A study on the conformation of the title compound, C-C-A, and on its constituent dinucleotides is presented. 1H-NMR spectra at 360 and 500 MHz were completely assigned by decoupling experiments. Computer simulation of the spectra yielded precise proton-proton and proton-phosphorus coupling constant values. The coupling constants are analyzed in terms of torsion angles and of Nand S-type sugar pucker.31P-NMR spectra gave some information about P-0 backbone torsion angles a and t. CD spectroscopy was used to obtain insight in the base-base interaction. The C(I) and C(2) unit in C-C-A show normal preference for N-type conformation of the sugar ring, whereas the A(3) residue appears rather biased towards the S-conformation. The C and a backbone torsion angles in the C-C phosphodiester linkage in C-C-A aDpear to assume normal g-, gconformation, the r, a combination in the C-A linkage is proposed to have a g+, t conformation. In the C-C fragment in C-C-A a regular stack is indicated; it is suggested that the C-A part adopts an unusual antiparallel base stack. INTRODUCTION Geometrical details of nucleic acid constituents can be investigated in considerable depth by a combination of various techniques. The judicious use of high-resolution proton NMR spectroscopy provides insight into the magnitudes of proton-proton and proton-phosphorus torsion angles and, by inference, into the spatial configuration of the sugar ring and part of the nucleotide backbone.''2 Temperature dependent 31P-NMR spectroscopy can yield at least qualitative information on the rotamer distribution about the P-O torsion angles, a distribution that cannot be monitored by 1H-NMR?,4, Circular dichroism of oligonucleotides can be used as a probe for base-base interaction; the spectral intensity and shaDe reflects general features of this interaction.5 The trinucleoside diphosphate sequence C-C-A is an invariable feature in tRNA molecules at the 3'-acceptor end. Crystal structures of yeast tRNAPhe molecules in two different crystallographic surroundings have been publish© I RL Press Limited, Oxford, England. 7517 Nucleic Acids Research ed.6-9 In these structures the two C-residues at the C(74)-C(75)-A(76) 3'acceptor end invariably partake in a regular stacking interaction, whereas the crucial A(76) points away from this normal C-C helix. However, the various tRNA structures appear to differ with respect to the geometry about the phosphate ester bonds 03'-P(C) and P-05'(a). The two monoclinic crystal structure density maps were alternatively interpreted in terms of t, g+ 7 and t, g9 conformations; in the orthorhombic structures t, g6 and g , g conformations were suggested. It should be realized that an unambiguous determination of such a small geometrical detail, in a molecule of the size of tRNA, from low resolution X-ray data is beset with difficulties. Furthermore the possibility of inadvertent loss of the 3'-terminal A-residue in part of the crystallized material should not be overlooked (R.S. Brown, personal communication). In order to throw more light on the conformational characterisitics of the 3'-acceptor end of tRNA we chose to study as simple model compounds the Ndimethylated analogon C-C-A (Fig. 1) and its constituents C-Cpm and mpC-A. From previous work on the N-dimethylated compounds A-A ° and A-U it is known that this particular modification strongly enhances the stacking proclivity without introducing significant changes in the geometry of the stacked species. '1° In other words, it is expected that anomalous behaviour, if any, will show up in an aggravated form in the modified compounds. Recently, Cheng et at.13 presented a proton NMR study on the conformational behaviour of the unmodified trimer C-C-A. Mainly on the basis of ring current H N calculations these authors propose a predominant OH _c}~ / conformer in which the three residues assume an 2-CpgK' /A-RNA-like regular right-handed stacking arI 'HIC\ CH3 rangement. The coupling constants reportedl3 OH 7 appear to contradict this proposal, however. We ° \ I purport to show in this paper that a regular -pm? Cp\O H O stack of all three residues in succession apIC\'C/ Hf pears less probable in C-C-A and, by inference, O0 OH L o\NINCH in C-C-A. In contrast, the total evidence points \o H_I_elA to a regular stack of the C residues.(c,a=g ,gj -P \ 0A/ N-\H concomitant with the presence of an unusual HC H&P\H antiparallel stacking arrangement in the C-A LHO OH fragment (~,a=g+,t). Fig. 1. Structure of C-C-A