Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

UNIT 7.22

Andre Dallmann1,2,3 and Michael Sattler1,2 1

Institute of Structural Biology, Helmholtz Zentrum M¨unchen, Neuherberg, Germany Center for Integrated Protein Science Munich and Chair of Biomolecular NMRSpectroscopy, Department Chemie, Technische Universit¨at M¨unchen, Garching, Germany 3 Present address: Division of Molecular Structure, National Institute of Medical Research, London, United Kingdom 2

NMR spectroscopy is a powerful tool to study the structure and dynamics of nucleic acids. In this unit, we give an overview of important experiments to determine and characterize hydrogen bonds in nucleic acids and provide detailed instructions for setting up recently developed sensitivity-improved NMR pulse sequences, i.e., BEST selective long-range HNN-COSY, selective BESTTROSY-HNNCOSY, and Py H(CC)NN-COSY. The strengths and limitations of these experiments will also be discussed. Detailed step-by-step protocols are provided for each of the three pulse sequences, with special emphasis on adjusting and setting of delays and shaped pulses. The NMR pulse sequences with example datasets and optimized, nonstandard adiabatic pulse shapes used for selective 15 N magnetization transfer are provided. These experiments enable NMR analysis of a broad variety of RNAs ranging from low to high molecular C 2014 by John Wiley & Sons, Inc. weight and complexity.  Keywords: NMR spectroscopy r hydrogen bonds r dynamics r RNA r sensitivity enhancement

How to cite this article: Dallmann, A. and Sattler, M. 2014. Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy. Curr. Protoc. Nucleic Acid Chem. 59:7.22.1-7.22.19. doi: 10.1002/0471142700.nc0722s59

INTRODUCTION RNA secondary structure determination remains one of the most challenging tasks in structural biology due to the flexibility and variety of RNA structural motifs. Techniques such as SHAPE (Low and Weeks, 2010) provide models of the secondary structure with reasonable accuracy for large systems. However, the experimental data are combined with computational predictions and provide indirect information where specific details may be incorrect. Although limited by adverse relaxation effects, NMR can provide highresolution information on the two- and three-dimensional structure of nucleic acids comprising up to 100 nt or higher (Tzakos et al., 2006; Lu et al., 2011; Duss et al., 2012). The development of NMR experiments such as HNN-COSY (Dingley and Grzesiek, 1998; Pervushin et al., 1998) made it possible to directly prove the existence of hydrogen bonds based on the observation of J-coupling (6 to 7 Hz) between donor and acceptor nitrogens in nucleic acid base pairs. Subsequently, additional experiments have been proposed for improving the sensitivity and providing additional correlations for structural analysis of nucleic acids (Table 7.22.1). We have recently developed novel experiments that provide improved sensitivity for the detection of base pairs in dynamic regions of nucleic acid structures (Dallmann et al., 2013). In the following, step-by-step protocols are provided

Current Protocols in Nucleic Acid Chemistry 7.22.1-7.22.19, December 2014 Published online December 2014 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142700.nc0722s59 C 2014 John Wiley & Sons, Inc. Copyright 

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Table 7.22.1 Overview of NMR Experiments for the Detection of Hydrogen Bonds in Nucleic Acids

Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

Experiment

Description

HNN-COSY (Dingley and Grzesiek, 1998)

The original standard HNN-COSY experiment for assigning any N-H . . . N-type hydrogen bonds in base pairs of helical RNA or DNA, as demonstrated for 69-nucleotide T1 domain of the PSTVd RNA. A sensitivity-improved version (BEST-TROSY-HNN-COSY) exists (Farjon et al., 2009).

TROSY-HNN-COSY (Pervushin et al., 1998)

TROSY version of the standard HNN-COSY, demonstrated on a 14-mer DNA duplex. Improved version (BEST-TROSY-HNN-COSY) exists (Farjon et al., 2009).

Long-range HN-HSQC (Dingley et al., 1999)

Long-range 1 H,15 N-HSQC exploiting the weaker 1h JHN long-range coupling rather than the 2h JNN couplings, demonstrated on a triplex-DNA. Rather insensitive compared to HNN-COSY experiment.

Amino-HNN-COSY (Majumdar et al., 1999a)

Version of HNN-COSY for measuring hydrogen bonds between amino groups as donors and endocyclic ring nitrogens as acceptors demonstrated on an A:A base pair in (GGAGGAT)2

H(CN)N(H)-HNNCOSY (Majumdar et al., 1999b)

First version of HNN-COSY that does not depend on detecting exchangeable imino protons. Limited mainly to G:C tetrads as focused on observation of H8-(C8-N7) . . . N2(-H2) hydrogen bonds.

Long-range H(N)CO (Dingley et al., 2000)

Long-range 1 H-(15 N)-C=O-HSQC exploiting the weak 2h JNC’ long-range couplings to detect base pairs involving carbonyl groups. Demonstrated on a quadruplex-DNA but rather insensitive due to very small coupling (< 1 Hz).

Long-range HNN-COSY (Hennig and Williamson, 2000)

Original version of the long-range HNN-COSY for detecting A:U base pairs in the absence of an observable imino proton. Demonstrated on the 31-nt HIV-2 transactivation response element–argininamide complex. An improved version (BESTsellr) is described in Basic Protocol 1 (Dallmann et al., 2013).

4

JNN -HN(N)TOCSY and CP-H(N)CO-(NN)-TOCSY (Liu et al., 2000)

HN(N)TOCSY and CP-H(N)CO-(NN)-TOCSY experiments for detecting H-N . . . C=O hydrogen bonds in DNA tetrads via the extremely weak 4h JNN -coupling (1 Hz). Extremely insensitive.

U-H3/A-H2 dual-detected HNN-COSY (Luy and Marino, 2000)

Combination of long-range and standard HNN-COSY in one experiment. Demonstrated on 30 nt CopA29 RNA hairpin. Reduced sensitivity as it compromises on the optimal delays for both transfers.

H2(N1N3)H3-, H5(N3N1) H1/H6(N3N1)H1- and H2(N2N7)H8 (Majumdar et al., 2001a)

A number of “through transfer” experiments (as compared to the usual “out-and-back” versions) demonstrated on the 27 nt HIV-1 TAR RNA. These experiments are in general less sensitive than their “out-and-back” counterparts and partly rely on detection of exchangeable imino proton as well, rendering them less efficient.

H2N6N3 and (H6)N6N3H2 (Majumdar et al., 2001b)

H2N6N3 and (H6)N6N3H2 are specific experiments designed to detect the N6-H6 . . . N3 hydrogen bond in sheared G:A base pairs via remote nonexchangeable protons. Both are highly specialized and insensitive experiments. continued

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Table 7.22.1 Overview of NMR Experiments for the Detection of Hydrogen Bonds in Nucleic Acids, continued

Experiment

Description

2

JNN -HNN-E. COSY (Luy et al., 2002)

E.COSY variant of the long-range HNN-COSY for measuring accurate 3 JH2H3 coupling constants, demonstrated for on 30-nt CopA29 RNA hairpin. Reduced sensitivity due to E.COSY approach.

