Unlabeled RNA

This chapter deals with the assignment strategy on a detailed step by step explanation, using an RNA which folds into a stem-loop structure as an example. The RNA will just be referred to as ‘24mer RNA’ and is a model construct with a 10-base-pair stem capped by a GAGA tetraloop (see Figure 7 for the sequence and secondary structure). The assignment procedure will describe the minimal set of spectra needed for unlabeled RNA and will refer to chapter “NMR assignment of ¹³C¹⁵N labeled RNA” where necessary. 

Experiment Setups for Unlabeled RNA:

Spectra needed: 
¹H Imino 1D recorded in 5 K steps in a range between 278 & 313 K generally (in rare cases temperatures above or below this range might also be helpful) 

1D measurements
As a start, it is essential to determine the optimal temperature at which to conduct further measurements. For this purpose, it is recommended to acquire a series of 1D ¹H spectra in a temperature range from 278 K to 313 K (with steps of 5 K) (Figure 7). As the resolution in the proton dimension is typically the limiting factor because of their small chemical shift range, this is a quick and simple method to resolve the temperature at which the signal dispersion and intensity are best.
We recommend to record the 1D ¹H spectra using two different experiments, the ¹H Imino 1D and the ¹H 1D all.

Two temperature optimizations are needed in general for RNA: For imino proton measurements usually a low temperature is beneficial due to slower solvent exchange, whereas for spectra of aromatic protons a higher temperature is usually preferable due to faster relaxation at low temperature. The choice of the temperature should consider a good signal resolution and high signal intensity; both factors must be weighed out against each other. When there are more signals observed than expected, it may indicate the presence of multiple conformations which are in slow exchange (timescale typically hundreds of ms or longer). Changes in temperature can be used to increase or decrease the exchange rates.

Recording a 1D ¹H temperature series shows temperature dependent chemical shift and intensity changes. For further investigation by 2D ¹H¹H NOESY and 2D ¹H¹⁵N HSQC/HMQC spectra with natural abundance it is crucial to optimize the temperature, at which the imino proton signals are most intense and best resolved. In our example the imino signal intensity for resonances at around 10.4, 12.7 and 13.2 ppm decreases at higher temperature, and the proton linewidths increase due to the exchange with water. Regarding signal resolution at low temperatures (278 & 283 K) three signals are resolved, whereas at temperatures above 288 K only two signals are resolved. Therefore, for this RNA a temperature of 278 K was chosen to be optimal. If spectral quality remains poor at all temperatures, Mg²⁺ / pH titrations should be performed.

Spectra needed:
2D ¹H¹H NOESY for imino-imino or
2D ¹H¹H NOESY for imino-all
2D ¹H¹⁵N HSQC/HMQC at natural abundance

The imino protons, G:H1 and U:13 are commonly observed between 10 and 15 ppm. Depending on the pH also a protonated A:H1 or C:H3 can be observed. When they are not protected in base-pairs or by steric hindrance, they exchange rapidly with H₂O, leading to exchange broadening of the signals. Typically, Watson-Crick (WC) paired U imino protons resonate between 13 and 15 ppm and WC paired G imino protons between 12 and 13.5 ppm. WC-paired imino protons are easily distinguishable by the ¹⁵N chemical shift of the imino nitrogen, which is typically around 147 ppm for G:N1 and around 162 ppm for U:N3. Imino protons paired in non-canonical base-pairs (e.g. G-U, G-A, typically experience a shift to lower ppm values in ¹H and ¹⁵N compared to the WC-paired resonances. 
The imino-proton assignment is used to determine the secondary structure of RNA. In helical regions the distance between sequential imino-protons is < 5 Ǻ and in a 2D ¹H¹H NOESY experiment cross-peaks are observed between sequential imino-protons. In addition, a natural abundance 2D ¹H¹⁵N HSQC or HMQC spectrum is used to determine whether an imino proton signal belongs to a G or U. 

The 2D ¹H¹H NOESY experiment (Nuclear Overhauser effect and Exchange SpectroscopY) is a two-dimensional NMR technique that can be used to detect the spatial proximity between protons (typically up to around 5 Å) in a molecule. Unlike COSY, which shows coupling through bonds, NOESY reveals the interactions through space via the nuclear Overhauser effect (NOE). This makes this experiment particularly valuable for determining the 3D structure of biomolecules in solution. The resulting spectrum shows cross peaks between the protons which are in close spatial proximity. The intensity (or volume) of the NOE cross peak is related to the distance (a strong signal indicates a closer proximity than a weak signal). In addition, a NOESY spectrum can be used to study transient interactions, ligand binding sites and conformational changes.

