NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low‐Gamma Detection

NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope‐labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear‐detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope‐labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo‐enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear‐detected NMR experiments including 13C‐detected experiments for ribose assignment and amino groups, the CN‐spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15N‐detected band‐selective excitation short transient transverse‐relaxation‐optimized spectroscopy (BEST‐TROSY) experiment. © 2020 The Authors.


INTRODUCTION
RNAs are macromolecules that play indispensable roles in the biological processes of all living organisms. Besides the well-known RNAs that are involved in coding and decoding of genes (mRNAs, tRNAs, and rRNAs), there is a plethora of functional, diverse classes of RNAs that are essential in regulation and expression, such as riboswitches (Winkler, Nahvi, & Breaker, 2002) and RNA thermometers (Altuvia, Kornitzer, Teff, & Oppenheim, 1989), to only name a few. To exert their biological function, RNAs have to adopt certain defined conformations described by distinct secondary and tertiary structures.
Among other techniques, NMR spectroscopy is one of the most powerful methods for studying RNA's structure and conformational dynamics in solution. This statement is illustrated by the fact that ∼40% of all current RNA structures were determined by NMR techniques (Berman et al., 2000). Besides structural information, information on dynamics (Dethoff, Petzold, Chugh, Casiano-negroni, & Al-hashimi, 2012), the interactions with other RNAs (Davis et al., 2005), proteins (Carlomagno, 2014), ions (Butcher, Allain, & Feigon, 2000), and small ligands (Reining et al., 2013) can be characterized by NMR spectroscopy. However, for RNAs, the NMR technique currently sets a size limitation to molecules of up to ∼150 nucleotides (nt) using site-selective labeling strategies (Alvarado et al., 2014).
For all NMR studies, the preparation of milligram quantities of RNA in an isotopelabeled form is a prerequisite. Isotope labeling can include 15 N-only, 15 N, 13 C as well as 15 N, 13 C, 2 H, which are NMR active but non-radioactive isotopes enriched above their natural abundance of 0.3%, 1%, and 0.01%, respectively. While the incorporation of 13 C, 15 N isotopes has little if any effect on RNA sample stability, 2 H incorporation can change for example the thermal stability of an RNA of interest (Katz, Crespi, & Finkel, 1964). For the synthesis of isotope-labeled RNAs, both biochemical and chemical methods have been developed in the past. However, due to the restricted access to isotope-labeled building blocks required for chemical solid phase synthesis, biochemical synthesis that relies on the enzymatic in vitro transcription with DNA-dependent RNA polymerases has become the method of choice in many laboratories, also due to easier setup requirements.
The exact workflow of the biochemical synthesis of an RNA of interest is variable depending on the RNA to be investigated and the aim of the NMR spectroscopic study. In any case the synthesis may include the preparation of a set of required enzymes in Schnieders et al. house, the design and synthesis of a proper DNA template, and finally the purification of the RNA in order to make an appropriate NMR sample. When the characterization of an RNA that is >100 nt is planned, selective or segmental labeling strategies may be favorable due to fewer signals that are more readily identifiable. This labeling can be, for example, conducted enzymatically with ligation-based approaches as was demonstrated for segmentally labeled RNA (Duss, Lukavsky, & Allain, 2012;Tzakos, Easton, & Lukavsky, 2007).
NMR experiments that are based on the excitation and detection of 1 H-nuclei (in the following referred to as "protons") represent the current experimental gold standard for detailed NMR spectroscopic characterizations. This decision of using proton excitation and detection experiments results from the high gyromagnetic ratio and natural abundance of the 1 H-isotope that concomitantly lead to a high sensitivity. On the other side, a low chemical shift dispersion due to the chemical similarity in building blocks, the small number of protons in the nucleobases of RNA as well as a rapid solvent exchange of, for example, imino protons, put limitations to proton-based NMR spectroscopy. Heteronucleardetected NMR experiments represent valuable alternatives to overcome these restrictions as they exhibit a higher chemical shift dispersion and are not participating in the exchange processes mentioned.
Within this protocol, we provide a detailed guide on the preparation of isotope-labeled RNA for NMR studies by in vitro transcription with T7 RNA polymerase (Guillerez, Lopez, Proux, Launay, & Dreyfus, 2005). Because this process is very elaborate, we omit description of some standard procedures (e.g., how to conduct a denaturing polyacrylamide gel electrophoresis) at the same level of detail that is provided for the rest of our protocols. This is indicated in the appropriate place with references to other Current Protocols in Nucleic Acid Chemistry protocols that describe these standard methods in a detailed manner. Our description within this protocol covers the synthesis of the DNA template (Support Protocols 1 and 2), the optimization of the transcription reaction (Basic Protocol 1), the purification of the RNA (Basic Protocol 1, Alternate Protocols 1 and 2) as well as the expression and purification of several required enzymes within this process (Support Protocols 3 and 4). Furthermore, we provide a general guide on the ligation-based chemo-enzymatic synthesis of an RNA that is labeled or modified at a single position (Basic Protocol 2; Keyhani, Goldau, Blümler, Heckel, & Schwalbe, 2018). Here, the chemical synthesis of the required 3 ,5 -nucleoside bisphosphate (Support Protocol 5), the ligation reactions with T4 RNA Ligases 1 and 2 as well as the expression and purification of T4 RNA Ligase 2 (Support Protocol 6) are described. Moreover, we provide protocols for carrying out heteronuclear-detected NMR experiments in general (Support Protocols 7 and 8) and describe how to set-up 13 C-detected NMR experiments for the ribose assignment, namely (H)CC-total correlation spectroscopy (TOCSY), (H)CPC and (H)CPC-HCC-TOCSY experiments (Basic Protocol 3; Richter et al., 2010). Furthermore, a guide to set up a so-called CN-spin filter heteronuclear single quantum coherence (HSQC) experiment for the determination of the status of hydrogen bonding is provided (Basic Protocol 4; Fürtig et al., 2016). For the characterization of amino groups in RNA, we provide a protocol for the 13 C-detected C(N)H-heteronuclear doublequantum correlation (HDQC) experiment (Basic Protocol 5, Support Protocol 9) as well as the "amino"-nuclear Overhauser effect spectroscopy (NOESY) experiment (Basic Protocol 6; Schnieders et al., 2019). With the first set of experiments, all amino resonances can be detected as sharp NMR signals. The latter experiment brings amino groups in direct structural context and yields correlations that are not accessible with 1 H-detected experiments. Lastly, the application of the 15 N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiment for the imino groups is described (Basic Protocol 7; Schnieders et al., 2017).

Figure 1
Schematic outline for the preparation of isotope-labeled RNA samples for NMR purposes including alternate purification pathways and sets of heteronuclear NMR experiments. All Basic, Alternate, and Support Protocols illustrated in this scheme are described within the scope of this publication. the considerably faster option. Nonetheless, we sometimes experience a higher transcription efficiency when using plasmid DNA over PCR amplified templates, which might be attributed to the polymerases' ability to bind the DNA upstream of the T7 promoter and then slide along the strand until it reaches the promoter sequence. In the case of a PCR product this sequential scanning of the T7 RNAP is not possible, so only more direct encounters with the T7 promoter lead to transcription.

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Isotope labeling
The labeling strategy for an RNA depends strongly on the objective of the study. For initial assessment of secondary structures via imino proton pattern or NOESY-based assignment of small RNAs, an unlabeled high-concentration sample (>250 μM) is often already sufficient. Moreover, the use of commercially available isotope-labeled rNTPs for NMR-scale RNA production can be rather expensive and should therefore only be considered when transcription and purification protocols are established, and scientific questions cannot be addressed with 1

H experiments.
With increasing RNA size, even proton-based assignments become more difficult due to spectral overlap. Here, more sophisticated heteronuclear NMR experiments can provide additional information on the nucleotide identity. 15 N labeling of guanine and uracil residues for example will advance base-specific assignment of resonances through the use of 15 N HSQC experiments, while 13 C labeled nucleotides can aid in resolving resonances from aromatic and sugar protons via 13 C HSQC experiments. Especially with RNAs of increasing length, it is advisable to selectively label only one or two types of nucleotides at a time to dramatically decrease signal overlap. In the case of more advanced structural analyses, full 13 C and 15 N labeling, sometimes combined with deuteration, is indispensable.
Furthermore, if your project requires a position-selective rather than a uniform isotope labeling strategy, we recommend utilizing the chemo-enzymatic synthesis approach described in Basic Protocol 2. This method not only allows for an incorporation of a single labeled nucleotide into an otherwise unlabeled RNA but also gives rise to RNA constructs that combine differently labeled segments into one strand.