H5(C5)NN-HNN-COSY (Pitt et al., 2004)

HNN-COSY using the nonexchangeable H5 proton of uracil for detection. Restricted to uracil due to use of a sizeable 3 JC5N3 coupling of  5 Hz that is only present in uracil and not cytosine. An improved version [Py H(CC)NN-COSY] is presented in Basic Protocol 3 (Dallmann et al., 2013).

BEST-TROSY-HNN-COSY (Farjon et al., 2009)

Improved version of the standard HNN-COSY experiment, implementing relaxation enhancement via BEST and TROSY for improved detection of high-molecular weight RNA. The version described in this unit (Basic Protocol 1) benefits from the additional use of selective 15 N pulses and improves sensitivity by 1.5 fold. Tested on 76-nt tRNAVal (Dallmann et al., 2013)

1

H-31 P long-range HSQC (Duchardt-Ferner et al., 2011)

1

Py H(CC)NN-COSY and BESTsellr HNN-COSY (Dallmann et al., 2013)

HNN-COSY experiments relying on nonexchangeable protons for detection, utilizing various optimization and sensitivity enhancement techniques. Both are described in this protocol.

H-31 P long-range HSQC experiment for detection of H . . . P hydrogen bonds in RNA. Demonstrated on 27-nt neoswitch-ribostamycin complex. So far the only available experiment for direct detection of H . . . P hydrogen bonds.

for setting up and troubleshooting the BEST selective long-range (BESTsellr) HNNCOSY (Basic Protocol 1), a selective (TROSY-BEST-)HNN-COSY (Basic Protocol 2), and the pyrimidine (Py) H(CC)NN-COSY (Basic Protocol 3). The pulse programs, a nonstandard 15 N-selective pulse (rna_phs8,0.7,4m.1) and sample datasets run on a Bruker Avance III 600 can be downloaded at http://www.currentprotocols.com/protocol/nc0722. Note that delays and variables printed in bold in the protocols below are experimental parameters that refer to settings in the supplied pulse programs.

BEST SELECTIVE LONG-RANGE HNN-COSY (BESTsellr) The BESTsellr experiment (Fig. 7.22.1) is an improved version of the original long-range HNN-COSY (Hennig and Williamson, 2000), which was developed for detecting base pairing in the absence of an observable imino proton (e.g., due to increased solvent exchange). The experiment transfers magnetization from the H2 proton of adenosine to the N1, then to a nitrogen across a hydrogen bond and back (Fig. 7.22.1A). When the Hoogsteen interface of guanine or adenine is involved in hydrogen bonding, magnetization is transferred from H8 to N7 over the hydrogen bond and back. The BESTsellr version of this experiment improves the sensitivity up to 8-fold through implementation of band-selective 15 N pulses (Fig. 7.22.2). This enables selecting the desired magnetization pathway and eliminating alternative pathways. In addition, band-selective pulses are used for 1 H spins to avoid excitation of the solvent signal and facilitate proton relaxation by longitudinal relaxation enhancement (Fig. 7.22.1B; Farjon et al., 2009). Due to these improvements, the BESTsellr experiment can be used to detect secondary structure

BASIC PROTOCOL 1

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Figure 7.22.1 BEST-selective long-range (BESTsellr) HNN-COSY. (A) The chemical-shift ranges of important nuclei are indicated. The different INEPT transfer steps are color-coded and indicated by arrows next to the respective couplings (in Hz) in the chemical structures of the A:U base pair. (B) Out-and-back pulse sequence for the BESTsellr HNN-COSY experiment. Magnetization transfer steps are color-coded as in (A). Narrow and thick bars represent high power 90° and 180° pulses, respectively. Unless indicated otherwise, the default phase for pulses is x. Phase-cycling: φ3 = x,-x φ13 = y,y,-y,-y, φrec =x,-x. Quadrature detection is achieved by applying the States-TPPI method to φ3 , φ13 . This figure has been reproduced from the supplemental information of Dallmann et al. (2013) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

in high-molecular-weight systems (demonstrated for 76 nt tRNAVal , see Fig. 7.22.3) and at room temperature. It is thus especially useful for studying dynamic and flexible nonhelical regions of nucleic acids.

Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

Strategic Planning Sample preparation. In order to obtain satisfactory results, the concentration of the RNA should be as high as possible. Since aggregation is rarely a problem for RNA, there is no upper limit. However, when studying hairpins, it should be considered that high concentrations of RNA will promote dimerization toward formation of doublestranded RNA (Duszczyk et al., 2008). Sample concentrations between 500 and 1000 μM are advisable. Results when using concentrations below 500 μM will depend on the field strength of the NMR spectrometer as well as sample size and thermodynamic stability. As a number of long shaped pulses and 15 N- as well as (potentially) 13 Cheteronuclear decoupling are employed, the sample should be measured in a low-salt buffer to minimize sample heating. Since the experiment does not detect exchangeable imino protons, there is no need to keep the pH as low as possible; any pH between 5.5 and 8 can be used. To achieve best results, the use of a Shigemi NMR tube matched to H2 O/D2 O is highly recommended, as this increases sample concentration about 2-fold and reduces B1 inhomogeneity imperfections. Please note that for this experiment a 15 Nlabeled RNA sample is needed (13 C labeling is not necessary, since no 13 C atoms are involved in the transfer). Depending on the nature of your RNA sample, heat to 85° to 90°C and either snap-cool (for hairpin RNA) or cool down slowly (duplex RNA) prior to putting the sample into the Shigemi tube.

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A

B

C

Figure 7.22.2 Overview of the bandwidth and center frequencies of the (A) 13 C and (B) 15 N selective pulses used in the Py H(CC)NN-COSY and (C) 15 N selective pulses in BESTsellr HNNCOSY. Below the chemical-shift scale, typical chemical-shift ranges for relevant RNA nuclei are given. This figure has been reproduced from the supplemental information of Dallmann et al. (2013) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

NMR spectrometer. The experiment is best run using a high-field magnet with a cryogenically cooled probe head. The pulse sequence has been tested on a Bruker Avance III 600 with a cryogenically cooled probe head, as well as a Bruker Avance III 800 with a cryogenically cooled probe head. As no exchangeable protons are detected, there is no need to record the experiment at low temperature, where a compromise between line broadening versus slower solvent exchange needs to be found. Accordingly, the measurement temperature should be chosen as high as possible (for obtaining the sharpest lines) without affecting the native structure of the RNA. Ideally, UV melting curves or equivalent thermodynamic data can be obtained on the RNA prior to the measurement.

Preparation of the NMR spectrometer 1. Adjust the sample temperature and let it equilibrate at least 10 to 15 min prior to carrying out further steps.

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Figure 7.22.3 Comparison of an imino-detected BEST-TROSY HNN-COSY experiment (left) with the BESTsellr HNN-COSY, recorded at 318 K on the 24 kDa tRNAVal in H2 O for 12 hr. Only one A:U base pair is observed in the HNN-COSY due to fast exchange of the imino protons with bulk water at elevated temperatures, whereas all expected A:U base pair correlations are observed for the long-range experiment. This figure has been reproduced from Dallmann et al. (2013) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

2. Tune, match, lock, and shim your sample as usual. 3. Load the experiment and set the pulse program to RNAbesthnnjlr_sel.

