Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

Numerous robust spectroscopic solutions have been proposed in the past to circumvent these coupling problems (33) (34) (35) (36) (37) (38) (39) (40) (41) . Unwanted splittings can be removed using constant time (CT) evolution (35) (36) (37) (38) , adiabatic band selective decoupling (39) (40) (41) , or a series of selective pulses. Constant time evolution limits the acquisition time that can be used to obtain adequate resolution. To improve resolution requires long constant-time delays that lead to significant signal loss for large RNA molecules (41) . Additionally, obtaining accurate relaxation parameters are problematic for 13 C-CPMG based relaxation dispersion rates for quantifying millisecond (ms) time-scale processes, as well as R 1 and proton-carbon hNOE (28, 42) important for quantifying ns-ps time-scale motions in RNA (43, 44) . Several precautions are needed to obtain accurate R 1 and R 1 measurements (28, (45) (46) : provided R 1 is derived from the initial slope of the relaxation decay curve, fairly accurate rates can be extracted for small RNAs; for R 1 experiments, distortions can arise from transfer between adjacent 13 C atoms with similar chemical shifts via a Hartmann-Hahn mechanism and these need to be minimized (28, (45) (46) ; to suppress the echo-modulation caused by the large scalar couplings during the 13 C relaxation delay, R 1 can be measured instead of CPMG (28, (47) (48) (49) . Still for nucleic acids high power spinlocks (>1 kHz) are needed to study isolated spin pairs such as C2 found in adenine and C8 in both adenine and guanine. For low spin lock power levels (<1 kHz), oscillations can be observed in the monoexponential decay of peak intensity, arising from residual scalar coupling interactions within neighbouring nuclei (48, (50) (51) .