RNA purification
For purification of the RNA to generate NMR samples of sufficient concentration and purity, three major routes are available.
Basic Protocol 1 describes a widely used chromatographic approach, where anion exchange fast protein liquid chromatography (FPLC) and "ion-pair" RP-HPLC are used to successively remove the T7 RNAP, DNA template, residual rNTPs and RNA byproducts including ribozymes from the RNA of interest. After the chromatographic steps, the RNA is freeze dried and desalted before being precipitated with LiClO 4 /acetone. Finally, the RNA is folded into ideally one homogenous conformation and transferred into a suitable NMR buffer. Note that refolding conditions may vary with the RNA of interest and therefore have to be determined individually. This purification strategy is applicable to RNAs within a wide range of sizes and structures.
If it is not possible to completely separate the ribozymes from your RNA of interest via RP-HPLC or if an HPLC instrument is not available, we recommend switching to a preparative polyacrylamide gel electrophoresis approach, as shown in Alternate Protocol 1. Here, the RNAs are separated in a large-scale denaturing gel electrophoresis and the RNA of interest is extracted from the gel matrix afterwards.
The quickest purification route is described in Alternate Protocol 2, where merely components of low molecular weight, such as Mg(OAc) 2 and residual rNTPs, are removed from the reaction mix and the buffer is exchanged by repeated washing cycles using centrifugal concentrators (Helmling et al., 2015). In this protocol, the conformation adopted by the RNA during transcription is largely maintained, because no denaturing purification steps are performed. Nonetheless, this method should only be utilized when the transcription produces a single RNA product, as byproduct RNAs like ribozymes are not separated from the RNA of interest. In this case, 3 -end homogeneity of the RNA is Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry achieved through the use of 2 -methoxy modified primers during PCR. A PCR product carrying the 2 -methoxy modification at the 3 end has shown to significantly increase product homogeneity for run-off transcription (Helmling et al., 2015). We recommend using this purification strategy for high throughput RNA structure analysis rather than titration experiments, because the presence of remaining T7 RNAP might interfere with ligand binding.

NMR buffer composition
RNA NMR samples are usually prepared in a buffer containing as few protons as possible (to avoid using deuterated buffer agents) and exhibiting a slightly acidic pH value (to guarantee long term stability of the sample and to reduce solvent exchange of labile protons). Typically, we use the following buffer composition as a starting point: 50 mM KCl, 25 mM K 2 HPO 4 /KH 2 PO 4 , and 5% (v/v) D 2 O at pH 6.2. Depending on the nature of your experiments and the investigated RNA, bivalent cations such as Mg 2+ can be added to stabilize the RNA's structure. As a reference substance for 1 H spectra we recommend sodium trimethylsilylpropanesulfonate (DSS), as its proton chemical shift is neither temperature nor pH dependent. Further adjustments in salt and buffer composition or pH might be required for individual studies, especially if the RNA is investigated as a part of an RNA protein complex.

PREPARATION OF ISOTOPE-LABELED RNA SAMPLES WITH IN VITRO TRANSCRIPTION USING T7 RNAP, DEAE CHROMATOGRAPHY, AND RP-HPLC PURIFICATION
The analysis of RNAs via NMR spectroscopy requires preparation of a sample with sufficient amount of pure RNA. Typically, samples between 50 and 500 μM in 300 μl buffer volume are used, but higher sample concentrations can be advantageous provided correct folding conditions can be established. In the following, we describe a standard protocol for the preparation of isotope-labeled RNA for NMR applications using the T7 RNAP. This polymerase can be purchased or prepared in house; the respective instructions for the preparation are described in Support Protocol 3. In a first step, the DNA template from which the RNA will be transcribed is amplified. For the amplification two methods are available. The first requires a DNA plasmid, which is amplified in cells, linearized, and subsequently purified (Support Protocol 1). A second approach uses a DNA template produced by solid phase synthesis in combination with a PCR using Phusion ® High-Fidelity DNA polymerase (Support Protocol 2). After DNA amplification, test-transcriptions are used to optimize reaction conditions to obtain the highest yield of the target RNA. Under these conditions the preparative transcription reaction can be performed and purified via diethylaminoethanol (DEAE) and RP-HPLC chromatography. Alternative procedures for the purification are shown in Alternate Protocols 1 and 2.    28. Centrifuge at 10,000 × g and 4°C for 30-60 min.

Materials
Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry You may centrifuge with higher speed if possible. Stick to the speed limit specified in the device manual of the reaction tube.
29. Remove supernatant and reconstitute pellet in ddH 2 O or NMR buffer depending on the folding method.
If you use a 20-ml centrifugal concentrator, you may reconstitute in a volume of ∼10 ml.

Folding and buffer exchange
The folding protocol might need to be adapted for each individual RNA construct. Therefore, some common folding pathways are introduced here. The RNA can be folded in water or in NMR buffer as well as at a high or low RNA concentration. Take  32. Fold RNA sample into a single conformation. Folding can either occur now or in step 40.
Do not freeze the sample after folding.
33. Prepare a centrifugal concentrator as described in the device manual.
Choose a molecular weight (MW) cutoff, which is about a third of the size of the target RNA. This way the RNA construct will not pass through the filter.
34. Transfer RNA sample into NMR buffer using the centrifugal concentrator and centrifuge at the recommended speed. 45. Seal the Shigemi tube with sealing film (e.g., Parafilm) and label the tube. Store sample at 4°C.

Sealing stabilizes the insert.
Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry 46. Perform essential NMR experiments (e.g., 1 H 1D, 1 H 2D NOESY, 1 H, for the assignment of the RNA. For basic NMR experiments for RNA, we refer to the review article: NMR spectroscopy of RNA (Fürtig, Richter, Wöhnert, & Schwalbe, 2003). For more sophisticated heteronuclear experiments see Basic Protocols 3-7.

PURIFICATION OF ISOTOPE-LABELED RNA FROM IN VITRO TRANSCRIPTION WITH PREPARATIVE PAGE
Alternate Protocol 1 describes a second approach for the purification of isotope-labeled RNAs from in vitro transcription. This method utilizes preparative PAGE instead of RP-HPLC and is therefore an alternative if no HPLC device is available or if the device does not separate the target RNA product sufficiently from byproducts (Current Protocols article: Hengesbach et al., 2008;Petrov, Wu, Puglisi, & Puglisi, 2013). This protocol starts after the successful in vitro transcription of the target RNA and yields a pure NMR sample. This method is not suitable if the NMR sample has to be free of any acrylamide impurities, which might be eluted together with RNA from the preparative PAGE. If acrylamide affects the results, an HPLC run to separate it from the RNA is inevitable. 2. Transcribe RNA as described in Basic Protocol 1, steps 1-15. 3. Determine amount of RNA and sample volume which has to be purified and check which PAGE size fits the RNA sample. Take into account the additional formamide (see step 9 below) and the required separation efficiency, e.g., the size difference between product bands. 10. Set gel into the PAGE device. If using a maxi gel, pre-run gel for 30 min.
Rinse wells thoroughly with running buffer directly after setting the gel and prior to loading the gel to prevent sedimentation of urea.
11. Add 20 μl dye-containing loading buffer in one spare gel pocket to determine gelrunning progress.
12. Load denatured RNA sample into free gel pockets. 14. Remove gel from glass plates and place it onto a sterile surface. 17. Cut the desired band out of the gel and then cut into small pieces.
18. Mix gel pieces with 10-20 ml elution buffer and press the mix through a syringe.
This process increases the gel surface for better elution.
19. Shake gel pieces at 4°-25°C overnight to elute RNA into the elution buffer.
Optionally freeze and thaw gel slices to disrupt the gel matrix and to increase the elution efficiency of the RNA. After the overnight elution, incubation at 65°C for 15 min and additional shaking for 30 min at 1,300 rpm can help to increase the amount of eluted RNA.
20. Filter elution buffer with a sterile filter (0.2 μm) and keep gel pieces for further elution.
In some cases, the elution of RNA is difficult and it may not be completed overnight. Therefore, add fresh 0.3 M NaOAc to the separated gel pieces and repeat steps 19 and 20.
21. Add two-and-a-half to four volumes of ice-cold absolute ethanol to precipitate RNA and place mixture at −20°C overnight.
Incubation at −80°C for 2 hr may also be sufficient.
You may centrifuge at higher speed if possible. Stick to the speed limit specified in the device manual of the reaction tube.

Remove supernatant and keep it for further precipitation.
In some cases, the RNA does not precipitate entirely. In this case, incubation and centrifugation may be repeated.

PURIFICATION OF ISOTOPE-LABELED RNA SAMPLES FROM IN VITRO TRANSCRIPTION VIA CENTRIFUGAL CONCENTRATION
This Alternate Protocol 2 describes a fast purification method for isotope-labeled RNAs from in vitro transcription using a centrifugal concentrator (see Fig. 4). It is the quickest purification method among the purification strategies described within this protocol. However, this method should only be applied when the transcription produces a single RNA product, as byproduct RNAs or other transcribed RNAs such as ribozymes are not separated. Therefore, it is important to generate 3 -end homogeneity of the transcribed RNA by using PCR DNA templates with 2 -O-methyl-modifications at the last two nucleotides of the 5 end (Helmling et al., 2015;Kao, Zheng, & Rüdisser, 1999;Support Protocol 2). We also advise that this purification procedure not be used for NMR titration experiments, because remaining enzymes in solution might interfere with ligand binding.

Materials
Transcribed RNA (see Basic Protocol Choose a MW cutoff, which will be about a third of the size of your target RNA. This way the RNA will not pass through the filter.