For our exemplary 24mer RNA, the overlay of the 1D ¹H imino spectra and the 2D ¹H¹⁵N HSQC spectra reveal eleven distinct imino N-H cross-peaks. The predicted secondary structure (Figure 8, right) shows however only 10 WC base-pairs, three G-C and seven A-U base-pairs. Each of these WC base-pairs contains a single, exchange-protected imino-proton accounting for ten of the resonances. The 2D ¹H¹⁵N HSQC/HMQC spectrum reveals that seven U and four G signals are observed reflecting the predicted three G-C and seven A-U base-pairs. A single G signal is unaccounted for by the predicted stem, indicating that one of the nonhelical G nucleotides is either sterically shielded or base-paired within the loop. In the following parts, the assignment procedure of imino resonances is done by sequential assignment in 2D ¹H¹H NOESY spectra.

In a helical structure the imino-protons point to the center of the helix and are hydrogen-bonded to form base-pairs. This geometry brings imino-protons of the adjacent G and U nucleotides within 5 Ǻ of each other, which results in cross-peaks in a NOESY spectrum. A particularly strong cross-peak is observed for non-canonical G-U base-pairs, because of the close proximity of both imino-protons. 

For our exemplary 24mer RNA, no non-canonical U signals are observed in the 2D ¹H¹⁵N HSQC. These signals are expected at < 12.5 ppm in 1H and < 160 ppm in ¹⁵N. In addition, no extremely intense cross-peaks are observed in the imino-imino 2D ¹H¹H NOESY, therefore a potential G-U base-pairs is ruled out. As a result, all observed cross-peaks are due to the proximity of adjacent base-pairs. To start the resonance assignment the cross-peak at ~ 13.9 ppm in F1 and ~ 12.4 ppm in F2 is chosen (Figure 9). Opposite the diagonal peaks the inverse cross-peak at ~ 12.4 ppm in F1 and ~ 13.9 ppm in F2 can be directly observed. From here on, the focus of this tutorial will be put on the cross peaks below the diagonal peaks to avoid confusion. The 15N chemical shift in the 2D ¹H¹⁵N HSQC reveals that the resonance at ~12.4 ppm is a G, and the resonance at ~ 13.9 ppm is a U. The U will be denoted as U(i) and the G as G(i±1) with the number indicating adjacent base-pairs either in the 5’- or 3’-direction. A single sequential correlation does not allow unambiguous assignment yet, as there are three different positions within this helix that show sequential G-C and A-U base-pairs. (Figure 9, marked red in sequence). To achieve unambiguous assignment, it is necessary to assign additional cross-peaks from either one of the two resonances. 

For U(i) a cross-peak is observed, which correlates it with another U(i±1) at 14.3 ppm. The U(i±1) itself shows another correlation to a G(i±2) (Figure 10). The sequential order based on these cross-peaks in combination with the nucleotide-type assignment (G or U) based on the 2D ¹H¹⁵N HSQC indicates a sequence of G-U-U-G. This sequence order is present only once in the helix (nucleotides G2, U22, U21 and G20, green). Since the sequence is palindromic, the nucleotide order in the assignment is still ambiguous. For this example, the subsequent cross-peaks will result in an unambiguous assignment. If there are no additional cross-peaks, it is recommendable to use chemical shifts prediction programs, such as LarmorD or CSmotif. The chemical shift depends on the neighboring bases, which can differ for palindromic sequences. 

For the 24mer RNA construct, there is an additional cross-peak between G(i±1) and another G, which allows for the assignment of G(i±1) as G2 (Figure 11). The additional G is assigned to G1. Because G1 is part of the stem-closing base-pair, only a single cross-peak is observed indicating that there is just a single neighboring base-pair. Resonances from stem-closing base-pairs are often not observed, especially at higher temperatures, due to increased solvent exchange rates leading to line broadening beyond detectability. Based on the concepts applied above, all of the cross-peaks within the stem can be assigned (Figure 12).

