Experiments

Parameter set: ZGDCESGP

Pulse sequence: zgdcesgp

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• SW: 26 ppm (full proton range in H₂O)
• O1P: 4.7 ppm (set on water resonance)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• p12: The selectivity of the water selective 180-degree shaped pulse (p12:sp1) can be increased by increasing its duration (p12). This leads to more selective suppression of the water signal and increases the intensity of RNA signals close to the water signal. The power level of the pulse (sp1) needs to be reduced accordingly.
• p16: to increase the sensitivity for larger RNAs, the homospoil/gradient pulse length can be reduced to 300 µs at the expense of water suppression efficiency.
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.

Additional options:

• ¹⁵N-decoupling: For RNAs containing uniform ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_N to apply ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• ¹³C-decoupling: For RNAs containing uniform ¹³C labeled nucleotides use the ZGOPTNS -DLABEL_C to apply ¹³C-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.

Description:

This experiment is used to record a 1D ¹H spectrum of all signals for RNA samples in H₂O.

The pulse sequence employs water suppression by excitation sculpting with gradients. Excitation sculpting is a robust technique which uses a gradient echo sequence to achieve very efficient suppression of the strong water signal while retaining the desired signals of the RNA sample. Therefore, this sequence is well suited for recording a 1D proton spectrum over the full chemical shift range.

As an option, this pulse program can be used with decoupling for either the ¹³C or ¹⁵N atoms. In addition to the imino peaks (which can be observed with higher sensitivity using the na_zggpjrse), this spectrum also shows the aromatic proton signals (A:H2, C/U:H6 and A/G:H8) around 7.5 ppm. Flanking this region are the amino peaks of A:H61/H62, C:H41/H42 and G:H21/H22. The H1’ ribose signals together with the H5 aromatic signals of C and U appear typically in the range of 6 to 5 ppm and the remaining ribose signals (H2’, H3’, H4’, H5’1 and H5’2) are found between 5 and 4 ppm.

Some signals may not be observable, because they overlap with the suppressed water signal. The excitation sculpting 1D ¹H spectrum is also used for proper chemical shift referencing of the ¹H signals and to verify the amount and quality of the RNA and whether other wanted and unwanted components are present in the sample, such as buffer signals or impurities.

Literature:

T.-L. Hwang & A.J. Shaka, J. Magn. Reson., Series A 112 275-279 (1995)

Parameter set: NA_ZGGPJRSE

Pulse sequence: na_zggpjrse

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• SW: 26 ppm (full proton range in H₂O)
• O1P: 4.7 ppm (set on water resonance)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• CNST19: 12 ppm, set to the center of the imino region. It defines the offset for maximum signal intensity.
• D1: 0.5 s, experiment allows short D1 due to preservation of H₂O magnetization.

Additional options:

• ¹⁵N-decoupling for RNAs containing ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_N to apply ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case and it is advised to use more dummy scans (set O3P to 150 ppm).

Description:

This experiment is used to record a 1D ¹H spectrum of the imino region of RNA (~10-15 ppm). It features a jump return echo sequence for binomial water suppression with the added benefit of increasing the imino signal intensity by preservation of the water magnetization. Most imino peaks appear between 15 and 10 ppm, but some up- or down‑field shifted exceptions to this rule might occur for imino protons in non-helical regions. The signal intensity (I) in this a jump return experiment is modulated on the chemical shift (CS) according to the following equation: I = sin³((Pi*/2) *(CS-o1p)/(CNST19-o1p). Maximum intensity is observed at the chemical shift set by CNST19. This scheme efficiently preserves the water magnetization along +Z. This allows for the use of a short relaxation delay D1 (as the imino proton magnetization is restored via chemical exchange with water).

Due to exchange with water protons the linewidth and number of observable imino proton signals are highly dependent as temperature influences base-pair stability and exchange rates. It is therefore advisable to optimize the experimental temperature specifically for imino proton spectral region (typically between 283 and 308 K).

Literature:

V. Sklenar & A. Bax, J. Magn. Reson. 74, 469 (1987)

Parameter set: ZGDCPR

Pulse sequence: zgdcpr

Sample requirements:

• Solvent: D₂O
• Labeling: any

Spectral settings:

• SW: 12 ppm (full proton range in D₂O)
• O1P: 4.7 ppm (set on water resonance, optimize to improve water suppression)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• Plw9: use weak RF-Power for presaturation of remaining H₂O, increase to improve water suppression (must be <0.001 W).
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.

Additional options:

• ¹³C-decoupling: For RNAs containing uniform ¹³C labeled nucleotides use the ZGOPTNS -DLABEL_C to apply ¹³C-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• ¹⁵N-decoupling: ¹⁵N-decoupling is possible with this experiment, but usually not useful for RNA samples in D₂O as all ¹⁵N bound protons rapidly exchange with D₂O.

Description:

This experiment is used to record a 1D ¹H spectrum of all signals for RNA samples in D₂O.

The pulse program uses presaturation of the water signal during the relaxation delay. This allows for very selective suppression of the water signal and allows detection of ribose ¹H signals close to the water.

As an option, this pulse program can be used with decoupling for either the ¹³C or ¹⁵N atoms. Moreover, the ¹H 1D spectrum is also used for proper chemical shift referencing of the ¹H signals and to verify the amount and quality of the RNA and whether other components are present in the sample, such as buffer signals or impurities.

Parameter set: NA_NOESYESGPPH

Pulse sequence: na_noesyesgpph

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• SWF1: 12 ppm (reduced spectral width for indirect proton dimension)
• SWF2: 26 ppm (full range in H₂O for direct proton dimension)
• O1P: 4.7 ppm (set on water resonance)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• D8: 100-150 ms, to obtain NOESY buildup spectra for efficient use in NMR structure calculations, the NOE mixing time can be varied from about 50 ms to 200 ms; below this range hardly any NOE cross peak may be visible and above the mixing time may be too long and therefore the signals are prone to direct relaxation effects, spin diffusion and secondary NOEs. Yet, NOESY spectra acquired with a mixing time of around 300 ms can still be useful for assignment purposes.
• CNST22: 9 ppm, defines the center for the indirect ¹H-dimension. The offset shift allows for a reduced spectral width in the indirect proton dimension and thereby a concomitant resolution increase in the proton range (from 3.0 to 15.0 ppm).
• p12: The selectivity of the water selective 180-degree shaped pulse (p12:sp1) can be increased by increasing its duration (p12). This leads to more selective suppression of the water signal and increases the intensity of RNA signals close to the water signal. The power level of the pulse (sp1) needs to be reduced accordingly.
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.

Additional options:

• ¹⁵N-decoupling: For RNAs containing uniform ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_N to apply ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• ¹⁵N-decoupling and ¹³C-decoupling: For RNAs containing uniform ¹³C and ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C and ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• To use a zero-quantum filter at the end of the mixing time (Thrippleton et al.) use option -DZQF.
• The option -DAWS enables apodization weighted sampling (Simon et al.). Useful for NS > 32 and be aware not to process the spectrum with linear prediction.

Description:

This pulse sequence uses excitation sculpting to suppress water. For RNA in H₂O, the spectral widths should be chosen so that the correlation of the imino protons with the aromatic and ribose signals can be observed (26 ppm). For increased resolution in the indirect dimension, the frequency can be shifted (via CNST22) so that the spectral width can be reduced to 12 ppm.

Literature:

M.J. Thrippleton & J. Keeler, Angew. Chem. Int. ed. 42, 3938-3941 (2003)

B. Simon & H. Koestler, J. Biomol. NMR 73, 155-165 (2019)

Parameter set: NA_NOESYGPPHPR

Pulse sequence: na_noesygpphpr

Sample requirements:

• Solvent: D₂O
• Labeling: any

Spectral settings:

• SWF1: 6.0 ppm (reduced spectral width for indirect proton dimension)
• SWF2: 9.0 ppm (full range in D₂O for direct proton dimension)
• O1P: 4.7 ppm (set on water resonance, optimize to improve water suppression)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• D8: 100-150 ms, to obtain NOESY buildup spectra for efficient use in NMR structure calculations, the NOE mixing time can be varied from about 50 ms to 200 ms; below this range hardly any NOE cross peak may be visible and above the mixing time may be too long and therefore the signals are prone to direct relaxation effects, spin diffusion and secondary NOEs. Yet, NOESY spectra acquired with a mixing time of around 300 ms can still be useful for assignment purposes.
• CNST22: 5.5 ppm, defines the center for the indirect ¹H-dimension. The offset shift allows for reducing the ppm range and thereby efficient full range recording of the indirect dimension with high resolution.
• Plw9: Use weak RF-Power for presaturation of remaining H₂O, increase for better water suppression (must be <0.001 W).
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.

Additional options:

• ¹⁵N-decoupling: For RNAs containing uniform ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_N to apply ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• ¹⁵N-decoupling and ¹³C-decoupling: For RNAs containing uniform ¹³C and ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C and ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• The option -DPP is used to apply a purge pulse after the acquisition (this is recommended for when using a short relaxation delay d1).
• To use a zero-quantum filter at the end of the mixing time (Thrippleton et al.) use option -DZQF.
• The option -DAWS enables apodization weighted sampling (Simon et al.). Useful for NS > 32 and be aware not to process the spectrum with linear prediction.

Description:

The NOESY with presaturation is useful for RNA samples in D₂O where imino and amino protons are anyway invisible. It operates with minimal water suppression for the residual H₂O signal. The absence of amino protons reduces spectral crowding in the region between 6 - 8 ppm, and thus is well suited for the sequential correlation of aromatic and H1’ /H2’ protons and identification of individual ribose spin systems. In combination with the 2D ¹H¹H NOESY – for all protons (in H₂O), the H5-H6 correlations for base-paired cytidines serve as anchor points for the sequential assignment.

Literature:

M.J. Thrippleton & J. Keeler, Angew. Chem. Int. ed. 42, 3938-3941 (2003)

B. Simon & H. Koestler, J. Biomol. NMR 73, 155-165 (2019)

Parameter set: NA_SFNOESYSF2D

Pulse sequence: na_sfnoesysf2d

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• SWF1: 6.0 ppm (imino range for indirect proton dimension)
• SWF2: 6.0 ppm (imino range for direct proton dimension)
• O1P: 12.0 ppm (set to the center of the imino region)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• D8: 100-150 ms, NOESY mixing time.

Additional options:

• ¹⁵N-decoupling and ¹³C-decoupling: For RNAs containing uniform ¹³C and ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C and ¹⁵N-decoupling during acquisition. The maximum acquisition time is restricted to 250 ms in this case.
• The option -DAWS enables apodization weighted sampling (Simon et al.). Useful for NS > 32 and be aware not to process the spectrum with linear prediction.

Description:

The SOFAST-SOFAST NOESY uses selective pulses that only affect the imino proton region and leave the H₂O magnetization unperturbed. This allows the use of short D1 delays (0.3-0.5 s). Since only imino protons are recorded in the experiment, the spectral width can be chosen small to increase the resolution. With O1 set to 12 ppm (the center of the imino region) and both spectral widths set to 6 ppm; the spectrum covers in both dimensions the essential range for the imino-to-imino signals from 9 to 15 ppm.

Literature:

B. Simon & H. Koestler, J. Biomol. NMR 73, 155-165 (2019)

Parameter set: NA_SFNOESY2D

Pulse sequence: na_sfnoesy2d

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• SWF1: 12.0 ppm (indirect proton dimension)
• SWF2: 14.0 ppm (direct proton dimension)
• O1P: 12 ppm (set to center of imino proton region)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• D8: 150 ms, NOE mixing time.