2.
Remove precipitated magnesium pyrophosphate by centrifuging the transcription mixture at 3,000 × g for 5 min at room temperature. Then, transfer supernatant to the centrifugal concentrator.
4. Concentrate solution to 1.0-0.3 ml and wash with 60-160 ml NMR buffer depending on the transcription scale.
Mix sample carefully between the steps with a pipet, without touching the membrane. Stick to the speed limit specified by the manufacturer otherwise the membrane could be damaged. The temperature inside the centrifuge should be in a range of 4°-25°C, depending on type and stability of the target RNA. Higher temperature will increase the flowthrough.
5. Analyze flowthrough and the solution above the membrane (target RNA) by UV/vis absorption spectroscopy and analytical denaturing PAGE to ensure that no RNA has passed through the membrane of the centrifugal concentrator.
6. In the last washing step, concentrate solution to 200-300 μl and remove RNA from the centrifugal concentrator with a pipet.
The RNA can stick to the membrane and therefore has to be removed vigorously with a pipet. It is recommended that the RNA solution is pipetted along the inside of the membrane and that the membrane is washed with a small additional amount of NMR buffer (∼50 μl) after removal.

Determine RNA concentration via UV/vis absorption spectroscopy.
Take into account MgCl 2 , D 2 O, and DSS which will be added afterwards and will dilute the sample.
8. Proceed with folding and NMR sample preparation as described in Basic Protocol 1, steps 30-46.

PREPARATION OF DNA TEMPLATE FROM PLASMID
Every DNA-dependent RNA polymerase used in an in vitro transcription requires a DNA template from which the sequence can be read and an RNA strand can be synthesized. A commonly used template is a linearized DNA plasmid encoding the RNA transcription cassette. With the linearization directly downstream of the transcription cassette no transcription terminator is required but instead the polymerase will create run-off transcripts (Diaz, Rong, McAllister, & Durbin, 1996). In this Support Protocol 1, we provide step-by-step instructions on how to prepare a plasmid DNA template.

Transformation and amplification
This protocol assumes the previous successful insertion of the RNA transcription cassette into a suitable bacterial plasmid. Any plasmid with a (class III) T7 promoter sequence and ampicillin resistance should be suitable.
1. Perform plasmid transformation into competent DH5α E. coli cells according to manufacturer instructions.
Add ampicillin directly prior to use.
3. Pick an isolated clone from the LB-agar plate (e.g., with a sterile pipet tip) and transfer into the 5-ml culture flask.
You can also inoculate with a previously prepared glycerol stock. For that, either briefly thaw the glycerol stock and add 20-50 μl to your 5-ml culture or transfer part of the frozen glycerol stock directly using, e.g., a sterile pipet tip.
4. Grow cells ∼4-6 hr at 37°C and 120 rpm before transferring the 5-ml starter culture into 50 ml starter culture. Incubate the 50-ml culture at 37°C and 120 rpm overnight.
5. Measure OD 600 of the starter culture and transfer the appropriate volume into the main culture to adjust the initial OD 600 to ∼0.1.
For long-term storage and fast inoculation, we recommend preparing a glycerol stock. Therefore, during the exponential phase of cell growth, take 0.5 ml cell culture and mix it thoroughly with 0.5 ml 50% glycerol. Freeze the cell suspension in liquid nitrogen and store at −80°C. Use the glycerol stock for future inoculations.
7. Centrifuge culture medium at 4,000 × g for 15 min and 4°C to harvest cells.
8. Isolate plasmid DNA according to the instructions of a plasmid DNA purification kit.
Linearization 9. Perform a restriction digestion on an analytical scale of 50 μl. For this, prepare a reaction mix according to the protocol in Table 4.
While the total volume of enzyme needed depends on the concentration set by the manufacturer, make sure that the enzyme volume does not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol. If necessary, increase the volume of the reaction mix.

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Current Protocols in Nucleic Acid Chemistry 11. Incubate digestion reaction 1 hr at the temperature optimal for the restriction enzyme.
Check enzyme specifications; usually it is 25°C or 37°C.
12. Verify linearization efficiency with a gel electrophoresis using a 1% agarose gel. Apply ∼150 ng of DNA per sample.
13. After successful test linearization, prepare a preparative scale digestion. Calculate the amount of enzyme required to digest the total amount of plasmid (e.g., 1 U/μg DNA). Consult the manufacturer specifications and protocols of your individual enzyme if necessary. The enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.

Phenol-chloroform-isoamyl alcohol extraction
14. Add one volume of PCI solution to the digestion reaction solution and mix vigorously before centrifuging for 10 min at 9,000 × g at 4°C.
15. Carefully transfer aqueous phase (top phase) into a new tube and repeat extraction again with one volume of fresh PCI. Transfer aqueous phase again into a new tube.
16. Add one volume of chloroform to the aqueous phase. Mix thoroughly and centrifuge for 10 min at 9,000 × g. Carefully transfer aqueous phase (top phase) into a new tube.
In this chloroform step, residual traces of phenol are removed.
17. Add one-tenth volume of 3 M NaOAc to the aqueous phase, mix, and precipitate with 0.7 volumes of 2-propanol or 2.5 volumes of absolute ethanol. Incubate at −20°C for 1 hr.
20. Determine concentration via UV/vis absorption at 260 nm and perform an agarose gel electrophoresis with linearized plasmid and undigested plasmid as a control.
See Figure 5 for reference. The linearized plasmid should run differently (usually higher) in the agarose gel than the supercoiled plasmid.

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Current Protocols in Nucleic Acid Chemistry For both plasmids the typical band shift from supercoiled to linear conformation can be observed. Note that only linearized DNA accurately matches the band height of a reference ladder.

PREPARATION OF PCR DNA AS TEMPLATE
PCR is a fast and convenient method for the amplification of DNAs (Mullis et al., 1986). The template DNA for this method is usually purchased or synthesized in house by solid phase synthesis. The correct design of this template DNA is described in Strategic Planning. If 3 homogeneity is required, using 2 -O-methyl modified reverse primers is an alternative solution to the application of ribozymes. The methoxy group at the 5 end of the PCR template DNA stops the transcription because it destabilizes the complex between the polymerase and the template, preventing non-templated RNA synthesis (Kao et al., 1999).
For a maximum amount of pure PCR product, reaction conditions such as the primer, template, and MgCl 2 concentrations have to be optimized. Furthermore, the annealing temperature has to be optimized in order to obtain a high yield of product DNA. All optimizations and the procedure for a successful PCR are described within this support protocol.

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Current Protocols in Nucleic Acid Chemistry Figure 6 6% native PAGE for DNA of PCR annealing temperature, primer, and buffer optimization. Methoxy primer yields less product. At higher annealing temperatures the amount of product increases. The effect of GC buffer instead of HF buffer is negligible. 3. Reconstitute lyophilized primers with ddH 2 O to obtain a concentration of 100 μM.
4. Check the following optimization steps on a native PAGE for DNA or an agarose gel.
A native PAGE for DNA will reveal sharper bands than an agarose gel. However, for larger DNA constructs (>1 kbp) an agarose gel is more suitable, as the DNA migrates faster through the gel matrix.

5.
Optimize annealing temperature and the number of cycles (Fig. 6). Optimize elongation time according to the template length. An exemplary program is shown in Table 5.
3°C above the primer melting temperature is a good choice for an initial annealing temperature. Optimize in a range of ±10°C around the primer melting temperatures. For the number of cycles, use up to 30 cycles.
7. Optimize PCR template DNA concentration to maximize the yield of target DNA and to avoid additional byproduct (see Troubleshooting). Try 1-3 ng/μl template.
8. Optimize DMSO concentration in case of byproducts to facilitate the binding of the primers. Use up to 3% DMSO.

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Current Protocols in Nucleic Acid Chemistry  9. Determine amount of DNA required for the preparative transcription reaction.
10. Set a master mix with all reagents in a 2-ml reaction tube as shown in the pipetting scheme (Table 6) and distribute to PCR reaction tubes. Each tube should contain 50 μl reaction mixture.
11. Perform a PCR with the optimized conditions.
12. Combine samples from the individual PCR reaction tubes and run an analytical agarose gel electrophoresis or a native PAGE for DNA to confirm a successful reaction.
13. Use PCR product directly for transcription without any purification steps in case of single pure product.
If byproducts are formed but are not transcribed, the PCR product can be used for further transcription without purification. Store at −20°C if not used directly.

PREPARATION OF T7 RNA POLYMERASE (T7 RNAP)
Although T7 RNAP is commercially available, purchasing the amount required for an in vitro RNA transcription in NMR scale can be quite expensive. This support protocol provides a cost-saving and straightforward alternative to the commercial product. Furthermore, we recommend using the P266L mutant of T7 RNAP since it significantly Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry improves in vitro transcription, particularly from templates carrying unfavorable initial sequences (Guillerez et al., 2005). 3. Measure OD 600 of the starter culture and transfer the appropriate volume into the main culture to adjust initial OD 600 to ∼0.1.

Materials
If you are planning to add antifoam emulsion to the main culture to prevent foam formation, consider that antifoam will increase the optical density of the culture medium and should be added to the main culture prior to any blank measurements.
4. Incubate main culture at 37°C and 120 rpm until OD 600 of 0.6-0.8 for LB medium or 1-2 for TB medium is reached.
Due to the higher maximum cell density in TB medium, the ideal induction point within the exponential growth is at a higher OD 600 value. OD 600 of 0.6-0.8 is usually reached within 3-4 hr, OD 600 of 1-2 within 4-5 hr.

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Current Protocols in Nucleic Acid Chemistry 5. Add IPTG to a final concentration of 0.5 mM to induce protein expression.
6. Grow cells at 37°C until OD 600 of 10-15 is reached. This usually takes 3 hr more.
7. Centrifuge culture medium at 4,000 × g and 4°C for 15 min to harvest cell pellet.