Setting general parameters 4. Set the 15 N transmitter frequency offset to 188 ppm (O3P) in order to center the pulses on the chemical shifts of the endocyclic nitrogens, the 13 C transmitter frequency offset to 154 ppm (O2P) in order to decouple the C2 atoms most effectively, and the 1 H transmitter frequency offset to the resonance frequency of the water at about 4.7 ppm (O1P), with respective sweep widths (SW) of 100 ppm for 15 N (to cover all endocyclic nitrogen chemical shifts) and 10 ppm for 1 H (to include the chemical-shift range of adenine H2). 5. Set the acquisition times to 127.8 msec and 16.4 msec [time domain (TD) sizes to 1532 × 200 points] in F2 and F1 dimensions, respectively, for reasonable resolution in the indirect dimension and to reduce sample heating during decoupling in t2 . Use >16 dummy scans (DS), to give sufficient time for equilibration of the sample with the pulse sequence prior to the experiment.

Setting the delays 6. Set the recycling delay. Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

The optimal recycle delay has been found to be about 300 msec (i.e., set d1 = 0.3 sec), which is a compromise between optimal T1 relaxation recovery and faster recycling. However, further optimization of the recycling delay might be necessary, depending on the sample.

7. Set the H-N transfer delay.

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The optimal H-N transfer delay (d4 in the pulse sequence) is theoretically calculated to be 1/(4 2 JHN ) = 16.67 msec—with 2 JHN = 15 Hz—while empirically the authors have found 9.5 msec to be optimal (set d4 = 0.0095 sec). This is supported by theoretical calculations taking T2 relaxation during the delay time into account. The actual delay times δ 1 - δ 3 in the pulse program (Fig. 7.22.1) are much shorter, since the chemical-shift evolution during the shaped pulses as well as during the gradient pulse and recovery delay have to be taken into account. They are calculated as: δ1 = δ2 = d4 − τrecoveryDelay − τGradient − (0.529 × τPC9 ) δ3 = d4 − τGradientrecoveryDelay − τGradient with the factor of 0.529 for chemical-shift evolution during the polychromatic pulse taken from Farjon et al. (2009) and Lescop et al. (2010). Chemical-shift evolution during the simultaneous REBURP pulses (Geen and Freeman, 1991) on 1 H and 15 N (p22 and p19) has been found to be negligible. The delay time may have to be adjusted depending on sample size and conditions, with larger samples requiring shorter delays to account for the resulting relaxation losses. However, in our experience, variations in the delay time when studying a 24-mer or a 76-mer RNA did not affect the results significantly.

8. Set the N-N transfer delay. The optimal N-N transfer delay (d25 in the pulse program) is theoretically calculated to be 1/(4 2 JNN ) = 38.46 msec—with 2 JNN = 6.5 Hz—but strongly depends on the 15 N T2 relaxation time and thus on the molecular weight, with higher-molecular-weight molecules requiring shorter delays to account for the resulting relaxation losses. Set delay d25 = 22.5 msec for a 24-mer RNA hairpin and d25 = 17.5 msec for a 76-mer tRNAVal , as was found optimal in our hands. The actual delay times δ 4 and δ 5 in the pulse program (Fig. 7.22.1) are much shorter, since the chemical-shift evolution during the shaped pulses has to be considered. They are calculated as: δ4 = d25/2 − (0.5 × τrna δ5 = d25 − τrna

phs8,0.7,4m.1 )

− (2 × τ90◦

hardpulse /π )

phs8,0.7,4m.1

A separate optimization for each sample may be performed.

Adjusting shaped pulses 9. Next, set and adjust the 1 H BEST-selective pulses. Calculate the shaped pulse power based on the experimentally calibrated 1 H hard pulse length, assuming linear (or linearly corrected) amplifiers. These parameters can be obtained, for example, using the Shapetool implemented in the Bruker Topspin software. All of the 1 H BEST-selective pulses are automatically centered on the aromatic region at 7.5 ppm and cover a bandwidth of 2 ppm (i.e., comprising the region from 6.5 to 8.5 ppm). Pulses p21 and p23 (in the pulse sequence) are polychromatic pulses (Kupce and Freeman, 1994) with 1000 points each used for excitation, while pulse p22 is a Reburp pulse (Geen and Freeman, 1991) with 1000 points used for refocusing. The pulse lengths with all important information are given in Table 7.22.2 for an NMR spectrometer operating at 600 MHz 1 H Larmor frequency. Pulse p24 is not part of the BEST-selective pulse scheme, but instead is used for decoupling the imino proton during the 15 N chemical-shift evolution time. Thus, it is centered on the imino proton region (fixed at 13.5 ppm in the pulse program) and covers a bandwidth of 6 ppm.

10. Use 13 C (15 N) GARP decoupling during acquisition, employing a 1.25 (1.04) kHz radiofrequency field. Set the power levels of the 13 C pulses to 0 W when the RNA is not 13 C-labeled. 11. Two different shaped pulses are applied during the pulse sequence on 15 N. During the H-N transfer, use a Reburp.1000 pulse shape. This unorthodox application of Reburp for inversion rather than refocusing was chosen due to its narrower inversion

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Table 7.22.2 List of Pulses (as Numbered in the Accompanying Pulse Sequence) and their Important Parameters

Pulse length (μsec) at 600 MHz

Effective bandwidth covered (ppm)

6000

7.5 ± 1

Pulse

Channel

Pulse shape

Degree of rotation

p21

1

Pc9_4_90.1000

90

p22

1

p23

1

H

Pc9_4_90.1000

90

6000

7.5 ± 1

p24

1

H

Reburp.1000

180

1000

13.5 ± 6

p19

15

N

Reburp.1000

180

4800

228 ± 8

p29

15

N

rna_phs8,0.7,4m.1

180

4000

188 ± 50

H

Polychromatic pulse (Kupce and Freeman, 1994) with 1000 points H

Reburp.1000

180

4800

7.5 ± 1

REBURP pulse (Geen and Freeman, 1991) with 1000 points

Constant adiabaticity hyperbolic secant–shaped pulses (Tann´us and Garwood, 1996) with a total sweep width of 8000 Hz, β=5.3, Amplitude Power Index (n) of 0.7 sweeping from low to high field.

profile as compared to the Iburp2.1000. Center this pulse at 228 ppm. During the N-N transfer, use a pair of constant adiabaticity hyperbolic secant–shaped pulses (Tann´us and Garwood, 1996) (rna_phs8,0.7,4m.1). Applying a pair of adiabatic pulses for refocusing is important to cancel pulse shape imperfections and phase errors (Zweckstetter and Holak, 1998). Power levels can be calculated based on the 15 N hard pulse.

