Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids

Triple resonance HCN and HCNCH experiments are reliable methods of establis hing sugar-to-base connectivity in the NMR spectra of isotopicaly labeled o ligonucleotides. However, with larger molecules the sensitivity of the expe riments is drastically reduced due to relaxation processes. Since the polar ization transfer between C-13 and N-15 nuclei relies on rather small hetero nuclear coupling constants (11-12 Hz), the long evolution periods (up to 30 -40 ms) in the pulse sequences cannot be avoided. Therefore any effort to e nhance sensitivity has to concentrate on manipulating the spin system in su ch a way that the spin-spin relaxation rates would be minimized. In the pre sent paper we analyze the efficiency of the two known approaches of relaxat ion rate control, namely the use of multiple-quantum coherence (MQ) and of the relaxation interference between chemical shift anisotropy and dipolar r elaxation - TROSY. Both theoretical calculations and experimental results s uggest that for the sugar moiety (H1'-C1'-N1/9) the MQ approach is clearly preferable. For the base moiety (H6/8-C6/8-N1/9), however, the TROSY shows results superior to the MQ suppression of the dipole-dipole relaxation at m oderate magnetic fields (500 MHz) and the sensitivity improvement becomes d ramatically more pronounced at very high fields (800 MHz). The pulse scheme s of the triple-resonance HCN experiments with sensitivity optimized perfor mance for unambiguous assignments of intra-residual sugar-to-base connectiv ities combining both approaches are presented.