Purification
Perform all purification steps at 4°C.
9. Lyse cell suspension via high-pressure homogenization. 13. Load protein solution onto the column.
14. Wash column with ten CV T7 RNAP buffer A.
17. Apply a gradient of 100% T7 RNAP buffer B over 100 ml and collect eluate in fractions of 5 ml (see Fig. 8A for reference).
18. Run a 12% SDS-PAGE of all fractions and pool fractions that contain T7 RNAP (see Fig. 9A for reference).
20. Load T7 RNAP onto the size exclusion column.
The maximum volume to be loaded depends on the column size and specifications. Check the manufacturer's recommendations to ensure optimal separation. If necessary, reduce the volume of the enzyme solution via centrifugal concentration.
21. Elute with one CV of 2.5× T7 RNAP buffer C and collect eluate in fractions of 5 ml (see Fig. 8B

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PREPARATION OF YEAST INORGANIC PYROPHOSPHATASE (YIPP)
The RNA yield of a transcription is strongly dependent on the Mg 2+ ion concentration in the reaction mixture. The formation of pyrophosphate leads to a precipitation of magnesium pyrophosphate concomitantly decreasing the concentration of Mg 2+ ions in the solution. Because the folding of ribozymes and the activity of the T7 RNAP is dependent on Mg 2+ ions, their concentration should be kept stable during the transcription (Karlsson, Baronti, & Petzold, 2020). To achieve this stability, 9.6 μg/ml YIPP can be used to decompose pyrophosphate to monophosphate, thereby preventing the removal of magnesium by precipitation (Kunitz, 1952). Shaking incubator UV/Vis spectrophotometer (NanoDrop One/One; Thermo Fisher Scientific) Centrifuge (Megafuge 8R; Thermo Fisher Scientific) High-pressure homogenization (e.g; Microfluidizer ® M-110 P; Microfluidics) FPLC system 5-ml Ni-NTA affinity column (e.g., GE HisTrap HP 5 ml column) SDS casting chamber and glass plates (XCell SureLock Mini-Cell Electrophoresis System) Preparative size exclusion column (e.g., GE HiLoad 26/600 Superdex 75 pg gel filtration column) Freezer (−20°C/−80°C)

Expression and cell harvest
Based on previous expressions, 1 L LB medium yields between 35-40 mg of purified protein.
Add ampicillin directly prior to use.
3. Measure OD 600 of the starter culture and transfer the appropriate volume into the main culture to adjust initial OD 600 to ∼0.1.

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Current Protocols in Nucleic Acid Chemistry OD 600 of 0.6-0.8 is usually reached within 3-4 hr.
5. Add a final concentration of 1 mM IPTG to induce protein expression.
6. Grow cells for another 5 hr at 37°C.
7. Centrifuge culture medium at 4,000 × g for 30 min and 4°C to harvest cell pellet.

Purification
To maintain the thermostability of the enzyme, it is recommended that all purification steps are performed at 4°C.
9. Lyse cell suspension via high-pressure homogenization. 11. Equilibrate a 5-ml Ni-NTA column with ten CV of YIPP buffer A.
12. Load protein solution onto the column.
13. Wash column with ten CV of YIPP buffer A.
15. Run a 15% SDS-PAGE of all fractions and pool fractions that contain YIPP (33.2 kDa; see Support Protocol 3, Fig. 9A for reference).
The following size exclusion step is not compulsory but recommended.
16. Equilibrate size exclusion column with 1.5 CV of YIPP buffer C.
17. Load YIPP solution onto the size exclusion column.
The maximum volume to be loaded depends on the column's size and specifications. Check the manufacturer's recommendations to ensure optimal separation. If necessary, reduce the volume of the enzyme solution via centrifugal concentration.
18. Elute with one CV of YIPP buffer C and collect eluate in fractions of 5 ml (see Support Protocol 3, Fig. 8B

PREPARATION OF SITE-SPECIFIC LABELED RNAS USING A CHEMO-ENZYMATIC SYNTHESIS
The NMR spectroscopic characterization of increasingly long RNAs is hampered by an increased resonance overlap. This problem can potentially be avoided by site-specific labeling methods. However, the most commonly used method for these purposes, solid phase synthesis, is limited to ∼50 nt (Jud & Micura, 2017). Herein, we provide a protocol for the chemo-enzymatic synthesis of RNAs that contain a single modified/labeled nucleotide at a desired position without any size limitation. By the site-specific incorporation of 13 C, 15 N labeled or unnatural modified nucleosides, such as 19 F or 13 C-methoxy groups, the signal abundance is dramatically reduced and spectra with single signals can be obtained (Keyhani et al., 2018). Using this method, it is even possible to incorporate modified nucleosides carrying sterically demanding modifications such as photoremovable protecting groups (e.g., ortho-nitrophenylethyl) or photoswitches (e.g., azobenzene).
The site-specific incorporation of a modified nucleotide into an RNA involves three enzymatic steps (enzymatic pathway in Fig. 10). The first step is a 3 extension of the acceptor RNA strand (RNA 1) with the modified nucleoside 3 ,5 -bisphosphate (for synthesis see Support Protocol 5) using T4 RNA Ligase 1 (T4 Rnl1). Further extensions are blocked by the phosphate group at the 3 position of the modified nucleotide. The ligation mixture can be either purified by RP-HPLC or the non-ligated RNA can be oxidized by NaIO 4 to remove it from the enzymatic pathway. The phosphate group at the 3 position is removed by a phosphatase and the RNA is (splint) ligated with the 5 -phosphorylated donor RNA strand (RNA 2) in presence of a DNA splint by T4 RNA Ligase 2 (T4 Rnl2; for enzyme preparation see Support Protocol 6). The RNA is purified, e.g., by RP-HPLC. The enzymes can be purchased commercially, however, we recommend preparing T4 Rnl2 in house due to the significant cost factor and observed higher ligation efficiency as described in Support Protocol 6.

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Current Protocols in Nucleic Acid Chemistry Figure 10 Schematic overview of the enzymatic site-specific incorporation of modified nucleotides employing periodate oxidation. Through the oxidation, the non-ligated RNA (after ligation) is removed from the enzymatic pathway. No byproducts remain after the splinted ligation. The acceptor RNA strand (RNA 1) requires an OH group at the 3 end and the donor RNA strand (RNA 2) requires a phosphate group at the 5 end for the chemo-enzymatic incorporation. 4. Perform a 5 -phosphorylation of the donor RNA strand (RNA 2), e.g., with T4 polynucleotide kinase (T4 PNK). Follow the manufacturers protocol.
As an alternative, a short (<30 nt) 5 -phosphorylated RNA 2 strand can be purchased commercially.
5. We recommend purchase of T4 RNA Ligase 1 (T4 Rnl1) and the phosphatase commercially and preparation of T4 RNA Ligase 2 (T4 Rnl2) in house as described in Support Protocol 6.

extension of the acceptor RNA (RNA 1)
6. Prepare ligation mixture that includes all components from Table 7. Incubate ligation mixture at 37°C and 300 rpm overnight. In addition, prepare a negative control without T4 Rnl1.
The negative control is necessary to track whether the non-ligated RNA is removed from the enzymatic pathway during the oxidation (step 9). Therefore, it will be treated as all the other samples in the following steps and will be referred to as "step 1 negative control" over the course of this protocol. The ligation efficiency depends on the type of the modification. Scaling up the reaction may lead to a decreased ligation efficiency. It is recommended that several batches are prepared in parallel instead of one large batch.

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Current Protocols in Nucleic Acid Chemistry  8. Add one volume of PCI solution to the reaction solution and mix vigorously. Centrifuge 10 min at 10,000 × g and 4°C.
9. Carefully transfer aqueous phase (upper phase) into a new tube and repeat extraction with one volume of fresh PCI solution.
Two extractions are typically sufficient.
10. Add one volume of chloroform to the aqueous phase (to remove phenol traces) and mix thoroughly. Centrifuge 10 min at 10,000 × g and 4°C. Carefully transfer aqueous phase (upper phase) into a fresh reaction tube.
11. Prepare centrifugal concentrator as described in the device manual.
Choose a MW cutoff which is about a third of the size of your target RNA. This way the RNA will not pass through the membrane.
12. Transfer RNA in the centrifugal concentrator and centrifuge at the recommended speed to remove ligation buffer.
ATP and DTT in the ligation buffer will prevent the oxidation by NaIO 4 . Repeated ethanol precipitations (approximately three) could also be sufficient to remove the ligation buffer.
13. Perform oxidation with NaIO 4 to remove non-ligated RNA from the enzymatic pathway. Oxidize the "step 1 negative control." The pipetting scheme for the oxidation is demonstrated in Table 8. Incubate reaction 1 hr and 300 rpm at room temperature (23°C).
NaIO 4 is sensitive to light. Therefore, it is recommended that the reaction mixture is protected from light, e.g., with aluminum foil.

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Current Protocols in Nucleic Acid Chemistry 19. Analyze RNA by UV/vis absorption spectroscopy and analytical denaturing PAGE.
Take into account that the UV/vis absorption may be higher due to leftover ATP and nucleoside 3 ,5 -bisphosphate, which may not have been entirely removed in step 3.
We recommend the following protocol with the pipetting scheme given in Table 9. Incubate at 37°C for a minimum of 6 hr. The dephosphorylation should lead to a nearly quantitative turn over. Scale up of the reaction may lead to a decrease in ligation efficiency. Therefore, it is advisable to prepare several batches in parallel instead of one large batch.
21. Perform PCI extraction to remove phosphatase as described in steps 3-6.