Gradient values 12. Use SMSQ10.100 (a smooth chirped amplitude shape with 100 points) as shape for all gradients in this pulse program, with a fixed gradient pulse duration of p16=1000 μsec and gradient amplitude set to G0,1,2,3 = 33.0, 22.0, 38.5, 22.5 G/cm (60%, 40%, 70%, and 50% for GPZ0, GPZ1, GPZ2, and GPZ3), respectively. Use a gradient recovery delay d16=200 μsec. BASIC PROTOCOL 2

Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

BEST TROSY-HNN-COSY The original HNN-COSY experiment (Dingley and Grzesiek, 1998) has been improved through the use of relaxation enhancement techniques such as BEST and TROSY (Farjon et al., 2009), making it applicable to high-molecular-weight RNA such as the 76 nt tRNAVal . The version that is presented in this second protocol has been further improved by employing the same pair of 15 N-selective broadband adiabatic pulses that are also used in the BESTsellr experiment described in Basic Protocol 1 (Figs. 7.22.1 and 7.22.2). This implementation of 15 N-selective pulses has been combined with a standard HNN-COSY (Fig. 7.22.4; RNAhnnjwgs_dec_sel, downloadable from http://www/currentprotocols.com/protocol/nc0722), as well as the BEST-TROSYHNN-COSY (RNAbesttrosyhnn). As the setup of the HNN-COSY has been described before (Dingley and Grzesiek, 1998; Dingley et al., 2008; Farjon et al., 2009), and the implementation of the 15 N-selective broadband adiabatic pulses is comparable, only the setup for the selective BEST-TROSY-HNN-COSY is described here, briefly. Note that, through the use of the 15 N-selective pulses, the amino nitrogens are decoupled effectively, precluding observation of any wobble base pairs involving NH2 . . . N. Notably, NH . . . N–type hydrogen bond correlations (including e.g., Hoogsteen-type) are observed with 1.5-fold increased sensitivity compared to the nonselective version of the

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Figure 7.22.4 Selective BEST-TROSY-HNN-COSY. (A) The different INEPT transfer steps are color-coded and indicated by arrows next to the respective couplings (in Hz) in the chemical structures of the A:U base pair as an example. (B) Out-and-back pulse sequence for the Selective BEST-TROSY-HNN-COSY experiment. Magnetization transfer steps are color-coded as in (A). Narrow and thick bars represent high-power 90° and 180° pulses, respectively. Unless indicated otherwise, the default phase for pulses is x. Phase-cycling: φ5 = x,y,-x,-y, φ6 = y,-x,-y,x,-y,x,y,-x, φ7 = 2(-y,x,y,-x), 2(y,-x,-y,x) and φrec = x,-y,-x,y. Quadrature detection is achieved by applying the States-TPPI method to φ5 , φ6 .

experiment (Fig. 7.22.5). The inability to observe hydrogen bonds involving amino protons in this experiment is acceptable, considering that in the standard version of the experiment, rectangular pulses are employed, which cannot simultaneously invert exocyclic and endocyclic nitrogens with good efficiency, thus reducing sensitivity.

Strategic Planning Sample preparation and NMR spectrometer. The same general guidelines as described for the BESTsellr experiment (Basic Protocol 1) are applicable. However, please bear in mind that unlike the BESTsellr experiment, the BEST TROSY-HNN-COSY relies on the exchangeable imino proton for detection. Thus it has to be run in aqueous solution, and temperature has to be carefully chosen to provide the best compromise between narrower line width (at high temperature) and slower imino proton exchange with water (at low temperature). In our experience, temperatures between 278 K for short constructs (24 nt RNA stem loop) and 298 K for larger RNAs (76 nt tRNAVal- ) seem to work best. For limiting exchange of the imino proton with water, the pH should be chosen as low as possible, with pH = 5.5 to 6.5 yielding good results. A spectrometer field strength below 600 MHz proton Larmor frequency will require optimization and adaptation of the band-selective pulses, as the currently implemented selective pulse durations would exceed the available delays in the pulse sequence. Preparation of the NMR spectrometer 1. Adjust the sample temperature and let it equilibrate at least 10 to 15 min prior to carrying out further steps. 2. Tune, match, lock, and shim your sample as usual. 3. Load the experiment and set the pulse program to RNAbesttrosyhnn.

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Figure 7.22.5 Standard and selective HNN-COSY spectra recorded on 18a stem RNA in H2 O at 278 K. The three nonterminal G:C base pairs and the stable A:U base pair are observed with 16 scans (see Fig. 7.22.7 for secondary structure). The 1-D slices for each cross-peak show that the gain in sensitivity by using a pair of adiabatic selective refocusing pulses during the N-N INEPT transfer is on average 50%. Experimental times were 40 min each. This figure has been reproduced from the supplemental information of Dallmann et al. (2013) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

Setting general parameters 4. Set the 15 N transmitter frequency offset to 188 ppm (O3P) in order to center the pulses on the chemical shifts of the endocyclic nitrogens, the 13 C transmitter frequency offset to 154 ppm (O2P) in order to decouple the C2 atoms most effectively, and the 1 H transmitter frequency offset to the resonance frequency of the water at about 4.7 ppm (O1P), with respective sweep widths (SW) of 100 ppm for 15 N (to cover all endocyclic nitrogen chemical shifts) and 20 ppm for 1 H (to include the chemical-shift range of the imino protons). 5. Set the acquisition times to 127.8 msec and 16.4 msec [time domain (TD) sizes to 1532 × 200 points] in the F2 and F1 dimensions, respectively, for reasonable resolution in the indirect dimension and to reduce sample heating during decoupling. Use >16 dummy scans (DS) to give sufficient time for equilibration of the sample with the pulse sequence prior to the experiment.

Setting the delays 6. Set the recycle delay. The optimal recycle delay was found to be about 300 msec (set d1 = 0.3 sec), which is a compromise between optimal relaxation to equilibrium and faster recycling. However, further optimization of the recycling delay might be necessary, depending on the sample. Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

7. Set the H-N transfer delay. The optimal H-N transfer delay (d26 in the pulse sequence) is theoretically calculated to be 1/(4 1 JHN ) = 2.4 msec with 1 JHN = 90 Hz, taking into account that T2 relaxation will shorten the optimal delay time. For the molecular weight range where the experiment

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is applicable, relaxation effects do not affect the optimal transfer time significantly; therefore, set d26 = 0.0024 sec. However, in the presence of chemical or conformational exchange broadening of the imino protons, the transfer time should be reduced. The actual delay times δ 1 - δ 3 in the pulse program (Fig. 7.22.4) are shorter than d26, as the chemical-shift evolution during the shaped pulses as well as during the gradient pulse and recovery delay have to be taken into account. They are calculated as: δ1 = d26 − τrecoveryDelay − τGradient − (0.529 × τPC9 ) − (0.5 × τReBurp ) δ2 = d26 − τrecoveryDelay − τGradient − (0.5 × τReBurp ) δ3 = (0.629 × τPC9 ) − 10 μsec with the factor of 0.529 for chemical-shift evolution during the polychromatic pulse, the factor of 0.5 for chemical-shift evolution during the polychromatic pulse, and 0.66 for chemical-shift evolution during the Eburp pulses (Farjon et al., 2009; Lescop et al., 2010).

8. Set the N-N transfer delay. The optimal N-N transfer delay (d25 in the pulse sequence) is theoretically calculated to be 1/(4 2 JNN ) = 38.46 msec—with 2 JNN = 6.5 Hz—but strongly depends on the 15 N T2 relaxation time and thus on the molecular weight, with higher-molecular-weight molecules requiring shorter delays to account for the resulting relaxation losses. Set delay d25 = 22.5 msec for a 24-mer RNA hairpin and d25 = 17.5 msec for a 76-mer tRNAVal , as was found optimal in our hands. The actual delay times δ 4 and δ 5 in the pulse program (Fig. 7.22.4) are much shorter, since the chemical-shift evolution during the shaped pulses has to be taken into account. They are calculated as: δ4 = d25/2 − (0.5 × τrna

phs8,0.7,4m.1 )

− (2 × τ90◦

hardpulse /π )

− τrecoveryDelay − τGradient − 20 μsec δ5 = d25 − τrna

phs8,0.7,4m.1

A separate optimization for each sample may be performed.