Additional options:

• ¹⁵N-decoupling and ¹³C-decoupling For RNAs containing ¹³C and ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C and ¹⁵N-decoupling during acquisition. Maximum acquisition time is 250 ms in this case.
• The option -DAWS enables apodization weighted sampling (Simon et al.). Useful for NS > 32 and be aware not to process the spectrum with linear prediction.

Description:

The SOFAST NOESY excites all protons before the t₁ time and the NOE mixing time. After the NOE transfer, only the selective pulses for the imino signals are used. This means that the spectrum shows the NOE cross peaks from all protons (in the direct dimension) to the imino protons (in the indirect dimension).

Literature:

B. Simon & H. Koestler, J. Biomol. NMR 73, 155-165 (2019)

Parameter set: NA_SFHMQCF3GPPH

Pulse sequence: sfhmqcf3gpph

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (set to center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 1024 for unlabeled or 8 for ¹⁵N-labeled samples, adjust to a multiple of 8 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST54: 12.5 ppm, center of imino proton region.
• CNST55: 6 ppm, bandwidth for imino selective pulses.

Additional options:

• CALC_SP: for calculation of all band selective Proton pulses based on CNST54 and CNST55 (recommended) use option -DCALC_SP.
• LABEL_CN: For ¹³C-decoupling in the ¹⁵N-dimension (for ¹³C labeled RNA) use option -DLABEL_CN.

Description:

This experiment is used to record a 2D ¹H¹⁵N correlation spectrum for imino protons. The SOFAST ¹H¹⁵N HMQC uses selective ¹H pulses that only affect the imino proton region and leave the H₂O magnetization unperturbed. ¹H¹⁵N correlations can be acquired most sensitively with the SOFAST HMQC because the inter-scan delay can be selected short and thus many scans can be accumulated in a short time. In general, 15 minutes (¹⁵N labeled RNA) or 18 hours (unlabeled RNA) should be sufficient for good S/N.

Literature:

P. Schanda and B. Brutscher, J. Biomol. NMR. 33, 199-211 (2005)

Parameter set: NA_SFHMQCGPPH

Pulse sequence: sf_mehmqcgpph

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: unlabeled

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 143 ppm (set to center of aromatic C2/C6/C8 ¹³C signals)
• SWF1: 25 ppm (indirect ¹³C dimension)
• SWF2: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 256, adjust to a multiple of 8 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST2: ¹J(CH) 185 Hz.
• CNST49: 8.5 ppm, center of aromatic H2/H6/H8 protons.
• CNST55: 4 ppm, bandwidth for H2/H6/H8 selective pulses.

Additional options:

• CALC_SP: for calculation of all band selective Proton pulses based on CNST54 and CNST55 (recommended) use option -DCALC_SP.

Description:

This experiment is used to record a 2D ¹H¹³C correlation spectrum for the aromatic H2, H6 and H8 signals. The SOFAST ¹H¹³C HMQC uses selective ¹H pulses that only affect the aromatic proton region and leave the H₂O magnetization unperturbed. ¹³C-¹H correlations can be acquired with particular high sensitivity with the SOFAST-HMQC because the relaxation delay can be selected short and thus many scans can be accumulated in a short time. In general, 5 h should be sufficient to acquire a high quality spectrum. For ¹H¹³C correlations, the SOFAST ¹H¹³C HMQC can only be used for the aromatic range (H2/H6 and H8), the remaining signals are too close to the water resonance. However, this region is the most important for RNA assignment and the experiment can be used to record the ¹³C-dimension with high resolution.

Literature:

P. Schanda and B. Brutscher, J. Biomol. NMR. 33, 199-211 (2005)

Parameter set: NA_HSQCETGPSP.4

Pulse sequence: hsqcetgpsp.4

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: any

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 107 ppm (set to center of all ¹³C signals)
• SWF1: 100 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 128 for unlabeled and 8 for ¹³C-labeled samples, adjust to a multiple of 4 to achieve desired S/N.
• CNST2: 170 Hz, ¹J(CH).
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.

Description:

The experiment is used for ¹³C/¹H correlations of all aromatic and ribose atoms. Plays a minor role in the detection of unlabeled RNA because the resolution is too poor. Helps when comparing different labeled RNAs. In addition, water suppression is very effective due to gradient selection. In general, 9 h should be sufficient to measure a good spectrum

Literature:

S. Asami, W. Kallies, J.C. Guenther, M. Stavropoulou, S.J. Glaser & M. Sattler, Angew. Chem. Int. Ed. Engl. 57(44), 14498-14502 (2018)

Parameter set: NA_DIPSI2ESGPPH

Pulse sequence: na_dipsi2esgpph

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: any

Spectral settings:

• SWF1: 6 ppm (indirect ¹H dimension)
• SWF2: 9 ppm (direct ¹H dimension)
• O1P: 4.7 ppm (set on water resonance)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 8 to achieve desired S/N.
• CNST22: 5.5 ppm, Center for the indirect ¹H-dimension.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D9: 60 ms, TOCSY mixing time.

Additional options:

• ¹³C-decoupling for RNAs containing 13C labeled nucleotides use the ZGOPTNS -DLABEL_C to apply ¹³C-decoupling during acquisition. Maximum acquisition time is 250 ms in this case.
• To use a zero-quantum filter at the end of the mixing time, the option -DZQF (Thrippleton et al.)
• The option -DAWS enables apodization weighted sampling (Simon et al.). Useful for NS > 32.
• The -DMOCCA option uses a different TOCSY mixing sequence with improved relaxation properties (see Furrer et al.)

Description:

The 2D ¹H¹H TOCSY experiment (Total Correlation Spectroscopy) is a two-dimensional NMR method used to identify through-bond correlations within a spin system, typically in coupled proton networks in small molecules, peptides or sugars. In contrast to COSY, which only reveals direct scalar couplings (J), TOCSY can reveal indirect couplings across entire spin systems - even if some nuclei are not directly coupled. COSY helps to establish connections between chemically bound protons, and TOCSY enables the identification of complete spin systems. For nucleic acids, TOCSY provides a cleaner spectrum to observe the aromatic cross signals of H5 and H6 in pyrimidines. The mixing time for the correlation can therefore be short. Unfortunately, TOCSY cannot be used to assign the proton spin system of the ribose because the coupling constant between H1' and H2' in canonical RNA is small and therefore the transfer of magnetization does not work well with the exception of a C2' endo conformation. In this case, the ³JH1'H2' coupling is at 8-10 Hz, and the signals are visible in this range. This pulse sequence also uses excitation sculpting to suppress water. Since only the non-exchangeable protons provide cross signals, the spectral width can be small, considering the frequency jump, with 9 ppm in the direct dimension and 6 ppm in the indirect dimension.

Literature:

M.J. Thrippleton & J. Keeler, Angew. Chem. Int. ed. 42, 3938-3941 (2003)

B. Simon & H. Koestler, J. Biomol. NMR 73, 155-165 (2019)

J. Furrer, F. Kramer, J.P. Marino, S.J. Glaser & B. Luy, J. Magn. Reson., 166, 36-49 (2004)

Parameter set: NA_HPDI

Pulse sequence: na_hpdi

Sample requirements:

• Solvent: D₂O
• Labeling: any

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: -1 ppm (set to center of ³¹P signals)
• SWF1: 25 ppm (indirect ³¹P dimension)
• SWF2: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D21: hetero-TOCSY mixing time, set to 20 ms for transfer to H2’/H3’/H4’ only or to 90 ms for transfer to all ribose protons.

Additional options:

• ¹³C-decoupling or RNAs containing ¹³C labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C-decoupling during acquisition.

Description:

The hetero-TOCSY technique, which uses a combination of heteronuclear cross-polarization and homonuclear coherence transfer, has been shown to offer higher sensitivity and resolution compared to traditional approaches such as INEPT-RELAY or INEPT-TOCSY due to the simultaneous coherence transfer and the in-phase and absorptive cross-peaks. In addition, the small coupling between ³¹P and protons (<12 Hz) and the relative sensitivity of ³¹P provide advantages in the application of hetero-TOCSY over techniques such as HMQC-TOCSY.

Literature:

G.W. Kellogg & B.I. Schweitzer, J. Biomol. NMR 3, 577-595 (1993)

G.W. Kellogg, J. Magn. Reson. 98, 176-182 (1992)

G.W. Kellogg, A.A. Szewczak & P.B. Moore, J. Am. Chem. Soc. 114, 2727-2728 (1992)

Parameter set: NA_HPDINO

Pulse sequence: na_hpdino

Sample requirements:

• Solvent: D₂O
• Labeling: any

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: -1 ppm (set to center of ³¹P signals)
• SWF1: 25 ppm (indirect ³¹P dimension)
• SWF2: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D8: 250-500 ms NOESY mixing time.
• D21: 90 ms hetero-TOCSY mixing time for transfer to all ribose protons.

Additional options:

• ¹³C-decoupling or RNAs containing ¹³C labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C-decoupling during acquisition.

Description:

The advantage of ³¹P-NMR is the 100% natural abundance of the NMR-active isotope. Unfortunately, the dispersion of ³¹P resonances is very limited. The application of the 2D hetero-TOCSY-NOESY method provides an approach for the sequential assignment of ³¹P resonances associated with H6, H8 and ribose protons of neighboring nucleotides. By adding a NOESY step to hetero-TOCSY, the aromatic and ribose protons can be correlated with the ³¹P resonances without the need for isotopic labeling. For overlapping signals use the 3D pulse sequence na_hpdino3d.

Literature:

G.W. Kellogg & B.I. Schweitzer, J. Biomol. NMR 3, 577-595 (1993)

G.W. Kellogg, J. Magn. Reson. 98, 176-182 (1992)

G.W. Kellogg, A.A. Szewczak & P.B. Moore, J. Am. Chem. Soc. 114, 2727-2728 (1992)

Parameter set: NA_ROESYADESJSGPPH

Pulse sequence: na_roesyadesjsgpph

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: any

Spectral settings:

• SWF1: 12 ppm (for samples in H₂O), 6 ppm (for samples in D₂O)
• SWF2: 26 ppm (for samples in H₂O), 9 ppm (for samples in D₂O)
• O1P: 4.7 ppm (set on water resonance)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D8: 150 ms, ROESY mixing time.
• CNST22: 9 ppm for samples in H₂O, 5.5 ppm for samples in D₂O.
• CNST26: 5000, RF field strength (gammaB1) for ROESY spinlock.
• CNST28: 45 degrees, tilt angle for ROESY spinlock.

Additional options:

• ¹⁵N-decoupling For RNAs containing ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_N to apply ¹⁵N-decoupling during acquisition. Maximum acquisition time is 250 ms in this case.
• ¹⁵N-decoupling and ¹³C-decoupling For RNAs containing ¹³C and ¹⁵N labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C and ¹⁵N-decoupling during acquisition. Maximum acquisition time is 250 ms in this case.
• The option -DAWS used apodization weighted sampling (Simon et al.). Useful for NS > 32.

Description:

2D ¹H¹H ROESY (Rotating-frame Overhauser Effect Spectroscopy) is an alternative NMR technique to 2D ¹H¹H NOESY and is also suitable for investigating the structure and dynamics of RNA and DNA molecules. Due to the pulse sequence, TOCSY artefacts are often present in the spectrum. With the EASY ROESY (Efficient Adiabatic SYmmetrized ROESY) approach, these TOCSY artifacts can be well suppressed using adiabatic spin-lock pulses used off resonance without loss of sensitivity. The advantage of ROESY is that the negative ROE signals can be easily distinguished from the positive signals due to chemical exchange.