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Current Protocols in Nucleic Acid Chemistry

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Current Protocols in Nucleic Acid Chemistry Figure 11 20% denaturing PAGE shows the first ligation step (1.lig), the dephosphorylation of the RNA (dp), the second splint ligation step (2.lig), and the purified target RNA (p). The 13-mer 1.lig RNA moves faster in the gel electrophoresis because of the additional negative charge of the 3 -end phosphate group.

Figure 12
Chromatogram of the preparative RP-HPLC purification of a splint ligated RNA, which was prepared via the chemo-enzymatic synthesis approach.
40. Fold RNA into a single conformation and prepare NMR sample as described in Basic Protocol 1, steps 30-46.

SYNTHESIS OF MODIFIED NUCLEOSIDE 3 ,5 -BISPHOSPHATES
The modified nucleosides require a phosphate group at the 3 ,5 positions for site-specific incorporation into the RNA as described in Basic Protocol 2. This support protocol describes the synthesis of a modified nucleoside 3 ,5 -bisphosphate (Barrio et al., 1978;Fig. 13).

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Current Protocols in Nucleic Acid Chemistry

7.
After complete conversion of the starting material (3-6 hr), slowly add ice (two to three pieces) to the reaction mixture.
In this step, the remaining diphosphoryl chloride is hydrolyzed. The ice cools the exothermic reaction, when 1.0 TEAB (pH 8) buffer is added.

In case of precipitation, add water to reconstitute precipitate. A smaller reaction volume facilitates the RP-HPLC purification.
10. Purify with RP-HPLC at room temperature.
Possible byproducts of the synthesis are mono-or triphosphates, which may not be separated by RP-HPLC.
11. Determine fractions containing product by mass spectrometry and pool them.

You may use a lyophilizer or centrifugal concentrator.
Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry 13. Repeat step 12 seven to ten times with an analytical amount of the substance that is analyzed via NMR spectroscopy. Ligation yield is not affected by triethylamine. To remove triethylamine, three to five cycles of co-evaporation are sufficient.
The removal of triethylamine is necessary for the NMR spectroscopic characterization.
15. Characterize product by mass spectrometry and NMR spectroscopy ( 1 H-1D, 13 C-1D, 31 P-1D, 1 Ho & Shuman, 2002) is used for Basic Protocol 2 and is commercially available. If purchasing, make sure the ligase has 2 ,3 cycP esterase activity. However, purchasing the ligase is very costly. Therefore, this straightforward protocol provides an alternative for an in-house preparation of the enzyme. Our tests showed that the in-house produced T4 Rnl2 is even more efficient than the purchased T4 Rnl2 (Fig. 15).

Expression and cell harvest
1. Prepare 200 ml starter culture and 1 L main culture containing LB medium and 100 μg/ml ampicillin.
Add ampicillin directly prior to use.
2. Inoculate starter culture with a glycerol stock of BL21 DE3 E. coli cells containing the pET-RNL2 plasmid and incubate at 37°C and 160 rpm overnight.
3. Measure OD 600 of the starter culture and transfer the appropriate volume into the main culture to adjust the initial OD 600 to ∼0.1.

4.
Incubate main culture at 37°C and 130 rpm until OD 600 of 0.8 is reached.
If you are planning to add antifoam emulsion to the main culture to prevent foam formation, consider that antifoam will increase the optical density of the culture medium and should be added to the main culture prior to any blank measurements.
5. Add IPTG to a final concentration of 1 mM to induce protein expression.
6. Grow cells at 37°C until OD 600 of 2 is reached. This usually takes 6 hr in total.
7. Centrifuge culture medium at 4,000 × g and 4°C for 15 min to harvest cell pellet.

Purification
Perform all purification steps at 4°C.

Heteronuclear-detected NMR experiments for RNA
The following protocols describe the implementation of several 13 C-and 15 N-detected NMR experiments for RNA. These include experiments for chemical shift assignment of 1 H and 13 C atoms of the ribose ring, the CN-spin filter HSQC experiment for imino and the C(N)H-HDQC as well as the "amino"-NOESY for amino groups. The selection is complemented with a protocol for execution of the 15 N-detected BEST-TROSY experiment. For all experiments, Topspin parameter sets are provided, which were generated at 600 MHz and need to be adapted using the command "paracon" at the respective spectrometer. Also note that the provided pulse sequences are not necessarily compatible with the "getprosol" command.

SETUP OF NMR SPECTROMETER FOR HETERONUCLEAR-DETECTED NMR EXPERIMENTS
This support protocol describes the general procedure, which should be conducted in advance every time when the acquisition of heteronuclear-detected NMR experiments is planned. Consequently, it is to be applied before the experiments described in Basic Protocols 3-7 are conducted.

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Current Protocols in Nucleic Acid Chemistry NMR spectrometer (equipped preferably with a z-axis gradient 13 C, 15  We usually determine the 1

H pulse automatically or in a simple one-pulse 1D experiment by conducting a 360°pulse and varying the pulse length until the remaining signal is at the zero crossing. Set the 1 H carrier frequency on the water resonance (4.7 ppm).
e. Determine 13 C and 15 N 90°pulses and set the carrier frequency on-resonant.
The determination of the correct 90°pulse length for 13 C and 15 N is based on SOFAST HMQC (Schanda & Brutscher, 2005). Read in parameter set pulse_calib13C with pulse sequence decp90_sfhmqc.ric ( 13 C)  3. Check the quality of your NMR sample and the shim by recording reference experiments like 1 H-1D and 13 C/ 15 N-HSQC experiments.

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Current Protocols in Nucleic Acid Chemistry and double IPAP (DIPAP) sequences, we will describe the general procedure on how to set them up in the following protocol. This Support Protocol is thus relevant for Basic Protocols 3-6.

Materials
≥0.5 mM RNA sample (300 μl) of interest (see Basic Protocols 1 and 2; Alternate Protocols 1 and 2) NMR spectrometer equipped preferably with a z-axis gradient 13 C, 15 Fiala & Sklenár, 2007. b. What is the size of the 1 J CC coupling constants (Fiala & Sklenár, 2007)? c. What is the chemical shift of the carbon nucleus that is detected ( 13 C on ) and what is/are the chemical shift(s) of the one(s) which is/are decoupled ( 13 C off / 13 C off ; Fiala & Sklenár, 2007)?
Based on this information, the IPAP parameters will be determined in step 4.

2.
If not already the case, include IPAP/DIPAP sequence in the pulse sequence of interest either during the last insensitive nuclei enhancement by polarization transfer (INEPT) step (IPAP, Fig. 16A; DIPAP, Fig. 16C) or as a separate element.
3. Set up NMR spectrometer according to Support Protocol 7.
4. Choose pulse shapes, carrier frequencies, and pulse length for on-and off-resonant carbon pulses. Those parameters are summarized in Table 13 (IPAP) and Table 14 (DIPAP) for all possible carbon atoms in RNA.
Note that pulse lengths and offsets need to be adjusted according to the magnetic field strength, when measuring at a field strength different from 800 MHz.
If there are different 1 J CC coupling constants present in nucleotides of interest, e.g., 1 J C4C5 (C) = 55 Hz and 1 J C6C5 (A) = 75 Hz, the IPAP time can be calculated using an intermediate coupling constant (e.g., T = 3.9 ms in Table 13). Note that this can only be applied if coupling constants and the chemical shifts of 13 C on and 13 C off are in the same range.
6. Record the experiment of interest. Conduct the experiment with double/quadruple of the desired points and half/quarter of the desired transients for IPAP/DIPAP, respectively.
The reason for this is that the experiments are recorded in an interleaved manner, which divides the number of points by two/four while processing doubles/quadruples the signal intensity.
7. Processing: As experiments with IPAP/DIPAP are recorded in an interleaved manner and the decoupling is based on a combination of in-phase and anti-phase doublets/quadruplets ( Fig. 16B and D), the in-phase (IP) and anti-phase (AP) spectra need to be split first. After splitting, they need to be processed and combined. Shift the resulting spectrum by 0.5 × 1 J CC to the correct chemical shift.  Geen & Freeman, 1991). If not indicated otherwise, pulses are applied along the x-axis. Delays are = 1/2J CN and T = 1/4J CC . The receiver phase ϕ rec has to be set individually depending on the phase cycling in the respective experiment. Decoupling for 15 N nuclei in this case can be achieved using state-of-the-art decoupling schemes like asynchronous (Shaka, Barker, & Freeman, 1985). (B) IPAP decoupling exemplary for a CN-HSQC experiment with 1 IP-(upper left) and 2 AP-spectrum (lower left), 1+2 the summation of IP-and AP-spectrum (upper right) and 1+2 shifted by 0.5*J CC (lower right). 1D traces are included in each spectrum. (C) Pulse scheme for a DIPAP decoupling during a CP-transfer. Narrow filled bars represent rectangular 90°pulses while wide-open bars illustrate rectangular 180°pulses. Semielliptic unfilled shapes represent selective 180°pulses. If not indicated otherwise, pulses are applied along the x-axis. Delays are = 1/2J CP , T = 1/4J CC while in this case the 1 J CC coupling constants to the two neighboring carbon atoms are of the same size. Pulse phases are ϕ 1 IPIP = 4(x), 4(-x), ϕ 1 IPAP = 4(y), 4(-y), ϕ 1 IPAP = 4(y), 4(-y), ϕ 1 APIP = 4(y), 4(-y), and ϕ 1 APAP = 4(x), 4(-x). The receiver phase ϕ rec has to be set individually depending on the phase cycling in the respective experiment but is identical for the different sub-spectra (IP-IP, IP-AP, AP-IP, and AP-AP). Decoupling in this case for 31 P and 1 H nuclei can be achieved using state-of-the-art decoupling schemes like asynchronous GARP4 sequences (Shaka et al., 1985) and WALTZ sequences (Zhou et al., 2007), respectively. (D) Illustration of DIPAP decoupling for a 13 C, 15 N-labeled UTP sample. Linear combination of all four spectra gives rise to the virtually decoupled signal on the right. IPAP, in-phase anti-phase; HSQC, heteronuclear single quantum coherence; DIPAP, double in-phase anti-phase.
Note that depending on the software, which is used for processing, there are programs available that do this automatically (e.g., splitcomb in Topspin). If multiple residues with different 1 J CC coupling constants are recorded in one experiment, process them differently using the corresponding 1 J CC coupling constant. For atoms where no homonuclear 1 J CC coupling is present, split the spectra, phase them correctly (AP spectrum is −90°phase shifted relative to the IP spectrum), and combine them.