9. The delay  is necessary for first-time point correction, and is calculated as: (1)  = 2 × t1 (0) for a non-13 C-labeled sample or (2)  = 2 × t1 (0) + τCrp60,0.5,20.1_13C for a 13 C-labeled sample. Note that the corresponding flag (-DLABEL_CN) in the pulse program should be set accordingly.

Adjusting shaped pulses 10. Next, set and adjust the 1 H BEST-selective pulses. Calculate the shaped pulse power based on the experimentally calibrated 1 H hard pulse length, assuming linearcorrected amplifiers. These parameters can be calculated, for example, using the Shapetool implemented in the Bruker Topspin software. All of the 1 H BEST-selective pulses are automatically centered on the imino proton region at 12.5 ppm and ideally should cover a bandwidth of 6 ppm (i.e., cover the region from 9.5 to 15.5 ppm). Unlike in the BESTsellr experiment (Basic Protocol 1), here an excitation Burp (Eburp) pulse with 1000 points and its time-reversed (tr) counterpart are used in the second H-N transfer (pulse p43) (Geen and Freeman, 1991), while in the first H-N transfer, the same polychromatic pulse as in BESTsellr is used (pulse p41) for excitation. In both transfers, the same Reburp pulse has been used for refocusing. For 15 N inversion during the H-N transfer, a standard rectangular-shaped pulse was used in order to invert the imino proton region. The pulse lengths with all important information are given in Table 7.22.3 for an NMR spectrometer operating at 600 MHz 1 H Larmor frequency.

11. During nitrogen chemical-shift evolution, a smoothed chirp pulse (p8) is applied on the 13 C channel to effectively decouple N-C J-coupling evolution. Its length is fixed

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Table 7.22.3 List of Pulses (as Numbered in the Accompanying Pulse Sequence) and their Important Parameters

Pulse length (μsec) at 600 MHz

Effective bandwidth covered (ppm)

Pulse

Channel

Pulse shape

Degree of rotation

p41

1

H

Pc9_4_90.1000

90

2750

12.5 ± 6

p42

1

H

Reburp.1000

180

1875

12.5 ± 6

p43

1

H

EBurp2(tr).1000

90

1750

12.5 ± 6

180

500

154 ± 100

180

4000

Excitation BURP pulse (Geen and Freeman, 1991) with 1000 points p8

13

C

Crp60,0.5,20.1 Smoothed Chirp pulse with a total sweep width of 60,000 Hz, 20% smoothing, and sweeping from high to low field.

p30

15

p31

15

N N

rna_phs8,0.7,4m.1 Squa100.10

180

188 ± 50 15

Same as N hard pulse

152 ± 50

at 500 μsec (as this is sufficient to decouple all aromatic carbons for all currently available spectrometers). Calculate the power level for this pulse as discussed above, or set it to 0 in case that the sample is not 13 C-labeled. 12. Use a pair of constant adiabaticity hyperbolic secant–shaped pulses (rna_phs8,0.7,4m.1) during the N-N transfer, as discussed above. Calculate power levels based on the 15 N hard pulse value. The second shaped pulse (p31) is a standard rectangular pulse shape with standard hard pulse settings, but centered on 152 ppm instead of 188 pp, to effectively invert the imino nitrogens during H-N transfer, eliminating the need to switch center frequencies during the pulse program.

Gradient values 13. Set SMSQ10.100 (a smooth chirped amplitude shape with 100 points) as shape for GPZ2, GPZ3, GPZ4, and GPZ5 with their strength set to G2,3,4,5 = 9.9, 24.2, 33.0, 16.5 G/cm (18%, 44%, 60%, and 30%) and their length fixed at p16 = 600 μsec. For GPZ1, use SMSQ10.32 (a smooth chirped amplitude shape with 32 points) with a strength G1 = 16.5 (30%) and a length of p19 = 300 μsec. Use a gradient recovery delay d16 = 100 μsec. BASIC PROTOCOL 3

Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

PYRIMIDINE (Py) H(CC)NN-COSY While the BESTsellr experiment (Basic Protocol 1) has improved sensitivity when compared to other pulse sequences for measuring hydrogen bonds via nonexchangeable protons, one drawback is that it only detects base pairs involving adenine in WatsonCrick type hydrogen bonding or adenine/guanine in Hoogsteen-type hydrogen bonds. This problem is overcome by the Py H(CC)NN-COSY experiment, which transfers magnetization from the nonexchangeable H5 proton of both uridine and cytosine through C5, C4, and N3, to its hydrogen-bonding partner and back (Fig. 7.22.6). While this compromises sensitivity due to the large number of magnetization transfer steps, it enables the simultaneous detection of G:C and A:U base pairs and, in general, any

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A

G:C base pair

A:U base pair 93-102

80-90

54.7

166-168

168

142-152

6-7

175.8 94-99 6.5 180-208 136-144

65.1 157-158

222-228

154-160

155

165-169

178.7 102-107

10.7 156-162 137-144

6-7 149-155 9

152-155 70-80

B

TP 0

1

2

3

4

φ3 τ

δ * 165.8

o2 (ppm) 98.7 15N

δ

8

y

y

δ

φrec

ε

δ

τ

τ

t2

φ4 φ5 GARP

* 98.7

y t1 t1 t1 4 4 4

9 Δ ΔΔ Δ 2 22 2

*

φ8 φ φ φ9 10 10 t1 δ4 δ5 δ4 4

7

y

φ7

y

y

τ

6

φ7

φ7

y 1H Δ Δ 2 2 13C

5

δ4 δ5 δ4

Gz gp3 gp4 Q3

Q3 offset 165.8 ppm

gp5

gp4 Q5

Q5tr

gp5

gp1 rna_phs8,0.7,4m.1

gp2

Crp60,0.5,20.1

*

Figure 7.22.6 Py H(CC)NN-COSY experiment. (A) The chemical-shift ranges for important spins are indicated. The different INEPT transfer steps are color coded and indicated by arrows next to the respective couplings (in Hz) in the chemical structures of the A:U and G:C base pairs. (B) Pulse sequence for the out-and-back Py H(CC)NN COSY experiment. Magnetization transfers are color-coded as in (A). Important time points are indicated above the pulse sequence, and the different transfer steps are color-coded. Narrow and thick bars represent high-power 90° and 180° pulses, respectively. Unless indicated otherwise, the default phase for pulses is x. Phase-cycling: φ3 = x,-x, φ4 = 4(x), 4(-x), φ5 = 4(-y), 4(y), φ7 = 4(x), 4(-x), φ8 = x,x,-x,-x, φ9 = y, φ10 = x and φrec = x,-x,-x, x, -x, x,x,-x. Quadrature detection is achieved by applying the States-TPPI method to φ9 , φ10 . This figure has been reproduced from the supplemental information of Dallmann et al. (2013) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

N-H . . . N-type hydrogen bonds involving a pyrimidine (Fig. 7.22.7). This renders the experiment a versatile tool for studying short- to intermediate-length RNA. Also, in this procedure, sensitivity is improved by using shaped pulses for decoupling alternative magnetization pathways. The implementation of longitudinal relaxation enhancement for this experiment is unfortunately not possible due to the proximity of the H5 proton resonance frequency to that of water.