Literature:

C.M. Thiele, K. Petzold & J. Schleucher, Chem. Eur. J. 15, 585-588 (2009)

J. Schleucher, J. Quant, S. Glaser & C. Griesinger, J. Magn. Reson A 112, 144-151 (1995)

B. Simon & H. Koestler, J. Biomol. NMR 73, 155-165 (2019)

Parameter set: NA_P31

Pulse sequence: zgig

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: any

Spectral settings:

• SW: 20 ppm (direct ³¹P dimension)
• O1P: -1 ppm (set to center of ³¹P signals)

Parameters to optimize:

• NS: Start 512, adjust to a multiple of 8 for desired S/N.
• D1: 1.5 s, relaxation delay, depends on ³¹P T1 relaxation times.

Description:

This experiment is used to record a 1D ³¹P spectrum of the RNA backbone phosphates. It includes ¹H-decoupling during acquisition.

Parameter set: NA_HMBCGPL2NDWG

Pulse sequence: hmbcgpl2ndwg

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: unlabeled

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (set to center of all ¹³C signals)
• SWF1: 60 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 256, adjust to a multiple of 4 to achieve desired S/N.
• CNST6: 170 Hz, minimum ¹J(CH) for low pass filter.
• CNST7: 210 Hz, maximum ¹J(CH) for low pass filter.
• CNST13: 8 Hz, ⁿJ(CH) for HC transfer.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.

Processing

• PH_mod(F1): pk (or no)
• use xfb and xf2m

Description:

The HMBC shows HC correlations via the ²J(CH), ³J(CH) and ⁴J(CH) couplings. For nucleic acids, the experiment is used for the assignment of A:H2 to A:H8 in adenine as both nuclei show a sizable (5-12 Hz) ⁴J(CH) coupling to A:C4. The ¹J(CH) correlation of A:H2 is suppressed by a low pass filter.

Literature:

M. J. P. van Dongen, S. S. Wijmenga, R. Eritja, F. Azorín, C. W. Hilbers, J. Biomol. NMR 1996, 8, 207-212.

Parameter set: NA_B_TROSYF3GPPH

Pulse sequence: b_trosyf3gpph.2

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4 for ¹⁵N-labeled or 1024 for unlabeled samples, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST54: 12.5 ppm, center of imino proton region.
• CNST55: 6 ppm, bandwidth for imino selective pulses.

Additional options:

• CALC_SP: for calculation of all band selective Proton pulses based on CNST54 and CNST55 (recommended) use option -DCALC_SP.
• LABEL_CN: For ¹³C-decoupling in the ¹⁵N dimension (for ¹³C-labeled RNA) use option -DLABEL_CN.

Description:

This experiment is an alternative to record a 2D ¹H¹⁵N correlation spectrum for imino protons. The 2D ¹H¹⁵N BEST-TROSY uses selective ¹H pulses that only affect the imino proton region and leave the H₂O magnetization unperturbed. ¹H¹⁵N correlations can be measured most sensitively with the BEST-TROSY because the interscan delay can be short and thus many scans can be accumulated in a short time. In general, 15 minutes (¹⁵N-labeled RNA) should be sufficient for good S/N. The TROSY experiment shows reduced T2 relaxation rates compared to the SOFAST-HMQC and allows for longer acquisition times and higher resolution in the ¹⁵N dimension. This is advantageous for larger RNAs.

Literature:

A. Favier & B. Brutscher, J. Biomol. NMR 49, 9-15 (2011)

E. Lescop, T. Kern & B. Brutscher, J. Magn. Reson. 203, 190-198 (2010)

Parameter set: NA_FHSQCF3GPPH

Pulse sequence: na_fhsqcf3gpph

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 115 ppm (center of imino region)
• SWF1: 109 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.8 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST19: 9.5 ppm, center of the region with maximum signal intensity.

Additional options:

• LABEL_CN: For ¹³C-decoupling in the ¹⁵N dimension (for ¹³C-labeled RNA) use option -DLABEL_CN.

Description:

Another alternative for a ¹H-¹⁵N correlation is the FHSQC, which is particularly suitable for protons that exchange rapidly with the solvent. It is usually used for recording a spectrum of both, the amino and the imino region.

Literature:

S. Mori, C. Abeygunawardana, M. O'Neil-Johnson & P.C.M. van Zijl, J. Magn. Reson. B 108, 94-98 (1995)

Parameter set: NA_B_TRHNNCOSYGPPH3D

Pulse sequence: na_b_trhnncosygpph3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹³C¹⁵N

Spectral settings:

• SWF1: 100 ppm (to cover hydrogen-bond acceptor [A:N1 and C:N3] and donor nitrogens spectral range)
• SWF2: 30 ppm (to cover hydrogen-bond donor nitrogen [U:N3 and G:N1] spectral range)
• SWF3: 21 ppm (to include imino protons)
• O1P:12 ppm (on imino ¹H resonances)
• O2P: 105 ppm (set in the middle of ¹³C resonances)
• O3P: 185 ppm (set in the center of hydrogen-bond donor [140-160 ppm] and acceptor [190-220 ppm] nitrogens)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• CNST55: 6 ppm, bandwidth for imino selective pulses.
• CNST21: the center of the hydrogen-bond donor nitrogens (~153 ppm for U-N3 and G-N1).
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.

Additional options:

• ¹³C-decoupling for RNAs containing ¹³C-labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C-decoupling during acquisition.

Description:

The experiment is used to record 2D ¹H¹⁵N spectra in which the imino proton is correlated with the A:N1 and U:N3, if the imino proton is involved in a stable, exchange-protected base-pair. The experiment is built up by two different magnetization transfers: First, the H-N INEPT transfers the magnetization from the imino proton to the hydrogen-bond donor nitrogen via the ¹J_N3H3 coupling. Subsequently, the N-N COSY transfers magnetization from the hydrogen-bond donor nitrogen to the hydrogen-bond acceptor nitrogen via ²J_N1N3 coupling. Thereby, the HNN-COSY provides direct proof for hydrogen-bonds in Watson-Crick base-pairs.

Literature:

A.J. Dingley & S. Grzesiek, JACS 120, 8293-8297 (1998)

Parameter set: NA_B_SLRHNNCOSYGPPH

Pulse sequence: na_b_slrhnncosygpph

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N and ¹³C¹⁵N

Spectral settings:

• SWF1: 100 ppm (to cover hydrogen-bond acceptor [A:N1 and C:N3] and donor nitrogens spectral range
• SWF2: 13 ppm (to cover H2 and H8 resonances while O1P is on water)
• O1P: 4.7 ppm (set on water resonance)
• O2P: 154 ppm (on H2 chemical shift offset)
• O3P: 200 ppm (set in the middle of hydrogen-bond donor and acceptor nitrogens, in contrast to HNN-COSY also purine N7 and N9 are included)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• CNST54: 7.5 ppm, H2/H8 chemical offset.
• CNST55: 2 ppm, bandwidth of H2/H8 selective pulses.
• CNST56: 225 ppm, center of the N1 region.
• CNST57: 20 ppm, bandwidth of N1 selective pulses.
• D1: 0.4 s, experiment allows short D1 due to preservation of H₂O magnetization.

Additional options:

• ¹³C-decoupling for RNAs containing ¹³C-labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C-decoupling during acquisition.
• For calculation of all band selective proton pulses based on CNST54 and CNST55 and nitrogen pulses based on CNST56 and CNST57 (recommended) use ZGOPTNS -DCALC_SP.

Description:

The experiment is used to record 2D ¹H¹⁵N spectra in which the aromatic A:H2 proton is correlated with the A:N1 and U:N3, if the A is involved in a stable, exchange-protected base-pair. Magnetization is transferred in two steps: 1) H-N INEPT via the ²J_N1H2 coupling 2) N-N COSY via the ²J_N1N3 coupling.

If the coupling constants are adapted, the LR HNN-COSY is also suitable to detect Hoogsteen interactions that involve the purine N7 side. Magnetization transfer to R:N7 is achieved starting on H8 via ²J_N7H8 coupling.

Literature:

A. Dallmann, B. Simon, M.M. Duszczyk, H. Kooshapur, A. Pardi, W. Bermel & M. Sattler, Angew. Chem., 52, 10487-10490 (2013)

Parameter set: NA_HSQCF3GPNOWGXY

Pulse sequence: na_hsqcf3gpnowgxy

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹³C¹⁵N

Spectral settings:

• SWF2: 22 ppm (to cover imino and amino proton spectral range)
• O1P: 4.7 ppm (set on water resonance)
• O2P: 105 ppm (center of ¹³C resonances)
• O3P: 116 ppm (center of imino and amino ¹⁵N resonances)
• CNST4: 93 (¹J coupling constant of imino NH and amino NH₂)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• CNST19: 9 ppm, center of imino and amino protons.
• SWF1: 103 ppm to cover the complete imino and amino nitrogen spectral range or ~32 ppm to use frequency jump (set with CNST29) and record amino nitrogen spectral range. Note: Imino nitrogens will be folded in resulting spectrum.
• CNST29: 86 ppm to use frequency jump (set SWF1 to 32 ppm) and center on amino nitrogen spectral range. Note: Imino nitrogens will be folded in resulting spectrum.
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.
• D8: 80-240 ms, NOESY mixing time.
• SPW1/PHCOR9: power level and phase correction of water-selective pulses (p11) in the soft WATERGATE acquisition scheme, optimize in gs mode.
• SPW11/PHCOR8: power level and phase correction of water-selective flip-back pulse (p29) during the mixing time, optimize in gs mode.

Additional options:

• ¹³C-decoupling for RNAs containing ¹³C-labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C-decoupling during acquisition.

Description:

The experiment is used to record 2D ¹H¹⁵N spectra in which the imino and amino protons are correlated through space to nearby protons via NOE. The experiment uses the Carr-Purcell-Meiboom-Gill ¹H-¹⁵N magnetization transfer for NH/NH₂ to minimize losses due to exchange. During the mixing time d8, magnetization from the ¹⁵N-edited imino and amino protons to any nearby RNA protons takes place. Notably, the C:N4-H5 correlation is frequently visible for base-paired C residues, provided the water suppression is good. Careful optimization of O1P, SPW1/PHCOR9 and SPW11/PHCOR8 is recommended. The CPMG-NOESY allows reduction of the f1 spectral (SWF1) width via a frequency jump (CNST29) for advanced users. Care must be taken when folding the imino resonances into the amino spectral region to avoid overlap between imino diagonal peaks with imino-amino cross-peaks.

Literature:

L. Mueller, P. Legault & A. Pardi, J. Am. Chem. Soc. 117, 11043-11048 (1995)

F.A.A. Mulder, C.A.E.M. Spronk, M. Slijper, R. Kaptein & R. Boelens, J. Biomol. NMR 8, 223-228 (1996)

Parameter set: NA_LRHSQCF3GPPH

Pulse sequence: na_lrhsqcf3gpph

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 200 ppm (center of imino region)
• SWF1: 100 ppm (indirect ¹⁵N dimension)
• SWF2: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST4: 20 Hz, relaxation-optimized coupling ²J(NH).
• CNST19: 8 ppm, center of the ¹H region with maximum signal intensity.

Additional options:

• LABEL_CN: For ¹³C-decoupling in the ¹⁵N dimension (for ¹³C-labeled RNA) use option -DLABEL_CN.