All the helical imino resonances can be assigned by sequential NOE contacts for this 24mer RNA. Additionally, for the remaining fourth G, a cross-peak to the loop-closing base-pair U15 is observed. This is assigned to G11 and indicates a base-paired or sterically shielded G within the loop. In summary, the combination of 2D ¹H¹H NOESY and 2D ¹H¹⁵N HSQC spectra could validate the predicted stem secondary structure and further reveal additional base-pairing/structure within the loop, which was not predicted by structure prediction programs.

The imino resonance assignment can be used as reporters to monitor structural changes induced by external factors, such as binding of small molecules or other biomolecules. Furthermore, it constitutes the basis for further assignment of the aromatic and ribose protons.

Spectra needed:

2D ¹H¹H NOESY - imino-all
2D ¹H¹H NOESY
2D ¹H¹³C HSQC at natural abundance
2D ¹H¹⁵N HMQC at natural abundance
2D ¹H¹H TOCSY

Of the typical NOE contact, depicted in Figure 13, only the sequential assignment of the imino protons, also called the imino-imino walk has been described so far. The other contacts will be described in the following passages:

The assignment of all imino-proton resonances usually reveals the secondary structure and base-pairing pattern within the RNA. On the other hand, dynamic elements, such as internal bulges, loops or junctions do not have to exchange protected imino-protons, leaving major parts of the RNA undescribed after the imino assignment. Therefore, it is necessary to assign aromatic and ribose protons as well to get a thorough description of the RNA. 
The imino-proton assignment presents still a valuable basis for this, as the first step is to transfer nucleotide specific assignment from imino-protons to aromatic-protons. It’s crucial to point out that for a ¹³C¹⁵N labeled sample there are additional experiments that make this assignment transfer a lot easier, which are not discussed at this point. A imino-all type 2D ¹H¹H NOESY experiment is used to observe and assign correlations, listed in Table 4, within a base-pair. Obviously, also correlations between adjacent bases are observed, but the signal intensity is significantly lower due to the larger proton-proton distances.

To assign resonance unambiguously, it is necessary to record a 2D ¹H¹³C HSQC and 2D ¹H¹⁵N HMQC spectrum at natural abundance (or make a preliminary assignment and validate when a labeled sample is available) and a 2D ¹H¹H TOCSY spectrum.

The imino-all type 2D ¹H¹H NOESY – imino all contains more information than just imino-imino resonances, which were addressed in the previous chapter. Here, also correlations of aromatic and ribose protons to the imino protons are observed. Complementary to the imino-imino type 2D ¹H¹H NOESY experiment, in which only the imino-imino NOEs within 10 and 15 ppm in both dimensions are observed. Further, there is a 2D ¹H¹H NOESY, which is used to dectect either a complete spectrum (Figure 14) with imino, aromatic and ribose protons observed in both dimensions or a better resolved spectrum, in which the imino-protons are not observed. The complete 2D ¹H¹H NOESY spectrum is shown in Figure 14 and the different spectral regions are annotated. 

Further, between 10 and 15 ppm in F2 and between 3 and 9 ppm in F1 NOEs from imino to aromatic, amino and ribose protons are observed. Within 5 to 9 ppm in both dimensions NOEs between amino, aromatic and H1’ protons are observed. NOEs from the aromatic/H1’/amino protons to the remaining ribose protons are observed between 5 and 9 ppm in F2 and between 3 and 5 ppm in F1. The imino-all type 2D ¹H¹H NOESY is thought to facilitate imino-amino and imino-aromatic assignment. For the assignment of aromatic and ribose protons including the sequential H1’-H6/H8 assignment a NOESY with decreased spectral width, ideally recorded on a D₂O sample, is recommended. The unambiguous assignment of all imino-resonances (see Chapter 2), will be used to show how to transfer the nucleotide specific assignment into the nucleobases.

As mentioned in the preceding paragraph, the ¹H chemical shift ranges allow for global differentiation of certain protons within the RNA. Additional resolution and information is obtained in the ¹³C-dimension of a 2D ¹H¹³C correlation spectrum (either HSQC or HMQC based) (Figure 15). All C-H bonded atoms can be distinguished in the HSQC spectra based on their ¹³C chemical shift. The ¹H and ¹³C chemical shift ranges of all C-H bonded atoms in RNA are listed in Table 5. In the following paragraphs it will be shown how combining the HSQC with other spectra (such as NOESY and TOCSY) enables the transfer of assignment from the imino to aromatic resonances. For educational purposes we used the complete 2D ¹H¹H NOESY spectra for visualization, but it can be recommended to record the 2D ¹H¹H NOESY – imino all spectra and a 2D ¹H¹H NOESY for aromatic and ribose correlations separately to increase the resolution for correlations between aromatic and ribose protons. 