Strategic Planning Sample preparation and NMR spectrometer. The same general guidelines as described for the BESTsellr experiment (Basic Protocol 1) are applicable. However, please bear in mind that unlike the BESTsellr experiment, the Py H(CC)NN-COSY is best run in deuterated solvent to avoid problems with water suppression and cancellation of signal (since the H5 protons used for detection resonate at 5.3 to 6 ppm, near the water signal at 4.7 ppm). Also, due to 13 C involved in the magnetization transfer, a 13 C,15 N-labeled sample is required. A spectrometer field strength below 600 MHz proton Larmor frequency will require optimization and adaptation of the band-selective pulses, as the currently implemented selective pulse durations would exceed the available delays in the pulse sequence. Preparation of the NMR spectrometer 1. Adjust the sample temperature and let it equilibrate at least 10 to 15 min prior to carrying out further steps.

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Figure 7.22.7 Pyrimidine (Py) H(CC)NN-COSY experiment for the simultaneous detection of A:U and G:C base pairs. (A) Out-and-back magnetization transfer steps for the standard (purple) and Py H(CC)NN-COSY (green) are illustrated for A:U and G:C base pairs. Details for the pulse sequence and experimental settings are provided in Fig. 7.22.4. (B) Imino region of the standard HNN-COSY recorded for the 18a stem RNA in H2 O and the H5 region of the Py H(CC)NN-COSY experiment recorded in D2 O, each measured for 3 hr at 298 K. Only the three nonterminal G:C base pairs and no A:U base pair are observed in the standard HNN-COSY experiment, while all Watson-Crick (4 G:C, 2 A:U) base pairs are observed with the new Py H(CC)NN-COSY, including the A:U base pair directly adjacent to the U:C mismatch (orange box). The signals at 157 ppm (*) are intra-residual correlations to the N1 atom of pyrimidine. As G:U base pairs lack N-H . . . N-type H-bonds, they are not detectable in the experiment. This figure has been reproduced from Dallmann et al. (2013) with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.

2. Tune, match, lock, and shim your sample as usual. 3. Load the experiment and set the pulse program to RNA_h5c5c4n3n1.

Setting general parameters 4. Set the 15 N transmitter frequency offset to 188 ppm (O3P) in order to center the pulses on the chemical shifts of the endocyclic nitrogens, the 13 C transmitter frequency offset to 98.7 ppm (O2P) in order to center pulses on the C5 atoms (though the center frequency is switched automatically within the pulse program), and the 1 H transmitter frequency offset to the resonance frequency of the water at about 4.7 ppm (O1P), with respective sweep widths (SW) of 100 ppm for 15 N (to cover all endocyclic nitrogen chemical shifts) and 10 ppm for 1 H (to include the chemical-shift range of adenine H2). 5. Set the acquisition times to 127.8 msec and 16.4 msec [time domain (TD) sizes to 1532 × 200 points] in the F2 and F1 dimensions, respectively, for reasonable resolution in the indirect dimension and to reduce sample heating during decoupling. Use >16 dummy scans (DS), to give sufficient time for equilibration of the sample with the pulse sequence prior to the experiment.

Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

Setting the delays All of the delays except the one for the N-N transfer period (d25 in the pulse sequence) are already predefined, and optimal values are set within the pulse program. The authors have tested these settings with a number of different RNAs and found the predefined values to be optimal in general. Consequently, it should normally not be necessary to change these predefined settings. If you nevertheless want to optimize and vary these settings for a specific application, explanations describing the role of these delays are

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given in the following. Please bear in mind that this experiment will not perform well on large RNAs (>50 nt) due to the high number of transfer steps, which accumulate relaxation losses quickly. 6. Since no longitudinal relaxation enhancement is applied, use a standard recycle delay (set d1 = 1 sec). This has been found to be optimal for a variety of samples. Further optimization might be necessary, depending on the sample.

7. Set the H5-C5 transfer delay. The optimal H5-C5 transfer delay was found to be Δ (d4) = 1.4 msec. It is unlikely that this value needs adjusting, since the delay is very short and no significant shortening of the optimal delay time due to relaxation is observed.

8. Set the C5-C4 transfer delay. The optimal C5-C4 transfer delay was found to be τ (d22) = 4.1 msec. It is unlikely that this value needs adjusting, since the delay is very short and no significant shortening of the optimal delay time due to relaxation is observed.

9. Set the C4-N3 transfer delay. The optimal C4-N3 transfer delay was found to be δ (d23) = 19 msec. This value was used for RNAs between 16 and 24 nucleotides in length, and may have to be adjusted to shorter times for longer RNAs to account for increased transverse relaxation.

10. Set the N-N transfer delay. The optimal N-N transfer delay (d25 in the pulse sequence) is theoretically calculated to be 1/(4 2 JNN ) = 38.46 msec—with 2 JNN = 6.5 Hz—but strongly depends on the 15 N T2 relaxation time and thus on the molecular weight, with higher-molecular-weight molecules requiring shorter delays to account for the resulting relaxation losses. Set a delay d25 = 22.5 msec for a 24-mer RNA hairpin and d25 = 17.5 msec for a 76-mer tRNAVal , as these settings were found to be optimal in our hands. The actual delay times δ 4 and δ 5 in the pulse program (Fig. 7.22.6) are much shorter, since the chemical-shift evolution during the shaped pulses has to be taken into account. They are calculated as: δ4 = d25/2 − (0.5 × τrna δ5 = d25 − τrna

phs8,0.7,4m.1 )

phs8,0.7,4m.1

− (2 × τhardpulse /π ) − τrecoveryDelay − τGradient

− τrecoveryDelay − τGradient

A separate optimization for each sample may be performed.

11. The delay ɛ is necessary for first time point correction and is calculated as: ε = 4 × t1 (0) + τCrp60,0.5,20.1

13C

+ (2 × τ90◦

hardpulse 1H )

Adjusting shaped pulses Since this experiment is less sensitive than the BESTsellr experiment, a careful calibration of the 1 H, 13 C, and 15 N hard pulses is important. As discussed above, the optimal shaped pulse power can be safely calculated using, for example, the shape tool (stdisp) implemented in the Bruker Topspin software. The pulse lengths with all important information are given in Table 7.22.4 for an NMR spectrometer operating at 600 MHz 1 H Larmor frequency. 12. No shaped pulses are used on the proton channel.