Description:

With the long-range FAST-HSQC, the non-exchangeable aromatics H2 and H8 are correlated with the N1/N3 and N7/N8 nitrogen atoms via the ²J_HN coupling. The experiment is based on the FAST-HSQC described above, whereby the excitation center is located on the aromatic and the HN transfer is significantly extended to 20 ms. It is advisable to record this experiment on ¹⁵N-only labeled samples, as ¹³C labeling speeds up T2 relaxation of H2/H8 due to dipolar interaction with ¹³C2/¹³C8.

Literature:

S. Mori, C. Abeygunawardana, M. O'Neil-Johnson & P.C.M. van Zijl, J. Magn. Reson. B 108, 94-98 (1995)

Parameter set: NA_LRSFHMQCF3GPPH

Pulse sequence: na_lrsfhmqcf3gpph

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 200 ppm (center of imino region)
• SWF1: 100 ppm (indirect ¹⁵N dimension)
• SWF2: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.4 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST4: 20 Hz, relaxation-optimized coupling ²J(NH).
• CNST54: 7.5 ppm, center of H2/H8 proton region.
• CNST55: 3 ppm, bandwidth for H2/H8 selective pulses.

Description:

In the LR-SOFAST-HMQC, the non-exchangeable aromatics H2 and H8 correlate with the nitrogen atoms via the ²J_NH coupling. The experiment is based on the SOFAST-HMQC described above, with the excitation center located on the aromatic protons and the HN transfer significantly extended to 20 ms.

Literature:

P. Schanda and B. Brutscher, J. Biomol. NMR. 33, 199-211 (2005)

Parameter set: NA_SF15NXF

Pulse sequence: na_sf15Nxf

Sample requirements:

• Solvent: H₂O
• Labeling: any

Spectral settings:

• SW: 26 ppm (full proton range in H₂O)
• O1P: 4.7 ppm (set on water resonance)

Parameters to optimize:

• NS: Start with 128, adjust to a multiple of 8 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST4: 93 Hz ¹J(NH).
• CNST54: 12.5 ppm, center of imino proton region.
• CNST55: 6 ppm, bandwidth for imino proton selective pulses.

Additional options:

• For calculation of band selective proton pulses based on CNST54 and CNST55 (recommended) use ZGOPTNS -DCALC_SP.

Description:

This experiment records the ¹⁵N-filtered 1D ¹H spectrum and the ¹⁵N-edited 1D ¹H spectrum in an interleaved manner as a pseudo-2D experiment. Use the AU program “split” with the option "ipap" to separate the spectra after acquisition. The first and second spectrum correspond respectively to the ¹⁵N-filtered and ¹⁵N-edited spectrum.

Literature:

P. Schanda and B. Brutscher, J. Biomol. NMR. 33, 199-211 (2005)

Parameter set: NA_NOESYSFHMQCF3GPPH3D

Pulse sequence: noesysfhmqcf3gpph3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹³C¹⁵N

Spectral settings:

• SWF1: 12 ppm (to cover the ¹H chemical shift range)
• SWF2: 25 ppm (to cover imino and amino nitrogen spectral range)
• SWF3: 14 ppm (to cover imino and amino proton spectral range)
• O1P: 8 ppm (center of all ¹H resonances)
• O2P: 110 ppm (center of ¹³C resonances)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• CNST10: Flip angle for 1^{st 1}H pulse in HMQC, 90-110.
• CNST54: 11-12.5 ppm, center of imino or amino+imino proton region.
• CNST55: 6-8 ppm, bandwidth for imino or amino+imino proton selective pulses.
• CNST22: 116 ppm, center of imino and amino ¹⁵N resonances for decoupling in F1.
• O3P: 153 ppm, center of F2 dimension (¹⁵N).
• SWF2: optimize according to the resonances intended to be observed (e.g. 25 to cover all imino nitrogens, or optimize down to ~9 ppm, some imino nitrogen resonances will be folded in resulting spectrum).
• D1: 0.5 s, experiment allows short relaxation delay due to preservation of H₂O magnetization.
• D8: 150 ms, NOESY mixing time.

Additional options:

• ¹³C-decoupling for RNAs containing ¹³C-labeled nucleotides use the ZGOPTNS -DLABEL_CN to apply ¹³C-decoupling in F1/F2.
• For calculation of all band selective proton pulses based on CNST54 and CNST55 and Nitrogen pulses based on CNST56 and CNST57 (recommended), use ZGOPTNS -DCALC_SP.

Description:

The 3D NOESY-HMQC expands the 2D imino-NOESY into the ¹⁵N dimension of imino nitrogens, thereby adding resolution. To minimize the number of pulses, the ¹⁵N dimension is recorded as an HMQC. The SOFAST (sf) acquisition scheme allows for short interscan delays, ultimately resulting in enhanced sensitivity. Folding of the ¹⁵N dimension is achieved via optimization of SWF2 and O3P and should be checked in 2D ¹H¹⁵N SOFAST-HMQC spectra. When setting up a 3D experiment, pseudo-3D test spectra should be recorded for the F3-F2 and F3-F1 dimension combinations, respectively, by setting TDF1 or TDF2 to 1.

Literature:

P. Rossi, Y. Xia, N. Khanra, G. Veglia & G. Kalodimos, J. Biomol. NMR 66, 259 (2016)

P. Schanda and B. Brutscher, J. Am. Chem. Soc. 127, 8014 (2005)

Parameter set: H2

Pulse sequence: zg2h

Sample requirements:

• Solvent: H₂O or D₂O

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• SW: 12 ppm

Parameters to optimize:

• NS: 4.

Description:

A 1D experiment for detection of the ²H signal. Used for measuring the splitting of the solvent signal in an aligned sample for RDC measurements.

Parameter set: NA_SFHMQCIA

Pulse sequence: na_sfhmqcia

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N
• CNST54: 12.5 ppm, center of imino proton region
• CNST55: 6 ppm, bandwidth for imino selective pulses
• D1: 0.4 s, experiment allows short relaxation delay due to preservation of H₂O magnetization

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN
• For calculation of band selective proton pulses based on CNST54 and CNST55 (recommended) use ZGOPTNS -DCALC_SP

Description:

The experiment measures two ¹H-¹⁵N SOFAST-HMQC spectra without ¹⁵N decoupling in the ¹H dimension (F2). The inphase and antiphase components are recorded in an interleaved fashion and can later be separated. This prevents the increasing peak overlap due to coupling evolution. If the sample is also ¹³C-labeled, decoupling can be selected as an option.

Processing:

• Split the IP and AP part in two spectra using “split ipap” in command line
• Process with 16k to 30k points in the F2 and 4k points in the F1

Literature:

Schanda et al. J Biomol NMR 33, 199-211 (2005).

Parameter set: NA_N_BTROSY

Pulse sequence: n_btrosy

Sample requirements:

• Solvent: H₂O
• Labeling: includes ¹⁵N (for the imino)

Spectral settings:

• O1P: 12.5 ppm (set on center of imino proton region)
• O3P: 153 ppm (center of imino region)
• SWF1: 8 ppm (indirect ¹H dimension)
• SWF2: 50 ppm (direct ¹⁵N dimension)

Parameters to optimize:

• NS: Start with 128, adjust to a multiple of 4 to achieve desired S/N.
• CNST55: 6 ppm, bandwidth for imino selective pulses.
• D1: 0.4 s, experiment allows short relaxation delay due to preservation of H₂O magnetization.

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN.

Description:

The ¹⁵N-detected BEST-TROSY is most useful for large RNA constructs, due to the lower relaxation of the ¹⁵N-magnetization, which allows higher resolutions. The ¹H-detected BEST-TROSY is however more sensitive and should be used for smaller RNAs. The excitation is performed on the protons, and detected on the nitrogens. The experiment requires a cryoprobe due to its inherent insensitivity. For even better sensitivity use TXO cryoprobes, as these are optimized for detection of ¹³C and ¹⁵N.

Literature:

Schnieders et al. J Biomol NMR 69, 31-44 (2017).

Parameter set: HSQCT1ETF3GPTCWG3D

Pulse sequence: hsqct1etf3gptcwg3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.
• VDLIST: Adjust the VDLIST entries to the T1 times.
• TD1: Set to the number VDLIST entries.

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN and set CNST21 (110 ppm) and CNST22 (110 ppm) and o2p to 110 ppm.

Description:

This experiment is used to record a series of 2D ¹H¹⁵N correlation spectra in a pseudo 3D fashion with varying relaxation delay times for the determination of ¹⁵N-T1. The experiment uses a watergate sequence for solvent suppression and temperature compensation for the series of spectra. A VDLIST is needed to set the variable delay times (commonly includes 8 or more relaxation delays ranging from 0 to 1-2 s). Set TD for the F1 (pseudo) dimension to the number of delays (with the acquisition mode (FnMODE) to QF(no-frequency)).

Parameter set: HSQCT2ETF3GPTCWG3D

Pulse sequence: hsqct2etf3gptcwg3d

Sample requirements:

• Solvent: H₂O
• Labeling: includes ¹⁵N (for the imino)

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.
• VCLIST: Adjust the VCLIST entries to the T2 times.
• TD1: Set to the number VCLIST entries.

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN and set CNST21 (110 ppm) and CNST22 (110 ppm) and o2p = 110 ppm.

Description:

This experiment is used to record a series of 2D ¹H¹⁵N correlation spectra in a pseudo 3D fashion with varying relaxation delay times for the determination of ¹⁵N-T2. The experiment uses a watergate sequence for solvent suppression and temperature compensation for the series of spectra. A VCLIST is needed to set the variable loop counter (commonly includes 8 or more numbers, determining the T2 delays by the amount of loops over a single CPMG element (with length d31). The counter numbers are set from the minimum value of 1 to where C × d31 is around 100-200 ms. Set TD for the F1 (pseudo) dimension to the number of delays (with the acquisition mode (FnMODE) to QF(no-frequency)).

Parameter set: NA_HSQCTRETF3GPSITC3D.3

Pulse sequence: na_hsqctretf3gpsitc3d.3

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1.5-2.5 s, a longer delay is advisable due to sample heating by the ¹³C spinlock.
• CNST27: set the T1rho spinlock field strength to 2000 Hz (usual: 1500-2500 Hz).
• CNST30: set the T1rho spinlock ¹⁵N offset to 1500 Hz (on a 600 MHz spectrometer).
• P33: set shaped pulse length for the adiabatic ramp to 3 ms.
• SP36: set gaussian ramp to Gaussramp+down.1 (when CNST30>0) or to Gaussramp-down.1 (when CNST30<0).
• SP37: set gaussian ramp to Gaussramp+up.1 (when CNST30>0) or to Gaussramp-up.2 (when CNST30<0).
• VDLIST: Adjust the VDLIST entries to the T1rho times.
• TD1: Set to the number VDLIST entries.

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN and set O2P to 110 ppm.

Description:

This experiment is used to record a series of 2D ¹H¹⁵N correlation spectra in a pseudo 3D fashion with varying relaxation delay times for the determination of ¹⁵N-T1rho. The experiment includes temperature compensation for the series of spectra. A VDLIST is needed to set the variable delay times (commonly includes 8 or more relaxation delays ranging from 10 to 200 ms). Set TD for the F1 (pseudo) dimension to the number of delays (with the acquisition mode (FnMODE) to QF(no-frequency)).

Parameter set: NA_HSQCNOEF3GPWG3D

Pulse sequence: hsqcnoef3gpwg3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• D1: 5 s or longer. A long relaxation delay is important for precise hetNOE values.

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN and set CNST21 (110 ppm) and CNST22 (110 ppm) and O2P 110 ppm.