C-DETECTED 3D (H)CC-TOCSY, (H)CPC, AND (H)CPC-CCH-TOCSY EXPERIMENTS FOR RIBOSE ASSIGNMENT
NMR spectroscopy of large RNAs but also proteins is often difficult as the signal-tonoise ratio (S/N) deteriorates with larger molecular weight of the RNA of interest, which is caused by the following main points. With an increased molecular weight the number of signals increases, which concomitantly leads to resonance overlap. For RNA, this resonance overlap is particularly pronounced for 1 H-nuclei due to a poor chemical shift dispersion. Furthermore, an increased transverse relaxation rate leads to signal broadening, which is especially prominent for 1 H-nuclei as well. When conducting 13 Cdetected NMR experiments these problems can be substantially reduced. 13 C nuclei feature a higher chemical shift dispersion and at the same time exhibit more favorable relaxation properties. To exploit these properties also in the assignment of the ribose moiety, where resonance overlap is particularly problematic, a set of experiments was developed by Richter et al., (2010). Here, we will provide a detailed guide on how to set up the Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry   Fig. 17).

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Current Protocols in Nucleic Acid Chemistry NMR tube (e.g., Shigemi) NMR spectrometer equipped preferably with a z-axis gradient 13 C, 15  2. Record a 1 H, 13 C-HSQC spectrum of the ribose region as a reference spectrum.
4. Use IPAP for homonuclear decoupling according to Support Protocol 8, applying the pulse lengths listed in Table 13.

Adjust TOCSY transfer length depending on the desired correlations.
For the observation of C1 -C5 use 15 ms and for C1 and C2 only use 3 ms.
6. Choose carrier frequencies according to the resonance distribution.

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Current Protocols in Nucleic Acid Chemistry 9. When the experiment is completed, process spectrum as described in Support Protocol 8 (sample data: Fig. 18A). 14. Set cnst2 to 166 Hz ( 1 J CH coupling).
16. Adjust number of transients and points and test the experiment by recording the first increment. Check whether the S/N is sufficient. If not, adjust number of transients.

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Current Protocols in Nucleic Acid Chemistry Figure 19 (A) Schematic coherence transfer pathways for the 13 C-detected 2D CN-spin filter HSQC experiment in Us (left) and Gs (right). The sizes of the 1 J CN , 1 J NH , and 1 J CC coupling constants are given (Fiala & Sklenár, 2007). Coupling constants that are only indirectly used for signal intensity modulation or for decoupling are depicted in gray. The carbon nuclei where the detection happens are marked with a circle. (B) Simulated C z N y coherence in dependence of the transfer time D during the NH spin filter element for different imino proton exchange rates, ranging from 0 s -1 (black) to 7,200 s -1 (red). The figure has been adapted from Fürtig et al., 2016 andSchnieders et al., 2020. HSQC, heteronuclear single quantum coherence.
Set the 1 H carrier frequency to the water resonance frequency (4.0 ppm). Set the 13  23. For IPAP, choose Q3 shapes for sp24 (C1 selective) and sp27 (C2 selective) as well as pulse lengths for p12 (2 ms, 600 MHz) according to the magnetic field strength.
24. Adjust number of transients and points and test the experiment by recording the first increment. Check whether the S/N is sufficient. If not, adjust number of transients.
26. Processing: Process spectrum as described in Support Protocol 8.

C-DETECTED 2D CN-SPIN FILTER HSQC EXPERIMENT
NMR spectroscopy of RNA usually focuses on the imino proton resonance as a reporter for base pairing. The reason is that it is only observable when protected through a hydrogen bond. Otherwise, it rapidly exchanges with solvent water and the resonance is broadened beyond detectability. While this allows quick secondary structure determination, any information about flexible regions of the RNA is not available. With the 13 Cdetected CN-spin filter HSQC experiment, however, those flexible regions are accessible as here the detection happens on carbon (Fürtig et al., 2016). Moreover, the status of hydrogen bonding of any imino-proton-carrying nucleobase (G or U) can be determined as signal intensities are modulated depending on the imino proton exchange rate (Fig. 19B).
The schematic coherence transfer in Figure 19A shows that this experiment is composed of a CN-transfer step that is succeeded by an NH spin filter element. The differences in signal intensity result from a modulation of the observed scalar 1 J NH coupling on the imino proton exchange rate. If the proton exchange is slow, the 1 J NH coupling can evolve during the spin filter element. However, if the imino proton exchange with solvent water Schnieders et al.

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Current Protocols in Nucleic Acid Chemistry is fast, scalar relaxation of the second kind leads to a decoupling of the scalar coupling which results to no effect on the signal intensity (Fig. 19B).

Materials
≥0.5 mM RNA sample with 13 C, 15 N-labeled imino group containing nucleotide of interest (G or U) or uniformly 13 C, 15 N-labeled RNA (see Basic Protocol 1) NMR buffer (see recipe) Other RNA specific requirements like Mg 2+ ions or small molecule ligands NMR tube (e.g., Shigemi) NMR spectrometer equipped preferably with a z-axis gradient 13 C, 15 N [ 1 H]-TXO cryogenic probe; alternatively, a z-axis gradient 1 H, [ 13 C, 15 N]-TCI cryogenic probe 1. Set up NMR spectrometer according to Support Protocol 7.
3. Record 13 C-detected CN-HSQC spectra in order to optimize the CN-transfer time, which depends on the desired correlations for G (C2N1 or C6N1) or U (C2N3 or C4N3).

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Current Protocols in Nucleic Acid Chemistry 4. Set up 2D CN-HSQC experiment using the optimized transfer time. Use IPAP sequences for homonuclear decoupling if needed (G-C6N1 and U-C4N3) and implement them according to Support Protocol 8. Set carrier frequencies and spectral windows according to the correlations of interest (Table 13). Check whether the number of transients is sufficient in the first increment of the CN-HSQC experiment. Compare spectra with a standard 1 H, 15 N-HSQC spectrum of the imino region ( Fig. 20A and B).
5. Load parameter set C_CON_SQXF or C_CON_IASQXF (IPAP) with the pulse sequences c_con_sqxf and c_con_iasqxf, respectively. Set the delay for the CN-transfer as optimized in the CN-HSQC experiment. If necessary, use IPAP sequences as conducted for the CN-HSQC experiment or according to Support Protocol 8. Use a 1 J NH coupling constant of 90 Hz to calculate spin filter delay (1/ 1 J NH ). (Table 15). Check in the first increment whether the S/N is sufficiently large.

Set carrier frequencies based on the CN-HSQC spectrum according to the correlations of interest
6. Processing: If IPAP schemes were used, process spectra according to Support Protocol 8. If this was not the case, simple 2D processing can be applied.
7. Data interpretation: Based on the signal intensities in this 2D experiment, the status of hydrogen bonding can be estimated.
If the correlations to the imino nitrogen atoms appear inverted in sign with respect to other non-imino CN-correlations, the exchange of the imino proton is slow. This is for example the case for U11 in the 14-nt UUCG hairpin RNA (see Fig. 21). If the imino proton is not protected from hydrogen exchange, the signal intensities are not affected, and the correlation appears with the same sign as other non-imino CN-correlations (e.g., U7 Fig. 21). Signals, which are close to zero crossing correspond to imino protons that are partly protected from solvent exchange either through a weak hydrogen bond or due to steric hindrance (e.g., U6 Fig. 21). If the exact imino proton exchange rate is to be determined, the CN-spin filter HSQC experiment can be modified to a pseudo 3D experiment according to Fürtig et al., 2016.

