RNA duplex (24). In particular, nucleotides in the stem region displayed stronger intra-residue than inter-residue H8/H6–H10 crosspeaks in NOESY spectra acquired with a 50 ms mixing time, as well as characteristic sequential H20–H10 and very strong sequential H20–H8/H6 crosspeaks. In a typical A-form duplex RNA, sequential H20–H8/H6 distances are <2.5 s, intra-residue H10–H8/H6 distances are in the range 3.5– 4.0 s and sequential H10–H8/H6 distances are 1 s longer. Except for the closing U27:A43 base pair, all ribonucleotides in the stem had small 3JH10–H20 as detected in DQF COSY experiments, thus intra-residue H10–H20 correlations were determined from NOESY spectra with short mixing times. In the loop, several NOE crosspeaks are observed that are not A-like: a correlation of U33:H10 to A35:H8 and numerous crosspeaks between A35 and t6A37. The sequential crosspeaks between atom pairs U36:H10–t6A37:H8 and U33:H10–C34:H6 are absent but U33:H20 and C34:H6 is clearly present. The typical NOE pattern between residues U33, C34 and A35 is a requisite feature of a U-turn ‘ﬁngerprint’ (33). Important Scheme 1. Secondary structure of the studied ASL (left) and structure of the 6 t A modification (right). Nucleic Acids Research, 2006, Vol. 34, No. 10 2881 NOE-contacts were observed for threonine protons to sugar and base moieties in the loop region of the hairpin (Figure 2). Assignment of imino–proton resonances was based on sequential imino–imino interactions and imino–aromatic con- tacts observed in a NOESY spectrum collected in 90% H2O/10% D2O (34). No signal was observed for the terminal U27:A43 base pair, which is attributed to fraying at the helix end. In addition to imino-signals from the stem, the H11 amide proton in the threonine moiety of t6A was observed at 10.32 p.p.m. This amide proton shows NOE interactions with the purines H2, Ha and Hb of the amino acid in t6A37. Such pattern is expected for a side-chain coplanar to the parent base and results in the modiﬁed nucleobase adopting a tricyclic structure (11). Phosphorus signals were assigned from a [31P,1H]Hetero- COSY experiment relating each phosphorus to intra-residue H50/500 and H30 signals of the preceding residue. A downﬁeld shift was observed for the C34:P signal which is typical in a U-turn motif (33). Carbon assignments are obtained from a [1H,13C] HSQC spectrum. This spectrum clearly shows that C5–H5 signals of C35, U33 and U36 are upﬁeld shifted to random coil chemical shift values. Such a shift is indicative for absent ring current effects from 50 neighbors induced by stacking interactions (35). A summary of the chemical shift assignments is listed in the Supplementary Data. Structure determination A total of 151 intra-residue and 108 inter-residue distance restraints were obtained for the 17 residue RNA oligomer. Most residues showed no H10–H20 crosspeaks in the 65 ms TOCSY spectrum, typical of N-type sugars (32). Intermediate TOCSY crosspeaks from H10 to H20 were observed for the fraying terminal residues (U27, A43) and 7 to 10 in the loop. Figure 2. (A) Zoom of the tricyclic t6A residue (upper left). Side (right) and top view (lower left) of the loop region from the studied hairpin. Nucleobases of modified residue and the anticodon are depicted in green and yellow, respectively. A ribbon is drawn through P atoms. NOE distance restraints derived from NOE interactions of the threonine moiety in t6A37, are indicated in yellow dashed lines. Red dashed lines connect atoms that are within hydrogen bonding distance. Red circles highlight hydrogen bonds of the threonine moiety in t6A37 with other residues in the loop. (B) Strip of a noesy spectrum in D2O (mix ¼ 150 ms) at the resonance frequency of t6A37:H15, depicting some important NOEs in the loop region.