Description:

This experiment is used to record two 2D ¹H¹⁵N correlation spectra in a pseudo 3D fashion, with and without proton saturation, for the determination of the ¹⁵N{¹H}-hetNOE. The experiment uses a watergate sequence for solvent suppression. Set TD for the F1 (pseudo) dimension to 2.

Parameter set: NA_B_HSQCEXF3GP3D

Pulse sequence: na_b_hsqcexf3gp3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹⁵N dimension)
• SWF2: 26 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 8 to achieve desired S/N.
• D1: 0.5 s, experiment allows short D1 due to preservation of H₂O magnetization.
• CNST54: 12.5 ppm, center of imino proton region.
• CNST55: 6 ppm, bandwidth for imino selective pulses.
• VDLIST: Adjust the VDLIST entries to the timescale of the exchange process.
• TD1: Set to the number VDLIST entries.

Additional options:

• LABEL_CN: for ¹³C-decoupling in the ¹⁵N dimension (for ¹³C¹⁵N-labeled RNA) use option -DLABEL_CN.
• For calculation of band selective proton pulses based on CNST54 and CNST55 (recommended) use ZGOPTNS -DCALC_SP.

Description:

This experiment is used to measure ZZ exchange between different states of an RNA molecule. For a qualitative assessment of the exchange, a single 2D spectrum can be recorded at one relaxation delay. In this case, set TD1 to 1 and use only a single delay value in the VDLIST.

For quantitative determination of the ¹⁵N ZZ-exchange rates, the experiment records a series of 2D ¹H-¹⁵N correlation spectra in a pseudo-3D fashion using variable relaxation delays. A VDLIST is required to define the delay times, typically consisting of eight or more values ranging from 0 to 1-2 s. Set TD for the F1 (pseudo) dimension to the number of delays specified in the VDLIST.

Parameter set: NA_NOESYF3GPPHXF19

Pulse sequence: na_noesyf3gpphxf19

Sample requirements:

• Solvent: H₂O
• Labeling: selective ¹⁵N (G or U)

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 153 ppm (center of imino region)
• SWF1: 12 ppm (indirect ¹H dimension)
• SWF2: 26 ppm (direct ¹H dimension)
• D8: 150 ms, NOESY mixing time

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST19: 11 ppm, center of the region with maximum signal intensity in F2.
• CNST22: 9 ppm, center of NOESY dimension (F1).

Processing:

• Split the 4 sub-spectra using the command “split ipap 4” in command line.

Description:

This experiment combines isotope filtering and editing schemes to separate signals according to whether the observed protons are attached to ¹⁵N or ¹⁴N nuclei. Four phase-cycling combinations are recorded in an interleaved fashion and stored in a single dataset. This results in all combinations of editing and filter steps in four different subspectra:

Spectrum A contains signals from all protons that are bound to ¹⁴N. Spectra B and C contain correlations between ¹⁵N-bound and ¹⁴N-bound proton resonances. These spectra are particularly useful for identifying NOESY cross-peaks as diagonal signals are suppressed. They contain the same information, with the F1 and F2 dimensions exchanged. The differences between them are mainly related to digital resolution and potential artifacts near the F1/F2 diagonal. Spectrum D contains signals from all ¹⁵N-bound protons.

The combination of isotope filtering and editing greatly simplifies the interpretation of NOESY spectra by separating different classes of cross-peaks into individual subspectra. However, these additional filter and editing elements result in a significant loss of sensitivity compared to a conventional NOESY experiment.

Literature:

G. Otting & K. Wuethrich, J. Magn. Reson. 85, 586 - 594 (1989)

Parameter set: NA_NOESYGPPHXF15N

Pulse sequence: noesygpphwgxf15N

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O3P: 115 ppm (center of amino and imino region)
• SWF1: 8 ppm (indirect ¹H dimension)
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST19: 5.5 ppm, center of F1 dimension.
• D8: 150 ms, NOESY mixing time.
• SP1/11: Optimize carefully for better water suppression.

Additional options:

• Presaturation for water suppression (use ZGOPTNS PRESAT).

Description:

The 2D ¹H¹H NOESY sequence with a ¹⁵N x-filter in both dimensions. This removes the signal from ¹⁵N-bound protons in F1 and F2. For RNA, this means that all imino and amino proton signals are suppressed. Therefore, only NOE signals from aromatic and sugar protons are observed. The protons bound to ¹⁵N are eliminated using a purge element (Breeze 2000). The spectrum is similar to a NOESY in 100% D₂O. Water suppression is achieved via a standard WATERGATE sequence.

Literature:

A.L. Breeze, Prog. NMR Spectrosc. 36, 323-372 (2000)

Parameter set: NA_HSQCETGPSP.4

Pulse sequence: hsqcetgpsp.4

Sample requirements:

• Solvent: H₂O or D₂O         
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 105 ppm (set to center of all ¹³C signals)
• SWF1: 110 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.

Additional options:

• -DLABEL_CN: ¹⁵N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides.
• O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

The experiment is used for the ¹H¹³C correlations of all aromatic and ribose atoms. The experiment is optimized for large ¹³C bandwidth (broadband shaped pulses for all purposes on ¹³C). Experiments for recording only specific ribose or aromatic ¹H¹³C correlations selective HSQC experiments are available (for C1’, C5’, C2/C6/C8 or C5), that offer higher resolution due to selective ¹J(CC) decoupling.

Parameter set: NA_HSQCCTETGPSP

Pulse sequence: na_hsqcctetgpsp

Sample requirements:

• Solvent: H₂O or D₂O         
• Labeling: ¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 77 ppm (set to center of all ¹³C signals)
• SWF1: 40 ppm (indirect ¹³C dimension)
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• d23:12.5 ms for 1/(2*¹J(CC)) or 25 ms for 1/¹J(CC).

Additional options:

• -DLABEL_CN: ¹⁵N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides, set O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

The experiment is used for the ¹H¹³C correlation of all the ribose atoms. The cross-peaks appear as singlets in the F1 dimension because of a constant-time chemical shift evolution period in which the ¹³C-¹³C couplings are refocused. This enhances the resolution of the experiment, but decreases sensitivity due to T₂ relaxation during the constant-time delay. The constant-time HSQC is very useful for measuring all ribose carbon C2’, C3’ and C4’ atoms with high resolution. If only C1’ or C5’ are of interest, the HSQC versions specialized for these two nuclei offer better sensitivity.

Parameter set: NA_SHSQCETGPSP_C1PRIME

Pulse sequence: na_shsqcetgpsp

Sample requirements:

• Solvent: H₂O or D₂O         
• Labeling: ¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 90 ppm (set to center of the ribose C1’ signals)
• SWF1: 12 ppm (indirect ¹³C dimension)
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 170 Hz ¹J(CH).
• CNST20: 12 ppm, ¹³C refocusing bandwidth for C1’ selection.
• CNST22: 72 ppm, ¹³C decoupling offset for C2’.
• CNST25: 12 ppm, ¹³C decoupling bandwidth for C2’.

Additional options:

• -DLABEL_CN: ¹⁵N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides.
• O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

The experiment is used for the ¹H¹³C correlation of the ribose C1’-H1’ atoms. The experiment uses shaped pulses on ¹³C and a selective ¹³C-decoupling (C2’-region).

Parameter set: NA_SHSQCETGPSP_C5PRIME

Pulse sequence: na_shsqcetgpsp

Sample requirements:

• Solvent: H₂O or D₂O         
• Labeling: ¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 63 ppm (set to center of the ribose C5’ signals)
• SWF1: 10 ppm (indirect ¹³C dimension)
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times
• CNST2: 150 Hz, ¹J(CH).
• CNST20: 12 ppm, ¹³C refocusing bandwidth for C5’ selection.
• CNST22: 82 ppm, ¹³C decoupling offset for C4’.
• CNST25: 12 ppm, ¹³C decoupling bandwidth for C4’.

Description:

The experiment is used for the ¹H¹³C correlation of the ribose C5’-H5’ atoms. The experiment uses shaped pulses on ¹³C and selective ¹³C-decoupling (C4’-region).

Parameter set: NA_SHSQCETGPSP_C5

Pulse sequence: na_shsqcetgpsp

Sample requirements:

• Solvent: H₂O or D₂O         
• Labeling: ¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 99 ppm (set to center of the C and U C5 signals)
• SWF1: 15 ppm (indirect ¹³C dimension)                                     
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 180 Hz ¹J(CH).
• CNST20: 12 ppm, ¹³C refocusing bandwidth for C5 selection.
• CNST22: 150 ppm, ¹³C decoupling offset for C4 and C6.
• CNST25: 40 ppm, ¹³C decoupling bandwidth for C4 and C6.

Description:

The experiment is used for the ¹H¹³C correlation of the aromatic C5-H5 atoms (C and U). The experiment uses shaped pulses on ¹³C and selective ¹³C-decoupling (range encompassing the C4-region and C6-region).

Parameter set: NA_SHSQCETGPSP_C2C6C8

Pulse sequence: na_shsqcetgpsp

Sample requirements:

• Solvent: H₂O or D₂O         
• Labeling: ¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 143 ppm (set to center of the C2 (A), C6 (C,U) and C8 (A,G) signals)
• SWF1: 30 ppm (indirect ¹³C dimension)
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 200 Hz ¹J(CH).
• CNST20: 30 ppm, ¹³C refocusing bandwidth for C2, C6 and C8 selection.
• CNST22: 99 ppm, ¹³C decoupling offset for C/U C5.
• CNST25: 15 ppm, ¹³C decoupling bandwidth for C/U C5.

Additional options:

• -DLABEL_CN: ¹⁵N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides, set O3P to 195 ppm (center of aromatic N region).

Description:

The experiment is used for the ¹H¹³C correlation of the region of the aromatic C2-H2 atoms (A), the C6-H6 atoms (C and U) and C8-H8 atoms (A and G). The experiment uses shaped pulses on ¹³C and selective ¹³C-decoupling (encompassing the C5 (C and U) region).

Parameter set: NA_C6NOESYHSQCGP3D.2

Pulse sequence: na_c6noesyhsqcgp3d.2

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (center of aromatics region)
• SWF1: 8 ppm (indirect ¹H dimension)
• SWF2: 30 ppm (indirect ¹³C dimension)
• SWF3: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D8: 150 ms, NOESY mixing time.
• CNST2: 195 Hz ¹J(CH) for aromatic protons.
• CNST16: 8.5 ppm, region with maximum signal intensity in F2, set to H6/H8 region.
• CNST19: 6 ppm, center of NOESY dimension F1.
• CNST28: 105 ppm, C5 chemical offset for C5 decoupling.
• CNST59: 35 ppm, bandwidth for C5 selective decoupling during t1.

Additional options:

•  -DLABEL_CN: ¹⁵N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides, set O3P to 195 ppm (center of aromatic N region).

Description:

The 3D NOESY-HSQC expands the 2D aromatic-NOESY into the ¹³C dimension of aromatic carbons, thereby adding resolution. Folding of the ¹³C dimension is achieved via optimization of SWF2 and O2P and should be checked in 2D ¹H¹³C HSQC spectra. When setting up a 3D experiment, 2D spectra should be recorded for the F3-F2 and F3-F1 dimensions, respectively, by setting TD1 or TD2 to 1.

Parameter set: NA_NOESY_SFHMQCHCH3D

Pulse sequence: na_noesy_sfhmqchch3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (center of aromatics region)
• SWF1: 12 ppm (indirect ¹H dimension)
• SWF2: 30 ppm (indirect ¹³C dimension)
• SWF3: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.5 s, experiment allows short D1 due to preservation of H₂O magnetization.
• D8: 150 ms, NOESY mixing time.
• CNST2: 200 Hz, ¹J(CH) for aromatic protons.
• CNST10: 90-110, Flip angle for 1^st pulse in HMQC.
• CNST22: 8 ppm, center of NOESY dimension (F1).
• CNST54: 8.5 ppm, center of aromatic proton region.
• CNST55: 5 ppm, bandwidth for aromatic proton selective pulses.