C-DETECTED C(N)H-HDQC EXPERIMENT FOR THE DETECTION OF AMINO GROUPS
Amino groups are important functional groups in RNA and are known to be involved in many different kinds of interactions due to their hydrogen bonding potential. However, NMR spectroscopy of RNA often sets a focus on the characterization of the imino protons as their resonances are well dispersed and allow a fast secondary structure determination. The resonances of amino groups on the other hand exhibit-especially for guanosines and adenosines-very broad lines, which are often beyond detectability. The reason for this is a partially restricted rotation around the C-NH 2 bond that is regularly   Fig. 22B) where 1 H-double quantum coherence is selected and evolved in the indirect dimension. As described in the Support Protocol 8, homonuclear 1 J CC coupling in adenosines and cytidines is decoupled using IPAP sequences. This protocol will explain step by step how to successfully set this experiment up and what to consider.

Materials
≥0.5 mM RNA sample with 13 C, 15 N-labeled amino group containing nucleotide of interest (G, C, or A) or uniformly 13 C, 15 N-labeled RNA (see Basic Protocol 1) NMR buffer (see recipe) Other RNA specific requirements like Mg 2+ ions or small molecule ligands NMR tube (e.g., Shigemi) NMR spectrometer equipped preferably with a z-axis gradient 13  The default phase is x. 15 N nuclei are decoupled during acquisition using asynchronous GARP4 sequences (Shaka et al., 1985). Gradient pulses were applied for 1 ms with a smoothed square amplitude (SMSQ10.100) and 100% gradient strength corresponds to 53 G/cm. The t 1 -time was incremented with half of the dwell time. Frequency discrimination in the indirect dimension ϕ 3 was cycled with 45°. Pulse phases, delays, and gradient strengths are as follows: 3. Record a 13 C-detected 2D CN-HSQC spectrum of the amino region as described in Support Protocol 9, to optimize the CN-transfer step and the IPAP for homonuclear CC-decoupling.

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Current Protocols in Nucleic Acid Chemistry Figure 23 (A) 15 N-HSQC spectrum of the amino region of a 34-nt GTP class II aptamer (Wolter et al., 2016(Wolter et al., , 2017. The experiment was recorded with 8 scans per increment and spectral widths of 24 ppm (2,048 complex points) and 40 ppm (128 complex points) in the direct and indirect dimensions, respectively. Carrier frequencies were 4.7 ppm, 150 ppm, and 85 ppm for 1 H, 13 C, and 15 N, respectively. With an inter-scan delay of 1 s the experiment was recorded for 40 min. (B, C, and D) C(N)H-HDQC spectra for the 34-nt GTP class II aptamer (secondary structure in part C) with optimized processing for C, A, and G, respectively. The spectra originate from the same experiment and only differ in their processing. The experiment was recorded with 80 scans per increment and spectral widths of 50 ppm (1,024 complex points) and 5 ppm (160 complex points) in the 13 C and 1 H dimensions, respectively. Carrier frequencies were 154 ppm ( 13 C), 7.1 ppm ( 1 H), and 90 ppm ( 15 N). With an inter-scan delay of 2.5 s, the experiment was recorded for 19 hr. B was processed using a 1 J CC coupling constant of 55 Hz and in C the 1 J CC coupling constant was 75 Hz. D was processed by splitting IP and AP spectra, phase shifting the latter (AP) by −90°, and adding them up. Signals marked with * are folded in the indirect dimension and resonances marked with ** appear in the spectra C and D as pseudo doublets and arise due to processing from guanosines and adenosines, respectively. The figure was taken from Schnieders et al., 2019. HSQC, heteronuclear single-quantum correlation; HDQC, heteronuclear double-quantum correlation; IP, in-phase; AP, anti-phase. Schnieders et al. experiment (for A;Simon, Zanier, & Sattler, 2001), 1 H, 1 H-NOESY experiments (mainly for C), and non-selective CN-HSQC experiments with different CN-transfer times can be acquired.

C-DETECTED CN-HSQC EXPERIMENT FOR AMINO GROUPS
In order to optimize parameters for the carbon direct-detected C(N)H-HDQC experiment and to assign resonances in this experiment, an amino selective CN-HSQC experiment should be conducted. Parameters which are to be optimized are the CN-transfer time and the IPAP sequence depending on the nucleotide type of interest (G, A, or C).

Materials
≥0.5 mM RNA sample with 13 C, 15 N-labeled amino group containing nucleotide of interest (G, C, or A) or uniformly 13 C, 15 N-labeled RNA (see Basic Protocol 1) NMR buffer (see recipe) Other RNA specific requirements like Mg 2+ ions or small molecule ligands NMR tube (e.g., Shigemi) NMR spectrometer equipped preferably with a z-axis gradient 13 C, 15

Choose selective 180°pulses.
We use Q3 shapes for 13 C and Reburp shapes for 15 N. Set pulse lengths so that the desired band widths are reached. In our hands, 400 μs ( 13 C, Q3) and 1,200 μs ( 15 N,Reburp) work well at 800 MHz.
4. Optimize CN-transfer. In theory, the time for the CN-transfer equals 1/2J CN .
With coupling constants of around 21 Hz for the different nucleotide types (G = 23 Hz, C = 21 Hz, and A = 20 Hz;Fiala & Sklenár, 2007), this transfer time would be 23.8 ms long. However, transverse relaxation leads to a constant decrease of the signal and therefore, depending on the rotational correlation time of the RNA, the time with maximum transfer efficiency is often shorter and needs to be determined experimentally (Fig. 24). Furthermore, transfer efficiencies also vary between the different amino group carrying nucleotides.
To determine the time with the maximum transfer efficiency, record the first increment of this 2D CN-HSQC experiment with a high number of transients (e.g., 1,024) for several transfer times (e.g., 12-40 ms). Comparison of integrals within the different regions then yields the optimized transfer times for the respective nucleotide type (Fig. 24). Optimized transfer times that we determined for a 14-nt RNA are depicted in Table 16.   Table 13 in Support Protocol 8). Use the 180°selective pulses as in the non-IPAP version. We use Q5 shapes for the selective 90°carbon pulse and in our hands 600 μs works well at 800 MHz. Set offset for the off-resonant 13 C-pulse (Table 13 in Support Protocol 8). Here, we use −11,000 Hz at 800 MHz.
6. Process spectra as described in Support Protocol 8 while using the correct 1 J CC coupling constants for C and A even if an intermediate IPAP delay was used. If no homonuclear coupling is present as is the case for G, spectra have to be processed differently as described in Support Protocol 8 (Fig. 25).

C-DETECTED "AMINO"-NOESY EXPERIMENT
Due to the inherently low proton density, structural characterization of RNA often suffers from a low number of inter-residual long-range nuclear Overhauser effect (NOE) contacts. Imino and amino protons are often involved in exchange processes that do not allow their detection, which in turn reduces the number of available NOE contacts even further. In amino groups, this exchange process is a restricted rotation around the C-NH 2 bond, which is often in an intermediate exchange regime on the NMR timescale. This broadens the signals beyond detectability as soon as the amino proton coherence is in the transverse plane. The "amino"-NOESY experiment partly removes this effect by moving away from 1 H-detection towards 13 C direct detection and by spin-locking the 1 H coherence in a HN-TOCSY transfer (Fig. 26). With this experiment, additional NOE contacts can be determined which are not accessible using any other conventional NOESY experiment.

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Current Protocols in Nucleic Acid Chemistry Especially, inter-residual H1 -to-amino-group contacts are of high interest and can improve RNA structure calculations where a limited number of NOE contacts is available. We will describe how to set up two variants of this "amino"-NOESY experiment, one with and one without a 13 C filter, in this basic protocol. The experiment with 13 C filter only selects for NOE contacts to 13 C-bound protons, while the other one is not selective.

Materials
≥0.5 mM RNA sample with 13 C, 15 N-labeled amino group containing nucleotide of interest (G, C, or A) or uniformly 13 C, 15 N-labeled RNA (see Basic Protocol 1) NMR buffer (see recipe) Other RNA specific requirements like Mg 2+ ions or small molecule ligands NMR tube (e.g., Shigemi) NMR spectrometer equipped preferably with a z-axis gradient 13 C, 15

Set NOESY mixing time (D8).
We typically use mixing times around 150 ms for this experiment.
6. Set carrier frequency and spectral window in the indirect dimension depending on the NOE contacts of interest.
If no 13 C filter is applied, imino protons have to be covered by the spectral window. In our hands a carrier frequency of 8.7 ppm with a spectral window of 12 ppm in the 1 H dimension worked well, but those parameters should be adapted to the RNA sample of interest. If the 13 C filter is included, the spectral window can be reduced to only cover aromatic and ribose protons, e.g., 5.5 ppm spectral window with a carrier frequency of 6 ppm.

Set number of transients and number of points.
Be careful to conduct the experiment with a sufficient number of transients because this experiment has a limited sensitivity. We use, for example, 128 transients for the standard "amino"-NOESY experiment and 256 transients for the one with 13  8. Start measurement and check whether the S/N is sufficient after a couple of increments.
9. Process spectra according to Support Protocol 8.
11. Include the newly determined NOE contacts in a structure calculation.
Note that signal intensities do not only depend on the proton-proton distances but also on transfer efficiencies and the rotational frequency around the C-NH 2 bond. Therefore, Schnieders et al.

