SEQUENTIAL BACKBONE ASSIGNMENT OF UNIFORMLY C-13-LABELED RNAS BY A 2-DIMENSIONAL P(CC)H-TOCSY TRIPLE-RESONANCE NMR EXPERIMENT

A new H-1-C-13-P-31 triple resonance experiment is described which allows unambiguous sequential backbone assignment in C-13-labeled oligonucleotides via through-bond coherence transfer from P-31 via C-13 to H-1. The approach employs INEPT to transfer coherence from P-31 to C-13 and homonuclear TOCSY to transfer the C-13 coherence through the ribose ring, followed by C-13 to H-1 J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of P-31(i) coherence via C4'(i) and C4'(i-1), because of the relatively large J(PC4) couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4'(i) and C4'(i-1) coherences are subsequently transferred to, amongst others, H1'(i) and H1'(i-1), respectively, leading to a 2D H-1-P-31 spectrum which allows a sequential assignment in the P-31-(1)H1' region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a C-13-labeled RNA hairpin with the sequence S'(GGGC-CAAA-GCCU)3'.