Additional options:

• -DLABEL_CN^{: 15}N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides, set O3P to 120 ppm (center of iminos and aminos).
• For calculation of band selective proton pulses based on CNST54 and CNST55 (recommended) use ZGOPTNS -DCALC_SP.

Description:

The 3D NOESY-HMQC expands the 2D aromatic-NOESY into the ¹³C dimension of aromatic carbons, thereby adding resolution. To minimize the number of pulses, the ¹³C dimension is recorded as an HMQC. The SOFAST acquisition scheme allows for short relaxation delays, ultimately resulting in enhanced sensitivity. Folding of the ¹³C dimension is achieved via optimization of SWF2 and O2P and should be checked in a 2D ¹H¹³C SOFAST-HMQC spectrum. When setting up a 3D experiment, 2D spectra should be recorded for the F3-F2 and F3-F1 dimensions, respectively, by setting TD1 or TD2 to 1.

Literature:

P. Rossi, Y. Xia, N. Khanra, G. Veglia & G. Kalodimos, J. Biomol. NMR 66, 259 (2016)

P. Schanda and B. Brutscher, J. Am. Chem. Soc. 127, 8014 (2005)

Parameter set: NA_NOESY_SFHMQCHCHXF3D

Pulse sequence: na_noesy_sfhmqchchxf3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (center of aromatic region)
• O3P: 120 ppm (center of iminos and aminos)
• SWF1: 12 ppm (indirect ¹H dimension)
• SWF2: 30 ppm (indirect ¹³C dimension)
• SWF3: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.5 s, experiment allows short D1 due to preservation of H₂O magnetization.
• D8: 150 ms, NOESY mixing time.
• CNST2: 200 Hz, ¹J(CH) for aromatic protons.
• CNST6: ¹J(CH)min for X-filter: 145 Hz.
• CNST7: ¹J(CH)max for X-filter: 200 Hz.
• CNST10: 90-110, flip angle for 1^st pulse in HMQC.
• CNST22: 9 ppm, center of NOESY dimension (F1).
• CNST54: 8.5 ppm, center of aromatic proton region.
• CNST55: 5 ppm, bandwidth for aromatic proton selective pulses.

Additional options:

• -DNO_FILTER: to turn off the x-filter use option. Useful to test the experiment setup.
• For calculation of band selective proton pulses based on CNST54 and CNST55 (recommended) use ZGOPTNS -DCALC_SP.

Description:

The xfilter 3D NOESY-HMQC is used to identify NOESY interactions between unlabeled binding partners and labeled RNA. It is also used to reduce signal overlap when assigning signals for selectively labeled RNA. To achieve this, a filter element is inserted before the t1 time, which suppresses signals bound to ¹³C or ¹⁵N.

A 3D NOESY-HMQC expands the 2D aromatic-NOESY into the ¹³C dimension of aromatic carbons, thereby adding resolution. To minimize the number of pulses, the ¹³C dimension is recorded as an HMQC. The SOFAST acquisition scheme allows for short relaxation delays, ultimately resulting in enhanced sensitivity. When setting up a 3D experiment, 2D spectra should be recorded for the F3-F2 and F3-F1 dimension combinations, respectively, by setting TD1 or TD2 to 1.

Literature:

A.L. Breeze, Prog. NMR Spectrosc. 36, 323-372 (2000)

P. Rossi, Y. Xia, N. Khanra, G. Veglia & G. Kalodimos, J. Biomol. NMR 66, 259 (2016)

P. Schanda and B. Brutscher, J. Am. Chem. Soc. 127, 8014 (2005)

Parameter set: NA_NOESYESGPPHXFED

Pulse sequence: na_noesyesgpphxfed

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 107 ppm (center of carbon resonances)
• O3P: 120 ppm (center of iminos and aminos)
• SWF1: 12 ppm (indirect ¹H dimension)
• SWF3: 24 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1.3 s, relaxation delay, depends on ¹H T1 relaxation times.
• D8: 150 ms, NOESY mixing time.
• CNST22: 9 ppm, center of indirect proton dimension.

 Additional options:

• -DAWS: enables apodization weighted sampling (Simon et al.). Useful for NS > 32 (process the spectrum without linear prediction in this case).
• -DNOFILTER: deactivate the ¹⁵N/¹³C-filter in F1. This sets ¹⁵N/¹³C pulse powers during the filter element to zero. Useful to test experimental setup.

Description:

A NOESY experiment with ¹³C/¹⁵N filtering in F1 and isotope editing in F2. The experiment is particularly useful for detecting ligand-RNA NOE contacts and for the analysis of selectively labeled RNA samples. Compared to a conventional NOESY experiment, the additional filtering and editing steps lead to a significant loss of sensitivity, typically requiring a substantially larger number of scans to obtain sufficient S/N.

The matched adiabatic pulse is generated using WaveMaker with the command wvm -a at the command line.

Literature:

C. Zwahlen, P. Legault, S.J.F. Vincent, J. Greenblatt, R. Konrat & L.E. Kay, J. Am. Chem. Soc 119, 6711-6721 (1997)

Parameter set: NA_B_TRHNCOGP3D

Pulse sequence: na_b_trhncogp3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 157 ppm (center of aromatics region)
• O3P: 153 ppm (center of imino region)
• SWF1: 25 ppm (indirect ¹³C dimension)
• SWF2: 25 ppm (indirect ¹⁵N dimension)
• SWF3: 24 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.3 s, experiment allows short D1 due to preservation of H₂O magnetization.
• D23: 8 ms, 1/(4*¹J(NC)) to adjust depending on the size of the RNA (5-10 ms).
• CNST54: 12.5 ppm, center of imino proton region.
• CNST55: 6 ppm, bandwidth for imino selective pulses.

Additional options:

• For calculation of band selective proton pulses based on CNST54 and CNST55 (recommended) use ZGOPTNS -DCALC_SP.

Description:

This 3D HNCO experiment is usually recorded as a 2D H/CO plane without chemical shift evolution of the ¹⁵N dimension. Imino protons (U:H3 and G:H1) of ¹³C-labeled RNA are directly correlated with U:C2 and U:C4 carbons or G:C2 and G:C6 carbons. If no C5 decoupling is applied, the U:H3C4 and G:H1C6 resonances appear as doublets in the indirect dimension, respectively.

Reference:

Z. Solyom, M. Schwarten, L. Geist, R. Konrat D. Willbold & Bernhard Brutscher, J. Biomol. NMR 55, 311-321 (2013)

A. Favier & B. Brutscher, J. Biomol. NMR 49, 9-15 (2011)

E. Lescop, P. Schanda & B. Brutscher,J. Magn. Reson. 187 163-169 (2007)

T. Schulte-Herbrueggen & O.W. Sorensen, J. Magn. Reson. 144, 123 - 128 (2000)

Parameter set: NA_C_CNHSQC

Pulse sequence: na_c_cnhsqc

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 159 ppm (center U:C2/C4 G:C2/C4/C6/C8)
• O2P: 9 ppm (center of aromatic and imino H)
• O3P: 153 ppm (center of imino region)
• SWF1: 40 ppm (indirect ¹⁵N dimension)
• SWF2: 50 ppm (direct ¹³C dimension)

Parameters to optimize:

• NS: Start with 512, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1.5 s, relaxation delay, depends on ¹³C T1 relaxation times.
• CNST4: ¹J(CN): 27 Hz.
• CNST5: ¹J(NH): 94Hz.

 Additional options:

• -DNO_FILTER: using no ¹H-filter (left figure).

Description:

In the CN-HSQC spectrum for imino groups, all nucleotides in the RNA can be observed, regardless of whether they are involved in hydrogen bonding or not. Using ¹³C direct detection, the correlation with the imino nitrogen is independent of exchange with water. Inserting a proton filter reverses the sign of the nitrogens with slow proton exchange rates (right spectrum). The filter can also be omitted (left spectrum); to do so, use the DNO_FILTER option. Due to the low sensitivity of the experiment, it is strongly advisable to use a cryogenic probe with a cooled ¹³C preamplifier. The experiment is even more sensitive when using a TXO cryoprobe in which the inner coil is optimized for ¹³C.

Literature:

B. Fuertig, R. Schnieders, C. Richter, H. Zetsche, S. Keyhani, C. Helmling, H. Kovacs & H. Schwalbe, J. Bio. NMR. 64 207-221 (2016)

Parameter set: NA_C_CN_IASQSP

Pulse sequence: na_c_cn_iasqsp

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 159 ppm (center U:C2/C4 G:C2/C4/C6/C8)
• O2P: 9 ppm (center of aromatic protons)
• O3P: 86 ppm (center of amino nitrogens)
• SWF1: 40 ppm (indirect ¹⁵N dimension)
• SWF2: 50 ppm (direct ¹³C dimension)

Parameters to optimize:

• NS: Start with 128, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1.5 s, relaxation delay, depends on ¹³C T1 relaxation times.
• CNST3: 75 Hz (A), 55 Hz (C) or 65 Hz (A and C), ¹J(CC).

Description:

The CN-HSQC for amino groups uses the in-phase/antiphase scheme for the virtual decoupling of A:¹J(C6,C4) = 75 Hz and U:¹J(C4,C5) = 55 Hz, so that a decoupled signal is recorded. Therefore, the following procedure is necessary for data processing

• C4/N4 (C):
  Use splitcomb command for C4/N4 (C): splitcomb ipap 2 target_expno 1 55 (where target_expno is the number of the expno where the splitcomb result is saved).
• C6/N6 (A):
  splitcomb ipap 2 target_expno 1 75
• C2/N2 (G):
  G:C2 has no neighboring carbon atom, no CC coupling is visible:
  Use split 2 target_expno and then change the phase in the second expno (target_expno+1) by -90 degree, process both subspectra with xfb and then use adsu to add both subspectra. 

Parameter set: NA_TRHCCHCO3D

Pulse sequence: na_trhcchco3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 142 ppm (center of C6/C8 and C2)
• O3P: 153 ppm (center of imino nitrogen region)
• SWF1: 60 ppm (indirect ¹³C dimension, C aromatic)
• SWF2: 25 ppm (indirect ¹³C dimension, C6/C8/C2)
• SWF3: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.

Description:

TR-HCCH-COSY is designed for the assignment of adenine bases and links the assignment of A:C2/H2 to A:C8/H8 via the bridging carbons at adenine C4 and C5. For small RNAs, around 20 nucleotides, it is usually sufficient to run the F1/F3 plane as a 2D measurement.

Literature:

B. Simon, K. Zanier & M. Sattler, J. Biomol. NMR 20, 173-176 (2001)

Parameter set: NA_H5C5C4

Pulse sequence: na_h5c5c4

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 166 ppm (center of C4)
• O3P: 180 ppm (center of C:N3 and U:N3)
• SWF1: 10 ppm (indirect ¹³C dimension, C4)
• SWF2: 10 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.

Description:

The H5(C5)C4 experiment correlates the U/C:H5 with U/C:C4 through INEPT transfer steps and offers high sensitivity.