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Table 7.22.4 List of Pulses (as Numbered in the Accompanying Pulse Sequence) and their Important Parameters

Pulse length (μsec) at 600 MHz

Effective bandwidth covered (ppm)

Pulse

Channel

Pulse shape

Degree of rotation

p13

13

C

Q5.1000/Q5tr.1000

90

1500

98.7 ± 11/165.8 ± 11

p14

13

C

Q3.1000

180

1000

98.7 ± 11/165.8 ± 11

Gaussian cascade pulses (Emsley and Bodenhausen, 1990) for excitation (Q5) and refocusing (Q3) with 1000 points. p8

13

C

Crp60,0.5,20.1

180

500

154 ± 100

p29

15

N

rna_phs8,0.7,4m.1

180

4000

188 ± 50

p30

15

N

rna_phs8,0.7,4m.1

180

4000

188 ± 50

13. The Q5.1000/Q5tr.1000 and Q3.1000 pulse shapes (p13 and p14 in the pulse sequence) are Gaussian cascade pulses for excitation and refocusing with 1000 points each. Use these for 13 C shaped pulses during the C5-C4 and C4-N3 transfers for 90° and 180° rotations respectively. Use the same shapes and lengths for selecting the C4 atom (centered on const21=165.8 ppm) as well as the C5 atoms (centered on const22=98.7 ppm), in order to decouple the C6 atoms effectively. 14. During nitrogen chemical-shift evolution, a smoothed chirp pulse (p8) is employed to effectively decouple N-C J-coupling evolution. Its length is fixed at 500 μsec (as this is enough to decouple all aromatic carbons for all currently available spectrometers). 15. Apply the same constant adiabaticity hyperbolic secant–shaped pulse (rna_phs8,0.7,4m.1) as discussed for the BESTsellr experiment in two places during the pulse sequence (p29 and p30), with the same parameters as discussed above. This is used for decoupling the exocyclic amino nitrogen (N4) in cytosine during the C4-N3 transfer and—as in the BESTsellr experiment—to decouple the exocyclic amino groups of adenine, guanine, and cytosine. Calculate power levels based on the 15 N hard-pulse value as discussed before.

Gradient values 16. Use SMSQ10.100 (a smooth chirped amplitude shape with 100 points) as shape for all gradients in this pulse program with their length fixed at p16 = 1000 μsec and their strength set to G1,2,3,4,5 = 44.0, 11.1, 16.5, 6.1, 3.9 G/cm (80%, 20.1%, 30%, 11%, and 7% for GPZ1, GPZ2, GPZ3, GPZ4, and GPZ5, respectively). Use a gradient recovery delay d16 = 200 μsec. COMMENTARY Background Information Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

The original HNN-COSY experiment was developed in 1998. In the following years, a number of variations, additional applications, and improvements of the experiment have been published (Dingley and Grzesiek, 1998; Dingley et al., 1999, 2000; Majumdar

et al., 1999a,1999b; 2001a,2001b; Hennig and Williamson, 2000; Liu et al., 2000; Luy et al., 2002; Majumdar and Patel, 2002; Pitt et al., 2004). With new techniques of sensitivity improvement such as relaxation enhancement through BEST and SOFAST (Schanda et al., 2005, 2006) at hand, the original HNN-COSY

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experiment has been significantly improved (Farjon et al., 2009). This SOFAST-TROSYHNN-COSY experiment has superior sensitivity to the original, and can be applied to highmolecular-weight RNA. However, like the original version, it suffers from the drawback of not being able to detect hydrogen bonds in the absence of an observable imino proton. Consequently, we set out to improve the original lr HNN-COSY (Hennig and Williamson, 2000) with relaxation enhancement and selective 15 N magnetization transfers, and were able to achieve an 8-fold sensitivity improvement and demonstrate its applicability to highmolecular-weight RNAs (Dallmann et al., 2013). This provides very useful information, since the experiment is best run at as high a temperature as the secondary structure of the RNA studied can tolerate. This makes it possible to record all experiments at the temperature at which the experiments for structure determination are acquired. An equally important point is that Py H(CC)NN-COSY eliminates the need to run multiple experiments, depending on which base pairs one needs to observe. It allows measuring transient A:U and G:C base pairs in dynamic regions of an RNA duplex at elevated temperatures simultaneously in a single experiment (Dallmann et al., 2013).

Critical Parameters and Troubleshooting There are a number of critical parameters, which need to be adjusted carefully. Calibration of hard pulses. While 15 N pulse lengths usually do not change significantly from sample to sample, an experimental calibration of the 15 N hard pulse is advisable, since even small losses due to miscalibration of about 5% quickly add up to total losses of about 20% given the high number and length of pulses. The same applies for the 1 H and 13 C hard pulses and their corresponding shaped pulses. Temperature. As discussed above, temperature is the most critical parameter. With smaller RNAs (24-mer), we found that acquiring data around room temperature gives optimal results for the Bestsellr and Py H(CC)NN experiment, and that in some cases deviations as low as 5 K from the optimal temperature can result in total loss of signal. This can be explained by the instability of some hairpin structures due to increased conformational dynamics or loss of base pairing and secondary structure at higher temperatures. At low temperatures (278 K), excessive line broadening due to slower molecular tumbling

and thus increased transverse relaxation may render NMR signals unobservable. For highermolecular-weight molecules, such as tRNAVal (74 nucleotides), the optimal temperature was at the limit tolerable for cryogenically cooled probe heads (318 K in our case). For the BESTTROSY-HNN-COSY, similar arguments apply, although it performs best at low temperatures (278 K for 24 nt RNA and 298 K for 76 nt RNA) because imino protons are detected. Salt concentration. Since a high number of very long selective pulses is applied, the salt concentration in the buffer should be as low as possible to avoid excessive sample heating and reduced sensitivity of cryogenically cooled probe heads. Salt concentrations above 150 mM ionic strength are discouraged.

Anticipated Results For the BESTsellr pulse sequence, a pair of cross peaks for each N-H . . . N type hydrogen bond from N1 or N7 of adenine to the imino proton of its hydrogen bonding partner is expected. A good way to check whether the experiment has worked is to count whether the number of pairs corresponds at least to the number of A:U base pairs that are also observed in the HNN-COSY experiment, or in a simple imino-NOESY experiment with the RNA. If this number is matched, no additional hydrogen bonds may exist involving adenines. If this number is exceeded, transient base pairing is observed. Assignments for the 15 N-resonances from the HNN-COSY can be transferred to the BESTsellr and Py H(CC)NN-COSY, thus also making this a valuable source for assigning H2 resonances of adenines in RNA without the need to assign all aromatic and anomeric protons first. However, to unambiguously assign more than one additional peak pair observed in either experiment, either full assignment of the aromatic and anomeric resonances or selective labeling schemes are necessary. For the selective HNN-COSY, the same correlations are expected to be observed as for the nonselective version, however with 1.5fold increased sensitivity. Note however that differences might occur in the detection of hydrogen bonds involving amino protons, as the 15 N spins are effectively decoupled with the selective pulses while residual effects might be picked up with the nonselective pulses (as discussed in the introduction to the selective HNN-COSY). For the Py H(CC)NN-COSY experiment, in principle any N-H . . . N type hydrogen bond from a pyrimidine to its partner can be

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detected, regardless of the nature of the partner. To check whether the experiment has worked, count the pairs of peaks with chemical-shift values typical of G:C and A:U base pairs, respectively. This number should match or exceed the number of G:C and A:U base pairs you expect to be present in your RNA. As discussed above, assignments of 15 N resonances can be transferred from an HNN-COSY experiment and used to assign H5 atoms of the pyrimidines of stable G:C and A:U base pairs. If the number of peak pairs in the experiment exceeds the number of base pairs expected and/or observed in the HNN-COSY experiment by more than one, assignment of the H5 atoms of pyrimidines is needed in order to unambiguously assign the extra peaks.