Starting the assignment transfer, it is best to look at the protons close to the imino protons, because these will show the most intense NOEs due to the short distance to the imino. In the canonical Watson-Crick base-pairs, these protons are A:H2 & A:H61/H62 in a A-U and G:H21/H22 & C:H41/H42 in a G-C base-pair. The aromatic A:H2 proton is covalently linked to a carbon, whereas the amino protons G:H21/H22, C:H41/H42 and A:H61/H62 are attached to a nitrogen. Due to rotation of the amino group around the C–N bond, the amino protons might be broadened beyond detection, depending on the rotation frequency. Further, a 2D ¹H¹³C HSQC is needed for unambiguous assignment of the aromatic A:H2-C2 and a 2D ¹H¹⁵N HMQC for unambiguous assignment of amino protons. If the HSQC spectra at the temperature optimized for imino-protons are missing several signals (e.g. only 3 A:H2 resolved out of 8), a compromise between resolved & sharp imino-signals and resolved & sharp aromatic signals has to be found. The assignment of A:H2 protons is based on the NOEs to U:H3 imino-protons. Because of the short distance between both protons a rather strong NOE is observed between U:H3 and A:H2 within a base-pair. To verify the A:H2 assignment the 2D ¹H¹³C HSQC is used, identifying the A:H2 by the ¹³C chemical shift ranging from 150 to 155 ppm (Figure 16). The 2D ¹H¹³C HSQC shown in Figure 16 looks rather different compared to the spectrum in Figure 15. Because the range in the ¹³C-dimension is rather large for RNA, often HSQCs are recorded with decreased spectral width to increase resolution drastically, focusing either on H2, H6 and H8 or H5 and all ribose protons. 

Looking at the most up field U8:H3 imino resonance, several NOEs are observed to be aromatic or amino protons. The most intense NOE is clearly observed to be a resonance at ~ 6.3 ppm (magenta). Fortunately, in the 2D ¹H¹³C HSQC there is no overlap in the ¹H-dimension and the ¹³C chemical shift of this resonance is determined to be 151.8 ppm. Unambiguously the NOE is observed between U8:H3 and A17:H2 within the U8-A17 base-pair. Subsequently, the A17:H2 resonance can be followed to check for additional NOEs to imino protons. A significantly weaker NOE from A17:H2 is observed to the resonance at 13.7 ppm, which was assigned to U7 previously. As U7 is directly preceding U8, U7:H3 and A17:H2 are within 5 Å of each other, a weaker NOE is observed. Like the assignment of A17:H2, all 7 U:H3-A:H2 cross-peaks within a base-pair (magenta) and between sequential base-pairs (green) were unambiguously assigned. In the 2D ¹H¹³C HSQC two A:H2-C2 cross-peaks are unassigned. These belong to the two remaining A nucleotides in the GAGA loop.

For the assignment of amino resonances, it is important to understand that the C-N bond is not rotating freely leading to separate signals for the two amino protons. By convention the downfield resonance is denoted as 2 (e.g. H22, H42 and H62) and the up-field resonance as 1 (e.g. H21, H41, H61). In the 2D ¹H¹⁵N HMQC, signals for both protons are expected at the same nitrogen resonance. The amino resonances can be differentiated by their ¹⁵N chemical shift ranges: with G:N2 typically around 75 ppm, A:N6 around 84 ppm and C:N4 around 98 ppm. Due to rotation on the intermediate timescale often not all aminos are observed. This can also be observed for our example RNA, where several signals for G and A are missing (Figure 17).

