Journal: Journal of Biomolecular Nmr
Article Title: 13 C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides
doi: 10.1007/s10858-010-9429-5
Figure Lengend Snippet: Pulse sequence of the carbon direct detected 3D-(H)CC-TOCSY-H1′C1′ experiment with a virtual decoupling scheme in t 3 . Narrow and wide filled bars correspond to rectangular 90° and 180° pulses, respectively. Selective pulses and gradients are indicated as semi - ellipses . The default pulse phase is x. The proton carrier frequency is centred at the water frequency (5.7 ppm). The values for the 13 C and 15 N offsets are set to 77 ppm ( 13 C Ribose ), 90 ppm ( 13 C C1′ ), 70 ppm ( 13 C C2′ ) and 160 ppm ( 15 N), respectively. Asynchronous GARP decoupling (Shaka et al. ) is used to suppress heteronuclear scalar coupling during acquisition. The pulse field gradients with a length of 1 ms have a smoothed square amplitude (Bruker Topspin 2.0, 2006). They are applied along the z -axis and have the following strengths: G 1 :16%, G 2 :16% (pulse length 300 μs), G 3 :80%, G 4 :70%, G 5 :10%, G 6 :50%, G 7 :60%. 100% of gradient strength corresponds to 53.5 Gauss/cm. Fixed delays are adjusted as follows: Δ = 3 ms (1/(2* 1 J HC )), T = 6.26 ms (1/(4* 1 J CC ). For the CC-TOCSY transfer the FLOPSY-16 mixing sequence (Kadkhodaie et al. ) was applied, optimized for a single transfer (τ M = 3 ms) or multiple transfers (τ M = 15 ms). For the virtual decoupling scheme, a band selective pulse 180° Q3 Gaussian cascade (Emsley and Bodenhausen ) of 2 ms (semi-ellipse) is applied either on C1′ or C2′. Phase cycling: φ 1 = x, − x, φ 2 = 2(x), 2(−x), φ 3 IP = 4(y), 4(−y), φ 3 AP = 4(x), 4(−x), φ rec = x, 2(−x), x, −x, 2(x), −x. Quadrature detection in the ω 1 and ω 2 dimensions is obtained by incrementing φ 1 and φ 2 in a States-TPPI manner (Marion et al. ). The in-phase and anti-phase components of the C1′C2′ coherences were recorded in an interleaved manner and afterwards combined with a standard Bruker c-program (au-prog: splitcomb) using the parameter: ipap, inphase/antiphase correction term of 1.09 and an average coupling constant 1 J(C1′C2′) = 40 Hz
Article Snippet: The double IPAP is applied in an interleaved manner and the linear combination of all four experiments (Fig. a) is performed after data acquisition by using the standard Bruker c-program splitcomb (included in Topspin 2.1).
Techniques: Sequencing