Time Considerations NMR sample preparation (with an isotopelabeled RNA sample at hand) takes a few minutes, and setting up the experiment on the spectrometer takes 1 hr. Depending on sample concentration, size of the RNA, and spectrometer quality, the experiment time may vary from as low as 15 min to up to 2 days.

Acknowledgment We acknowledge the Bavarian NMR Center (BNMRZ) for providing support and NMR measurement time. We thank Dr. Gerd Gemmecker for valuable discussions on the implementation of the BEST-TROSY-HNNCOSY and Sam Asami for reading the manuscript and discussion. Funding from the Deutsche Forschungsgemeinschaft grants SFB1035, GRK1721, G-NMR, and the Bavarian Ministry for Science and Education, is acknowledged.

Literature Cited Dallmann, A., Simon, B., Duszczyk, M.M., Kooshapur, H., Pardi, A., Bermel, W., and Sattler, M. 2013. Efficient detection of hydrogen bonds in dynamic regions of RNA by sensitivityoptimized NMR pulse sequences. Angew. Chem. Int. Ed. 52:10487-10490. Dingley, A.J. and Grzesiek, S. 1998. Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2 J NN couplings. J. Am. Chem. Soc. 120:8293-8297. Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy

Dingley, A.J., Masse, J.E., Peterson, R.D., Barfield, M., Feigon, J., and Grzesiek, S. 1999. Internucleotide scalar couplings across hydrogen bonds in Watson−Crick and Hoogsteen base pairs of a DNA triplex. J. Am. Chem. Soc. 121:6019-6027. Dingley, A.J., Masse, J.E., Feigon, J., and Grzesiek, S. 2000. Characterization of the hydrogen bond

network in guanosine quartets by. J. Biomol. NMR 16:279-289. Dingley, A.J., Nisius, L., Cordier, F., and Grzesiek, S. 2008. Direct detection of N-H[ctdot]N hydrogen bonds in biomolecules by NMR spectroscopy. Nat. Protoc. 3:242-248. Duchardt-Ferner, E., Ferner, J., and Wohnert, J. 2011. Rapid identification of noncanonical RNA structure elements by direct detection of OH . . . O=P, NH . . . O=P, and NH2 . . . O=P hydrogen bonds in solution NMR spectroscopy. Angew. Chem. Int. Ed. 50:7927-7930. Duss, O., Lukavsky, P.J., and Allain, F.H. 2012. Isotope labeling and segmental labeling of larger RNAs for NMR structural studies. Adv. Exp. Med. Biol. 992:121-144. Duszczyk, M.M., Zanier, K., and Sattler, M. 2008. A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat. Nucleic Acids Res. 36:7068-7077. Emsley, L. and Bodenhausen, G. 1990. Gaussian pulse cascades: New analytical functions for rectangular selective inversion and in-phase excitation in NMR. Chem. Phys. Lett. 165:469476. Farjon, J., Boisbouvier, J., Schanda, P., Pardi, A., Simorre, J.-P., and Brutscher, B. 2009. Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution. J. Am. Chem. Soc. 131:8571-8577. Geen, H. and Freeman, R. 1991. Band-selective radiofrequency pulses. J. Mag. Reson. 93:93-141. Hennig, M. and Williamson, J.R. 2000. Detection of N-H . . . N hydrogen bonding in RNA via scalar couplings in the absence of observable imino proton resonances. Nucleic Acids Res. 28:15851593. Kupce, E. and Freeman, R. 1994. Wideband excitation with polychromatic pulses. J. Magn. Reson. A 108:268-273. Lescop, E., Kern, T., and Brutscher, B. 2010. Guidelines for the use of band-selective radiofrequency pulses in hetero-nuclear NMR: Example of longitudinal-relaxation-enhanced BEST-type 1H-15N correlation experiments. J. Magn. Reson. 203:190-198. Liu, A., Majumdar, A., Hu, W., Kettani, A., Skripkin, E., and Patel, D.J. 2000. NMR detection of N-H . . . O=C hydrogen bonds in 13 C, 15 N-labeled nucleic acids. J. Am. Chem. Soc. 122:3206-3210. Low, J.T. and Weeks, K.M. 2010. SHAPE-directed RNA secondary structure prediction. Methods 52:150-158. Lu, K., Heng, X., Garyu, L., Monti, S., Garcia, E.L., Kharytonchyk, S., Dorjsuren, B., Kulandaivel, G., Jones, S., Hiremath, A., Divakaruni, S.S., LaCotti, C., Barton, S., Tummillo, D., Hosic, A., Edme, K., Albrecht, S., Telesnitsky, A., and Summers, M.F. 2011. NMR detection of structures in the HIV-1 5 -leader RNA that regulate genome packaging. Science 334:242245.

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Current Protocols in Nucleic Acid Chemistry

Luy, B. and Marino, J.P. 2000. Direct evidence for Watson-Crick base pairs in a dynamic region of RNA structure. J. Am. Chem. Soc. 122:80958096. Luy, B., Richter, U., DeJong, E.S., Sorensen, O.W., and Marino, J.P. 2002. Observation of H-bond mediated 3hJH2H3 coupling constants across Watson-Crick AU base pairs in RNA. J. Biomol. NMR 24:133-142. Majumdar, A. and Patel, D.J. 2002. Identifying hydrogen bond alignments in multistranded DNA architectures by NMR. Acc. Chem. Res. 35:1-11. Majumdar, A., Kettani, A., and Skripkin, E. 1999a. Observation and measurement of internucleotide 2JNN coupling constants between 15N nuclei with widely separated chemical shifts. J. Biomol. NMR 14:67-70. Majumdar, A., Kettani, A., Skripkin, E., and Patel, D.J. 1999b. Observation of internucleotide NH . . . N hydrogen bonds in the absence of directly detectable protons. J. Biomol. NMR 15:207-211. Majumdar, A., Gosser, Y., and Patel, D.J. 2001a. 1H-1H correlations across N-H . . . N hydrogen bonds in nucleic acids. J. Biomol. NMR 21:289306. Majumdar, A., Kettani, A., Skripkin, E., and Patel, D.J. 2001b. Pulse sequences for detection of NH2 . . . N hydrogen bonds in sheared G . A mismatches via remote, non-exchangeable protons. J. Biomol. NMR 19:103-113. Pervushin, K., Ono, A., Fernandez, C., Szyperski, T., Kainosho, M., and Wuthrich, K. 1998. NMR scalar couplings across Watson-Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 95:14147-14151.

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Key Reference Dallmann et al., 2013. See above. This is the original publication of the BESTsellr HNN-COSY and Py H(CC)NN-COSY experiments, including more theoretical background on the pulse sequences. The protocols presented here focus on practical aspects of the implementation of these experiments.

Biophysical Analysis of Nucleic Acids

7.22.19 Current Protocols in Nucleic Acid Chemistry

Supplement 59

Detection of hydrogen bonds in dynamic regions of RNA by NMR spectroscopy.

NMR spectroscopy is a powerful tool to study the structure and dynamics of nucleic acids. In this unit, we give an overview of important experiments t...
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