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Figure 27
Overlay of "amino"-NOESY spectra of the 34-nt GTP class II aptamer (Wolter et al., 2016(Wolter et al., , 2017 with (red) and without (gray) 13 C filter. Newly obtained NOE contacts are written in bold and are underlined. NOE contacts, which are obtained from the "amino"-NOESY with 13 C filter are written in red. Signals marked with ** appear in the spectra with optimized processing for adenosines and guanosines as pseudo doublets due to processing and arise from guanosines and adenosines, respectively. The dashed line marks the water resonance. The "amino"-NOESY experiment without 13 C filter (gray) was recorded with 128 scans per increment and spectral widths of 70 ppm (1,024 complex points) and 12 ppm (448 complex points) in the direct and indirect dimensions, respectively. Carrier frequencies were 160 ppm (  NOE contacts can only be determined qualitatively. We assumed that the maximum distance is 6.5 Å and thus included the contacts with this upper distance limit in the structure calculation.

N-DETECTED BEST-TROSY EXPERIMENT
Despite the disadvantage in loss of sensitivity, 15 N-direct detection can become interesting due to the favorable relaxation behavior of 15 N-nuclei when it comes to molecules with a large rotational correlation time (Fig. 28). Therefore, several 15 N-detected HN correlation experiments were tested on RNA and the effect of the molecular size on sensitivity, resolution, and relaxation behavior was investigated. The most sensitive 15 Ndetected HN correlation experiment, the 15 N-detected BEST-TROSY experiment, is described in this basic protocol.

Materials
≥0.5 mM RNA sample with 15 N-labeled imino group containing nucleotide of interest (G or U) or uniformly 15 N-/ 13 C, 15 N-labeled RNA (see Basic Protocol 1) NMR buffer (see recipe) Other RNA specific requirements like Mg 2+ ions or small molecule ligands NMR tube (e.g., Shigemi) NMR spectrometer equipped preferably with a z-axis gradient 13 C, 15  4. If the sample is not 13 C-labeled, leave out the carbon decoupling.

Set number of transients.
We used, for example, 576 transients for a 0.5 mM, 127-nt long RNA adapting three conformations.
8. Start experiment and check whether the S/N is sufficient in the first increment (Fig. 29).

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Background Information
Because this protocol covers a wide range of different methods from sample preparation to NMR experiments, the different historical backgrounds will be briefly summarized in the following sections. 13 C, 15 N-isotope labeling is usually inevitable for NMR structure determination and often also required if functional studies are conducted. There are two methods, which are used for these purposes, namely solid phase synthesis and in vitro transcription using T7 RNA polymerase. Solid phase synthesis requires phosphoramidites and was first conducted to yield an isotope-labeled RNA in 1994 using 13 C-labeled building blocks (Quant et al., 1994). As this method is limited in molecular size and needs a costly apparatus, Schnieders et al.

Basic Protocols 3-7: Heteronuclear-detected NMR experiments on RNA
As the major drawback in heteronucleardetected NMR experiments is the low sensitivity, it is very important to conduct the experiments under the best possible conditions. A parameter which can be easily controlled is the RNA concentration, which should be as high as possible, as the S/N scales directly with the sample concentration. In addition, the NMR spectrometer that is used for heteronuclear detection should at least be equipped with a cryogenic probe and best with a cryogenic probe optimized for heteronuclear detection. Along the same lines, the setup of the spectrometer should be always performed thoroughly, as the sensitivity drops with inaccurate settings.

Troubleshooting
For troubleshooting the protocols contained in this article, see

Basic Protocol 1
The analysis of the test transcriptions demonstrates which condition is ideal for the RNA to be transcribed at. The respective PAGE gel should show a band with the size of the target RNA and optimization should lead to a maximum of band intensity. It is favorable to optimize the byproducts to a low band intensity albeit it is not essential in case of eventual purification by HPLC or preparative PAGE (Basic Protocol 1 and Alternate Protocol 1). The transcription conditions have to result in a sharp band for the target RNA without any byproducts if the fast purification via centrifugal concentrator is used (Alternate Protocol 2). The preparative transcription and purification should finally yield 60 to 300 nmol in about 300 μl, depending on the type of labeling and the RNA sequence.

Support Protocol 1
The concentration of extracted DNA plasmid can be easily quantified via UV/vis absorption at 260 nm. Because the isolation procedure of plasmid DNA from bacterial cells can be performed with a highly optimized commercially available kit, the expected yields can range from 2 to 8 mg DNA per liter LB medium. Variability in the yield can occur based on the copy number of the specific plasmid used and the total amount of cells grown. The efficiency of the restriction digestion, which is analyzed visually via agarose gel, can be expected to give quantitative amounts of linearized plasmid. With careful execution, only ∼5% to 10% of DNA will be lost in the subsequent extraction.

Support Protocol 2
The exact amount of DNA after a successful preparative PCR is not determinable because the DNA must not be purified for further transcription reactions. But if necessary, it is possible to estimate the amount of DNA via a native PAGE. One may do so by adding a standard DNA ladder to the PAGE. An average amount of DNA amplified by preparative PCR is 1 nmol (about 60 μg of a 100bp DNA). A native PAGE further will show if the PCR results in pure target product or if Schnieders et al.

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Support Protocol 3
With a protein yield of ∼4 to 8 mg of pure T7 RNA polymerase per liter medium, this protocol provides an easy and cost-effective alternative to the commercially available en-zyme. Enzyme concentration can be measured via UV/vis absorption at 280 nm and enzyme activity should be tested in analytical scale transcriptions. Even after 1.5 hr of transcription incubation, an analytical RNA PAGE should show significant amounts of synthesized RNA.

Support Protocol 4
The protein purification protocol described here provides the possibility to express and purify highly active YIPP for in vitro transcription of RNA. Expected yields are 35 Schnieders et al.

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The protein activity can be tested via small scale in vitro transcription with transcription buffer as a reference. In the case of an active protein, the transcription reaction should be a clear solution in contrast to the cloudiness of the reference sample.

Basic Protocol 2
After the ligation with a modified nucleotide, the small acceptor RNA (RNA 1; <17 nt) will move faster during the gel electrophoresis because of the additional negative charge of the 3 -end phosphate group. A dephosphorylated RNA will move more slowly during the gel electrophoresis because of the additional nucleotide without a 3 -end phosphate group. For a larger acceptor RNA (>17 nt) no difference will be visible in the gel electrophoresis. The splint ligation with the donor RNA (RNA 2) will show a significantly larger RNA product, which should be detectable via gel electrophoresis.

Support Protocol 5
The yield depends on the type and position of the modification. The modified nucleoside 3 ,5 -bisphosphate is characterized by mass spectrometry and NMR spectroscopy. Byproducts like 2 ,5 -bisphosphate, mono-or triphosphate nucleosides often cannot be separated via HPLC. In case of characterization difficulties, a test ligation should be performed with T4 RNA Ligase 1 and a short acceptor RNA (<17 nt). A positive ligation result will indicate that the synthesis was successful. Byproducts will not be accepted by the T4 RNA Ligase 1.

Support Protocol 8
The implementation of an IPAP/DIPAP sequence should always lead to a completely decoupled singlet signal of the desired carbon atom.

Basic Protocol 3
The (H)CC-TOCSY experiment yields a spectrum in which the C1 atoms are correlated to all other carbon atoms of the same ribose ring. This information can be used for a sequential assignment in the (H)CPC and (H)CPC-CCH-TOCSY experiments. In the (H)CPC spectrum, the 31 P nucleus is correlated to C5 and C4 in 3 as well as to C3 and C4 in the 5 direction. The (H)CPC-CCH-TOCSY experiment yields correlations between the 31 P nucleus and both of the C1 atoms of the adjacent ribose moieties. Thus, by combining the information obtained in the three experiments, a full resonance assignment of the ribose carbon atoms can be obtained.

Basic Protocol 4
Using the CN-spinfilter HSQC information, the status of hydrogen bonding can be obtained. This information can be extracted from the signal intensities of the imino nitrogen resonances in the C-N correlated spectrum. In case of hydrogen bonding, the sign of the corresponding resonance is inverted as opposed to the remaining resonances. If the imino proton is not involved in hydrogen bonding, the resonance stays unperturbed. Consequently, it might happen that resonances of nucleobases in rather unstable hydrogen bonding do not give rise to a signal as they are at the zero crossing.

Basic Protocol 5
Using the C(N)H-HDQC experiment, a C-H correlated spectrum should be obtained. This spectrum should yield a sharp resonance for each guanosine, adenosine, and cytidine in the sequence. The experiment usually works most reliably for guanosines.

Support Protocol 9
In the amino-selective CN-HSQC spectrum, C-N correlations of all amino groups are obtained. If a correlation is not visible using this experiment (for possible reasons see Troubleshooting), neither will it be in the C(N)H-HDQC spectrum.

Basic Protocol 6
In the C-H correlated "amino"-NOESY spectrum, contacts to spatially close protons or 13 C-bound protons (filtered version) and amino groups are detected. In the version without carbon filter, broad single quantum diagonal peaks might appear. In helical parts of an RNA, inter-residual correlations between guanosine amino groups and preceding as well as cross-strand H1 protons are expected. Correlations might be used in structure calculations as upper distance restraints (6.5 Å).

Basic Protocol 7
The 15 N-detected BEST-TROSY experiment yields analogous information as compared to its 1 H-detected counterpart. Thus, one resonance for each imino proton, which is sufficiently protected from fast solvent exchange, is expected.

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Time Considerations
See Table 23 for times required to perform the protocols included in this article.