quantum correlation) and HMBC(heteronuclear
multiple bond correlation). These 2D experiments may be attached to COSY- or NOESY-type experiments
to produce 3D and 4D spectra. If isotopically labelled material is not available to ‘connect’a base to its own
sugar, it is necessary to look for NOESY cross-peaks from the H1 proton of the sugar to the H6 or H8 proton
of the base. In this way, different nucleotide residues may be identified, but their sequence relationship is
not yet established.
To assign a sequence position to each of the nucleoside residues, nOe connections have to be observed
between adjacent residues.^11 The pattern of inter-nucleotide nOes is analysed with reference to a model: a
B-type helix for DNA sequences, an A-type helix for RNA sequences. Analysis of the NOESY spectra
involves a ‘nOe-walk’, illustrated in practice in the DNA strand of a DNA/RNA hybrid (Figure 11.8). In
the example shown, the CH 3 (Me) of the middle T in the A 6 T 7 T 8 stretch displays a NOESY cross-peak not
only to its own (T 7 ) H6 but also to the H8 (or H6) of the residue 5 to it (A 6 in this instance). T 7 H6 on the
other hand should share a cross-peak with the CH 3 of the 3 residue (T 8 here) (highlighted in Figure 11.8)
and so on.
From this analysis, the sequence ATT is confirmed. If there is only one of these sequences in the fragment,
the residues may be numbered (A 6 T 7 T 8 ). The sequential assignment is further supported by observations
of NOESY connections amongst sugar and aromatic protons. Qualitative analysis of some key cross-peak
intensities can establish whether a sugar pucker is C3 - endoor C2 - endo(Section 2.1.4) and whether the
(^32) JHH13 7. cos 0 73. cos
Physical and Structural Techniques Applied to Nucleic Acids 435
Figure 11.7 (a) A section of a PE-COSY spectrum for a DNA/RNA hybrid– cross-peaks connecting A6 H1 to its
H2 and H2 are boxed in red. (b) A sketch of one of the boxed regions showing how the coupling
constants are measured