Sequential backbone assignment of uniformly ¹³C-labeled RNAs by a two-dimensional P(CC)H-TOCSY triple resonance NMR experiment

Summary

A new ¹H−¹³C−³¹P triple resonance experiment is described which allows unambigous sequential backbone assignment in ¹³C-labeled oligonucleotides via through-bond coherence transfer from ³¹P via ¹³C to ¹H. The approach employs INEPT to transfer coherence from ³¹P to ¹³C and homonuclear TOCSY to transfer the ¹³C coherence through the ribose ring, followed by ¹³C to ¹H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of ³¹P_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 ¹H−³¹P spectrum which allows a sequential assignment in the ³¹P−¹H1′ region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a ¹³C-labeled RNA hairpin with the sequence 5′(GGGC-CAAA-GCCU)3′.