Starting again on the U8 imino resonance (at 13 ppm), NOEs to A17:H61/H62 are to be expected. Two NOE cross-peaks at 7.7 and 8.1 ppm are in fact observable, which are also present in the 2D ¹H¹⁵N HMQC showing correlations to the same ¹⁵N chemical shift at ~85 ppm that is in the typical range for an A:N6. For a G-C base-pair NOEs to both G and C aminos are to be expected. For the G20 imino proton at ~13.5 ppm four rather intense NOEs can be observed. Checking the 2D ¹H¹⁵N HMQC, the chemical shifts at 6.3 and 8.5 ppm can be unambiguously assigned as G20:H21/H22, because they show correlation to the same nitrogen G20:N2 at ~75 ppm (Figure 17). The resonances at 7.1 and 8.4 ppm on the other hand are assigned to C5:H41/H42, because they show correlation to the same nitrogen C5:N4 at ~99 ppm. Further confirmation of this assignment is obtained by identifying sequential NOEs from G20 and C5 aminos to imino protons of U21 and U6. Due to the position in the center of the helix and stabilization by surrounding base-pairs the solvent exchange is reduced for these G20 and C5 aminos. 

In contrast, G1 is involved in the closing G1-C24 base-pair and experiences increased solvent exchange for the imino proton and faster rotation for the amino group. When compared to G20, G1 shows significantly less and weaker signals. Still, two decently intense NOEs are observed to resonances at 7.1 and 8.4 ppm, which can be identified as C amino protons in the 2D ¹H¹⁵N HMQC. In the same manner remaining amino protons can be assigned, although especially for G and A signals are missing.

After achieving unambiguous assignment for the amino protons in the last paragraph, we can transfer the assignment information further into the nucleobase. For G and A there are no protons within the base-pair near the aminos. On the other hand, for C, the H5 is near to the amino, often allowing for its assignment as well as the subsequent H6.

Depicted in Figure 17 is the 2D ¹H¹H NOESY, including also the imino/amino-imino/aromatic region, and the 2D ¹H¹³C HSQC. On the left, the previously assigned G and C amino resonances are annotated. Within the aromatic region for C5 and C24 strong NOEs are observed between both amino protons. Following the C:H42 resonance NOEs to other aromatics or aminos are observed. Having the 2D ¹H¹³C HSQC at hand the C:H5-C5 resonances can be easily identified (Figure 18). Since there are only three C nucleotides in the RNA, it is easy to check for the C:H42-H5 NOEs. Since the H5 resonance at 5.2 ppm has no overlap with any other H1’ or H5, the cross-peak to C5:H42 could be assigned unambiguously, as well as the C5:H5-C5 signal in the 2D ¹H¹³C HSQC. Similarly, out of the two remaining H5 resonances one shows a correlation with C24:H42, resulting in the assignment of C24:H5-C5.

Based on the C:H5-C5 assignment it is further possible to obtain the C:H6-C6 assignment (Figure 19). The C:H5-H6 cross peaks can be observed in 2D ¹H¹H NOESY, but this region is rather crowded. Therefore, it is more convenient to use a 2D ¹H¹H TOCSY spectrum, where only the pyrimidine H5-H6 cross peaks are observed in the aromatic region.

Within the last chapter it was shown how the nucleotide-specific assignment can be transferred from imino protons into the nucleobase for unlabeled RNA. It is to be noted that for the purines (A and G), the H8-C8 and for U, the H5-C5 and H6-C6 could not be assigned until yet. If it is a rather small RNA construct the assignment of A:H2 and C:H5/H6 and A/C/G aminos present a solid starting point to proceed with the sequential H1’-H6/H8 to assign all aromatic resonances, similar to the sequential imino assignment. For larger RNA constructs, this is often not possible, either due to severe signal overlap or the absence of NOE contacts within the sequential assignment. In this case, additional experiments, which require ¹³C¹⁵N labeled RNA, can be recorded that can directly correlate imino and aromatic protons.

While A:H2 and C:H5 can be determined over NOE cross-peaks to imino and amino protons, this is typically (some non-canonical base-pairs are an exception to the rule) not possible for assignment of the U:H6 and the purine (A and G) H8. The remaining aromatic protons need to be assigned by the so called H1’-aromatic walk (Figure 20), where the NOEs between aromatic H6/H8 and ribose H1’ are exploited for an sequential assignment, as in a helical, WC base-paired stem every H1’ should give NOE signals with the aromatic H6/H8 of the same residue as well as with the residue in 3’direction. Respectively every stem aromatic H6/H8 produces a NOE to the H1’ of its own residue and the one in 5’ direction. A short sequence of signals might look like that: N(i-1)H6/8-N(i-1)H1’-N(i)H6/8-N(i)H1’-N(i+1)H6/8-N(i+1)H1’. Note also the H5 gives rise to NOE signals with H1’ protons, but typically only to the H1’ in 5’ direction and not to the own residue. Loop and bulge regions can interrupt the sequential walk as H1’ and aromatic protons might be further apart, but don’t have to, as seen in Figure 20.