Parameter set: NA_H5C5C4N3N1

Pulse sequence: na_h5c5c4n3n1

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 99 ppm (center of C5 region)
• O3P: 185 ppm (center of N1/N3)
• SWF1: 100 ppm (indirect ¹⁵N dimension)
• SWF2: 10 ppm (direct ¹H dimension)  

Parameters to optimize:

• NS: Start with 256, adjust to a multiple of 4 to achieve desired S/N.
• CNST22: 22 ppm, bandwidth for 13C pulses.
• D25: 25 ms, N3/N1 transfer delay.
• SPNAM14: selective ¹⁵N 180 degree pulse generated by wavemaker using the command wvm -a.

Description:

The long-range HNN-COSY version for C and U detects H-bond-mediated ²J-NN couplings across base-pairs in the absence of observable imino protons. The crucial NN-transfer happens during 4*d25 and is typically ~100 ms, which can lead to severe signal intensity loss for larger RNAs and should thus be optimized.

Literature:

Dallmann, B. Simon, M. M. Duszczyk, H. Kooshapur, A. Pardi, W. Bermel & M. Sattler, Angew. Chem. Int. Ed., 52, 10487-10490 (2013)

Parameter set: NA_TRHCCNH3D

Pulse sequence: na_trhccnh3d

Sample requirements:

• Solvent: H₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 137 ppm (center of C6/C8)
• O3P: 153 ppm (center of imino region)
• SWF1: 24 ppm (indirect ¹H dimension)
• SWF2: 20 ppm (indirect ¹³C dimension, C6/C8)
• SWF3: 30 ppm (indirect ¹⁵N dimension, U:N3 and G:N1)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1.5 s, relaxation delay, depends on ¹H T1 relaxation times.
• D60: 28 ms TOCSY mixing time.

Description:

The TRHCCNH is used to correlate the assigned imino protons U:H3 and G:H1 with the aromatic carbons U:C6 and G:C8. The pulse sequence is very insensitive because the INEPT transfers occur via weak coupling (~10 Hz) in combination with a long CC-TOCSY. Sensitivity is enhanced by the TROSY transfer. Nevertheless, many scans must be acquired to achieve sufficient S/N. Typically, only the F3/F2 plane is recorded, which requires 2 days of measurement time. This experiment is only suitable for smaller RNAs up to a maximum of 30 nucleotides. The experiment uses ¹⁵N- and ¹³C-TROSY steps. Due to this the signals in the ¹³C dimension (F2) are shifted by ¹J(HC)/2 (G: 100Hz, U:90 Hz) and in ¹H dimension (F3) by ¹J(HN)/2 (47Hz).

Literature:

V. Sklenar, T. Dieckmann, S. E. Butcher & J. Feigon, J. Biomol. NMR, 7, 83 - 87 (1996)

The pulse sequence was developed by Frank Löhr (GUF), unpublished.

Parameter set: NA_HCNETGPSISP3D.2

Pulse sequence: na_hcnetgpsisp3d.2

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 113 ppm (center of C6/C8 and C1’ region)
• O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region)
• SWF1: 9 ppm (indirect ¹H dimension)
• SWF2: 40 ppm (indirect ¹³C dimension)
• SWF3: 40 ppm (direct ¹⁵N dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.

 Additional options:

• -DBASE: for magnetization transfer within the nucleobase only (H6/8-C6/8-N1/9). C5 decoupling is applied during t2 (set O2P to 138 ppm and SWF2 to 20 ppm).
• -DSUGAR: for magnetization transfer from the ribose only (H1'-C1'-N1/9). C2' decoupling is applied during t2 (set O2P to 89 ppm and SWF2 to 12 ppm).

Description:

The HCN experiment is used to transfer assignments from the aromatic C6 and C8 carbons to the ribose C1′ carbons. To achieve this, the nucleobase correlations C/U:H6-C6-N1 and A/G:H8-C8-N9 are recorded simultaneously with the ribose correlations C/U:H1′-C1′-N1 and A/G:H1′-C1′-N9. Since the chemical shifts of C/U:N1 and A/G:N9 differ significantly among U, C, A and G residues, a simple 2D ¹H/¹⁵N plane is often sufficient for resonance assignment in small RNAs.

For larger RNAs, it is recommended to record the aromatic and ribose correlations in two separate 3D experiments. For this purpose, use ZGOPTNS -DBASE for the aromatic correlations and ZGOPTNS -DSUGAR for the correlations to C1′. For both experiments, the ¹³C spectral width and O2P should be adjusted accordingly.

Literature:

V. Sklenar, R.D. Peterson, M.R. Rejante & J. Feigon, J. Biomol. NMR 3, 721 - 727 (1993)

Parameter set: NA_C_HCNCIASP

Pulse sequence: na_c_hcnciasp

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹⁵N¹³C

Spectral settings:

• O1P: 90 ppm (C1’ resonance)
• O2P: 7.5 ppm (center of aromatics H)
• O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region)
• SWF1: 12 ppm (indirect ¹³C dimension)
• SWF2: 40 ppm (indirect ¹⁵N dimension)
• SWF3: 40 ppm (direct ¹³C dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 0.5 s, experiment allows short relaxation delay due to preservation of H₂O magnetization.
• D3: 35 ms, C6/C8-N1/N9 transfer delay and CT-evolution in t1.
• D5: 40 ms. N1/9-C6/8 and N1/9-C1’ transfer delay N1/9 and CT-evolution in t2: ~.
• D35: 50 ms, C1’-N1/9 transfer delay.
• CNST2: 6 ppm, bandwidth for H6/H8 proton selective pulses.
• CNST19: 7.5 ppm, center of H6/H8 proton region.

Processing:

• use au-program splitcomb [ipap 4 new expno. 170 44 2 or 1] to process data.
• referencing: sr(F1)=sr(F3)+(o1p-CNST20)*bf1+0.5*1JCH6/8 (e.g. 100 Hz for J term as compromise for all nucleotide types).

Description:

In the (H)CNC experiment, assignments of the aromatic C/U:H6-C6 and A/G:H8-C8 resonances are transferred to the sugar C1′ resonances, analogous to the HCN experiment. Direct ¹³C detection often enables the observation of additional correlations that may not be visible in proton-detected experiments.Due to the low sensitivity the experiment can only be performed efficiently on a cryogenic probe equipped with a cold ¹³C preamplifier. Even higher sensitivity can be achieved with TXO cryoprobes whose inner coil is optimized for ¹³C detection.

For virtual decoupling of the ¹J(H1′C1)′ and ¹J(C1′C2′) couplings, double inphase/antiphase elements are incorporated in an interleaved manner during the refocusing of the C1′-N1/N9 correlations. Data processing is performed using the AU program splitcomb.

The (H)CNC experiment is a ¹³C-TROSY variant in which the signals in the ¹³C dimension (F3) are shifted by ¹J(HC)/2. A value of 100 Hz is typically used as a compromise for the different nucleotide types.

Literature:

V. Sklenar, R.D. Peterson, M.R. Rejante & J. Feigon, J. Biomol. NMR 3, 721 - 727 (1993)

The pulse sequence was developed by Frank Löhr (GUF), unpublished.

Parameter set: NA_HMQCNOESYHMQC4D

Pulse sequence: na_hmqcnoesyhmqc4d

Sample requirements:

• Solvent: D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (center of aromatic C region)
• O3P: 195 ppm (center of aromatic N region)
• SWF1: 4 ppm (indirect ¹H dimension)
• SWF2: 22 ppm (indirect ¹³C dimension, C6/C8)
• SWF3: 10 ppm (indirect ¹³C dimension, C1’)
• SWF4: 12 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 4, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D8: 150 ms NOESY mixing time.
• CNST16: 7.7 ppm, center of H2/H6/H8 region.
• CNST17: 5.7 ppm, center of H1’ region.
• CNST23: 4 ppm, bandwidth for H1’ selective ¹H pulses.
• CNST24: 6 ppm, bandwidth for H6/H8 selective ¹H pulses.

Description:

The 4D HCCH HMQC-NOESY-HMQC increases the resolution compared to the 3D NOESY experiments and helps in assigning the H1’-H6/H8 sequential walk. To ensure that the measurement time does not become too long, the experiment should be recorded using non-uniform sampling.

Literature:

J. Stanek, P. Podbevsek, W. Kozminski, J. PLavec, M.Cevev, J. Biomol. NMR 57, 1, (2013)

Parameter set: NA_SHCCHFLGPSP3D

Pulse sequence: na_shcchflgpsp3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 90 ppm (center of C1’)
• O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region)
• SWF1: 35 ppm (indirect ¹³C dimension, C ribose all)
• SWF2: 10 ppm (indirect ¹³C dimension, only C1’)
• SWF3: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST4: Multiplicity selection 4 for CH, 6 for CH + CH2.
• L1: loop for FLOPSY cycle, 1 for transfer from C2’, 3 for transfer from all ribose carbons.

Additional options:

• -DCALC_SP: for automatic calculation of P24/SP9 (C1’ selective) (recommended).

Description:

Using the selective (H)CCH-TOCSY, all ¹³C signals in the ribose can be assigned. To achieve this, the magnetization of all ribose carbons is transferred to C1', which is detected in the F2/F3 dimensions. Since the chemical shifts of C2' and C3' are very similar, a TOCSY with a short mixing time (l1=1, d31=4.7 ms) is recorded first; here, only the correlation with C2' is visible. In a second 3D experiment, l1 is set to 3 (d31=14.1 ms), and all ribose carbon signals are visible. By comparing the two 3D spectra, C2' and C3' can be distinguished.

Literature:

L.E. Kay, G.Y. Xu, A.U. Singer, D.R. Muhandiram & J. D. Forman-Kay J. Magn. Reson. B 101, 333 - 337 (1993)

Parameter set: NA_SHCCHFLGPSP3D2

Pulse sequence: na_shcchflgpsp3d2

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 90 ppm (center of C1’)
• O3P: 160 ppm (center of N9 (A and G) and N1 (C and U) region)
• SWF1: 4 ppm (indirect ¹H dimension, all ribose protons)
• SWF2: 10 ppm (indirect ¹³C dimension, only C1’)
• SWF3: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST4: Multiplicity selection: 4 for CH, 6 for CH + CH2.
• L1: loop for FLOPSY cycle, 1 for transfer from H2’, 3 for transfer from all ribose protons.
• CNST28: C(ribose) chemical offset: 78 ppm).

 Additional options:

• -DCALC_SP: for automatic calculation of P24/SP9 (C1’ selective) (recommended).

Description:

Using the selective (H)CCH-TOCSY, all ¹H signals in the ribose can be assigned. To achieve this, the magnetization of all ribose protons is transferred to C1', which is detected in the F2/F3 dimensions. To reduce overlap the experiment can be recorded with a short mixing time (l1=1, d31=4.7 ms). Here, only the correlation to the H2' is visible. By setting l1 to 3 (d31=14.1 ms) magnetization will be transferred from all ribose protons (H2’,H3’,H4’ and H5’).

Literature:

L.E. Kay, G.Y. Xu, A.U. Singer, D.R. Muhandiram & J. D. Forman-Kay J. Magn. Reson. B 101, 333 - 337 (1993)

Parameter set: NA_HCPETGPSI3D

Pulse sequence: na_hcpetgpsi3d

Sample requirements:

• Solvent: D₂O          
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 72 ppm (set to center of the ribose C3’,C4’,C5’ signals)
• O3P: -1 ppm (set to center of ³¹P signals)
• SWF1: 4 ppm (indirect ³¹P dimension)
• SWF2: 25 ppm (indirect ¹³C dimension)            
• SWF3: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST11: Multiplicity selection: 4 for CH, 6 for CH + CH2.

Description:

The 3D HCP triple-resonance experiment correlates the ribose C3’-H3’, C4’-H4’ and C5’-H5’# resonances on either side of the phosphorus group by transfer of magnetization via ^2,3J(CP) and ¹J(CH) coupling constants.