2D ¹H¹³C HMBC

The main challenge in assigning the adenosine (A) in nucleic acids is distinguishing between the aromatic protons A:H2 and A:H8, as these are often very close together in the ¹D ¹H spectrum. The 2D ¹H¹³C HMBC experiment solves this problem by exploiting their different long-range couplings to the A:C4 carbon atom. The HMBC (heteronuclear multiple bond correlation) is a 2D NMR technique for determining long-range correlations between protons (¹H) and heteronuclei (such as ¹³C or ¹⁵N), typically over two and three bonds, which is essential for determining molecular connectivity. For A, the three bond coupling 3J(H2,C4) is around 12 Hz. In contrast, the 3J(H8,C4) coupling is only 5 Hz. The long-range interval in the HMBC pulse sequence is set so that the desired long-range coupling can be developed efficiently. For the two couplings involved (either 12 Hz and 5 Hz), a value of 60 ms is a common compromise. In the 2D ¹H¹³C HMBC spectra two cross peaks are observed in the spectrum along the A:C4 shift (¹³C dimension), which are separated in the ¹H-dimension by the chemical shift difference between A:H2 and A:H8. This experiment (Figure 21 shows an example) enables the unambiguous assignment of A:H2 and A:H8 in unlabeled nucleic acids, which is often difficult with standard NOESY experiments alone.

2D ¹H¹H ROESY

The functional RNA molecule is often particularly flexible and dynamic and described by an ensemble of interconverting structures which involves interactions with the solvent and may also feature low-populated and short-lived conformational states. The underlying environmental and structural changes are referred to as either chemical and conformational exchange and the heterogeneity results in additional signals. In this respect, the ROESY (Rotating-frame Overhauser Effect SpectroscopY) experiment is very useful and can help to detect the signals arising from exchange processes. Alike the 2D ¹H¹H NOESY experiment, the 2D ¹H¹H ROESY measures the through-space correlations between protons, but is carried out in a rotating frame with spin-locked magnetization. Practically this means that the NOE in the transverse plane (ROE) always has the opposite sign as the diagonal peaks, regardless of the molecular-size (which is an advantage to the NOESY, where NOE for medium-sized molecules is near zero). The diagonal peaks are typically phased to be positive and therefore the ROE cross-peaks appear as negative peaks. Conveniently, the exchange peaks have the same sign as the diagonal peaks (positive) which makes it simple to differentiate them from the true ROEs. Note that just like for NOESY, to a greater or lesser extent, also in the ROESY experiment care has to be taken as false unwanted and distorted cross peaks can be observed either due to spin-diffusion (relayed ROEs at long mixing times) or spin-coupling and spin-system correlations (so called COSY and TOCSY artifacts).

2D ¹H³¹P hetero-TOCSY-NOESY (HP-DINO)

The HP-DINO experiment effectively resolves peak overlap by incorporating a 31P dimensions to provide enhanced spectral dispersion and resolution, which is often a challenge in conventional RNA NMR due to the spectral crowding. The experiment establishes correlations between the phosphorus nucleus (available at 100% natural abundance) of the phosphate group and the adjacent ribose (H2’, H3’, H5’1, H5’2)protons and base pyrimidine H6 or purine H8 protons by application of a hetero-TOCSY-NOESY approach. To further resolve overlapping signals, a 3D version can be used. This experiment is especially useful (crucial) for the sequential assignment of RNA regions that are unstructured (flexible) and where traditional NOE-based methods fail. The transfer from the ribose protons to the coupled phosphorus atoms Pi and Pi+1 is established via a hetero-TOCSY transfer using the DIPSI2 sequence. This transfer is more efficient at low TOCSY power and long mixing times. Subsequently, a NOE mixing time leads to the correlation of the aromatic H6 or H8 protons with the ribose H2' and H3' protons. This allows a small RNA (maximum 25 nucleotides) to be sequentially assigned. The experiment is very insensitive, so the sample concentration should be at least 1 mM and the measurement should be performed in D2O solvent. The hetero-TOCSY transfer can be optimized beforehand in a separate experiment with HPDI. 

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