Reference:

Marino et al. JACS 1994, 116, 6472-6473

Parameter set: NA_HCPDIGPJR3D.2 (for QCI ¹H{¹³C,¹⁵N,³¹P} probes)

Pulse sequence: na_hcpdigpjr3d.2

Sample requirements:

• Solvent: D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 90 ppm (set to center of C1’ region)
• O3P: -1 ppm (set to center of ³¹P signals)
• O4P: 160 ppm (set on N1/N9 signals)
• SWF1: 5 ppm (indirect ³¹P dimension)
• SWF2: 5 ppm (indirect ¹³C dimension)
• SWF3: 5 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• D15: 14 ms, C-C TOCSY mixing time.
• CNST16: 5.8 ppm, offset for maximum signal, set to the center of the H1’ region.

Description:

The 3D HCP-TOCSY triple-resonance experiment extends the 3D HCP experiment with a CC-TOCSY (using a DIPSI3 sequence), in which the ¹³C magnetization is further relayed to the neighboring ribose nuclei through their ¹J(CC) couplings. Efficient water suppression is accomplished using a 1-1 echo pulse sequence. This experiment includes ¹⁵N decoupling in F2 and F3 and requires a QCI ¹H{¹³C,¹⁵N,³¹P} probe.

Reference:

Marino et al. JBNMR 1995, 5, 87-92

Parameter set: NA_TRT1ETGPSITC3D

Pulse sequence: na_trt1etgpsitc3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 143 ppm (C2/C6/C8) or 102 ppm (C5) or 90 ppm (C1’)
• SWF1: 30 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 205 Hz for C2/6/8, 180 Hz for C5, 170 Hz for C'1, ¹J(CH).
• CNST28: 105 ppm (for C2/6/8), 150 ppm (for C5), 72 ppm (for C1’), offset for decoupling.
• VDLIST: Adjust the VDLIST entries to the T1 relaxation times.
• TD1: Set to the number VDLIST entries.

Additional options:

• -DLABEL_CN: ¹⁵N-decoupling for RNAs containing ¹³C¹⁵N-labeled nucleotides, set O3P to 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

This experiment is used to record a series of 2D ¹H¹³C correlation spectra in a pseudo 3D fashion with varying relaxation delay times for the determination of ¹³C-T1 relaxation times. A VDLIST is needed to set the variable delay times (commonly includes 8 or more relaxation delays ranging from several ms to 1-2 s). Set TD for the F1 (pseudo) dimension to the number of delays.

Literature:

A. L. Hansen & H. M. Al-Hashimi, J. Am. Chem. Soc. 129, 16072-16082 (2007)

Parameter set: NA_TRTRETGPSITC3D

Pulse sequence: na_trtretgpsitc3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 143 ppm (C2/C6/C8); or 102 ppm (C5) or 90 ppm (C1’)
• SWF1: 25 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 8, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1.5-2.5 s, a longer delay is advisable due to sample heating by the ¹³C spinlock.
• CNST2: 205 Hz for C2/6/8, 180 Hz for C5, 170 Hz for C'1, ¹J(CH).
• CNST27: 2000 Hz, T1rho spinlock field strength in Hz.
• CNST28: 105 ppm (for C2/6/8), 150 ppm (for C5), 72 ppm (for C1’), offset for decoupling.
• CNST30: T1rho spinlock offset +2500 Hz for C2/C6/C8, -2000 Hz for C5, +2000 Hz for C1’.
• SPNAM19: shaped pulse adiabatic ramp down (Gaussramp+down.1 or Gaussramp-down.1).
• SPNAM20: shaped pulse adiabatic ramp up (Gaussramp+up.1 or Gaussramp-up.1).
• VDLIST: Adjust the VDLIST entries to the T1rho relaxation times.
• TD1: Set to the number VDLIST entries.

Additional options:

• LABEL_CN: for ¹⁵N-decoupling, set O3P to 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

This experiment is used to record a series of 2D ¹H¹³C correlation spectra in a pseudo 3D fashion with varying relaxation delay times for the determination of ¹³C-T1rho relaxation times. A VDLIST is needed to set the variable delay times (commonly includes 8 or more relaxation delays ranging from around 10 to 100-200 ms). Set TD for the F1 (pseudo) dimension to the number of delays.

Literature:

A. L. Hansen & H. M. Al-Hashimi, J. Am. Chem. Soc. 129, 16072-16082 (2007)

Parameter set: NA_SELT1RGPF22D

Pulse sequence: na_selt1rgpf22d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: center on ¹³C chemical shift for the signal interest
• SWF1: 1 ppm (ignored for pseudo dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 16, adjust to a multiple of 8 to achieve desired S/N.
• D1: 1.5-2.5 s, a longer delay is advisable due to sample heating by the ¹³C spinlock.
• CNST10: ¹³C chemical shift difference for CSFILTER option.
• CNST20: 8000 Hz, B1 field for ¹H CW decoupling.
• CNST21: 100 Hz, B1 field for Hartman Hahn transfer.
• CNST22: 100-2000 Hz, B1 field for ¹³C spinlock in Hz.
• CNST28: ¹³C offset for spinlock in Hz.
• CNST30: ¹H chemical shift of signal of interest in ppm.
• VDLIST: Adjust the VDLIST entries to the T1rho relaxation times.
• TD1: Set to the number VDLIST entries.

Additional options:

• CSFILTER: for suppressing signals with similar ¹H but different ¹³C resonances, set CNST10 to chemical shift difference in [in Hz].
• LABEL_N: for additional ¹⁵N decoupling during acquisition.
• O3P: 160 ppm, for ¹⁵N-decoupling.

Description:

T_1ρ Relaxation dispersion (T1rhoD) is typically used to comprehensively quantify the parameters associated with chemical or conformational exchange on the microsecond-to-millisecond timescale. This window covers key structural refolding events in RNA at the individual base-pair level.

The experiment is designed to selectively excite a single peak. Therefore O2P and CNST30 need to be adjusted for every experiment, to achieve the excitation of the desired peak, based on its ¹H and ¹³C chemical shifts. The rates of the exchange process accessible by T1rhoD are determined by the field strength of the field applied in the rotating frame, i. e., the “spin-lock”, during the relaxation delay and range from 100 to 2000 Hz for ¹³C. The spin-lock is applied in a series of different field strengths and offsets relative to the resonance under investigation. The apparent T2 relaxation times show a dispersion profile if this resonance is involved in an exchange process on the respective timescale. At the same time, a dispersion profile is observed at the same field strength but at a distinct offset, corresponding to the chemical shift of the exchanging conformational state of the resonance.

Literature:

Hansen, A.L., Nikolova, E.N., Casiano-Negroni, A.& Al-Hashimi, H.M., JACS 131, 3818 (2009)

Parameter set: NA_HSQCNOEGPSPWG3D

Pulse sequence: na_hsqcnoegpspwg3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (center of aromatic region)
• SWF1: 30 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 5 s or longer. A long relaxation delay is important for precise hetNOE values.
• CNST2: 200 Hz, ¹J(CH) for aromatic carbons.

Additional options:

• LABEL_CN: for ¹⁵N-decoupling, set O3P to: 195 ppm (center of aromatic N region).

Description:

This experiment is used to record two 2D ¹H¹³C correlation spectra in a pseudo 3D fashion either with or without proton saturation during the relaxation delay, for the determination of the ¹³C{¹H} heteronuclear NOE (hetNOE). Set TD for the F1 (pseudo) dimension to 2. The experiment uses WATERGATE for water suppression and is therefore optimal for aromatic protons.

Parameter set: NA_HSQCNOEETGPSP3D

Pulse sequence: na_hsqcnoeetgpsp3d

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 90 ppm (center of C1’ region)    
• SWF1: 10 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 5 s or longer. A long relaxation delay is important for precise hetNOE values.
• CNST24: 12 ppm, bandwidth for ¹³C selective excitation.

Additional options:

• -DCC_DEC optional selective C2' ¹³C decoupling in F1.

Description:

This experiment is used to record two 2D ¹H¹³C correlation spectra in a pseudo 3D fashion either with or without proton saturation during the relaxation delay, for the determination of the ¹³C{¹H} heteronuclear NOE (hetNOE). Set TD for the F1 (pseudo) dimension to 2. Gradient selection enables detection of ribose proton signals for samples in H₂O.

Parameter set: NA_HSQCGPIASP_C2C6C8

Pulse sequence: na_hsqcgpiasp

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 145 ppm (center of aromatic region)
• SWF1: 30 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 200 Hz, ¹J(CH) for aromatic carbons.
• CNST20: 20 ppm, ¹³C refocusing bandwidth.

Additional options:

• LABEL_CN: for ¹⁵N-decoupling, set O3P to 195 ppm (center of aromatic N region).

Description:

The experiment measures two ¹H¹³C HSQC spectra without ¹³C-decoupling in the proton dimension (F2). The inphase and antiphase components are recorded in an interleaved fashion and can later be separated during processing. This prevents the increasing peak overlap due to coupling evolution. If the sample is ¹⁵N-labeled, decoupling can be selected as an option.

Processing:

• Split the IP and AP part in two spectra using “split ipap” in command line
• Process with 16k to 30k points in the F2 and 4k points in the F1

Parameter set: NA_HSQCGPIASP_C5

Pulse sequence: na_hsqcgpiasp

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 99 ppm (center of aromatic region)
• SWF1: 15 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 180 Hz, ¹J(CH) for C5.
• CNST20: 12 ppm, ¹³C refocusing bandwidth.

Additional options:

• LABEL_CN: for ¹⁵N-decoupling, set O3P to 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

The experiment measures two ¹H¹³C HSQC spectra without ¹³C-decoupling in the proton dimension (F2). The inphase and antiphase components are recorded in an interleaved fashion and can later be separated during processing. This prevents the increasing peak overlap due to coupling evolution. If the sample is ¹⁵N-labeled, decoupling can be selected as an option.

Processing:

• Split the IP and AP part in two spectra using “split ipap 2” in command line
• Process with 16k to 30k points in the F2 and 4k points in the F1

Parameter set: NA_HSQCGPIASP_C1PRIME

Pulse sequence: na_hsqcgpiasp

Sample requirements:

• Solvent: H₂O or D₂O
• Labeling: ¹³C or ¹⁵N¹³C

Spectral settings:

• O1P: 4.7 ppm (set on water resonance)
• O2P: 90 ppm (center of aromatic region)
• SWF1: 12 ppm (indirect ¹³C dimension)
• SWF2: 9 ppm (direct ¹H dimension)

Parameters to optimize:

• NS: Start with 64, adjust to a multiple of 4 to achieve desired S/N.
• D1: 1 s, relaxation delay, depends on ¹H T1 relaxation times.
• CNST2: 170 Hz, ¹J(CH) for C1’.
• CNST20: 12 ppm, ¹³C refocusing bandwidth.

Additional options:

• LABEL_CN: for ¹⁵N-decoupling, set O3P to 160 ppm (center of N9 (A and G) and N1 (C and U) region).

Description:

The experiment measures two ¹H¹³C HSQC spectra without ¹³C-decoupling in the proton dimension (F2). The inphase and antiphase components are recorded in an interleaved fashion and can later be separated during processing. This prevents the increasing peak overlap due to coupling evolution. If the sample is ¹⁵N-labeled, decoupling can be selected as an option.

Processing:

• Split the IP and AP part in two spectra using “split ipap 2” in command line
• Process with 16k to 30k points in the F2 and 4k points in the F1

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