We thank the protein science facility (PSF) at the Karolinska Institutet for expression and purification of T7 RNA polymerase and E. coli RNase H, Martin H&#228;llberg for the generous gift of the inorganic phosphatase, and the entire Petzoldlab for valuable discussions. We thank Luca Retattino for preparation of the U-bulge constructs and Emilie Steiner and Carolina Fontana for their contribution to macros and fitting scripts. We acknowledge the Karolinska Institute and the Dept. of Medical Biochemistry and Biophysics for the support of the purchase of a 600 MHz spectrometer and position financing (KI FoAss and KID 2-3707/2013). We are grateful for financial contribution from Vetenskapsr&#229;det (#2014-4303), Stiftelsen f&#246;r strategisk Forskning (ICA14-0023 and FFL15-0178) and The Ragnar S&#246;derberg Stiftelse (M91-14), Harald och Greta Jeansson Stiftelse (JS20140009), Carl Tryggers stiftelse (CTS14-383 and 15-383), Eva och Oscar Ahr&#233;ns Stiftelse, &#197;ke Wiberg Stiftelse (467080968 and M14-0109), Cancerfonden (CAN 2015/388), J.S. acknowledges funding through a Marie Sk&#322;odowska-Curie IF (EU H2020, MSCA-IF project no. 747446).

K.P. is consultant to Arrakis Therapeutics, a company that discovers small molecules targeting RNA.

The protocol presented herein is a synthesis of several protocols published previously in the form of research articles10,20,21,31. Hence, segments of the protocol can be applied, while others can be exchanged to the preference of the reader. For example, the R1&#961; measurements can be performed on an RNA sample produced with any method, given that folding and homogeneity of length are assumed. Furthermore, the protocol does not contain information on resonance assignment of the RNA sequence-a step required for RD experiments-as this has been covered extensively in previous literature19,37,38. Partial, segmental, or site-specific labeling schemes36,41,42,43,44&#160;are approaches to facilitate resonance assignment or reduce the overlap of resonances that are of interest in RD experiments and have been described at length in the literature. This method allows the use of uniform labeling of any nucleotide identity, which can already simplify resonance assignment significantly.
The IVT method presented here overcomes known issues with sequences and labeling, increases yield, and decreases cost and work time compared to other methods. The use of the viral initiation sequence reduces the need for reaction optimization, which is a known problem in the field that can be time-consuming to perform and yields only few copies of the transcript in the case of non-G initiation. The T7 IVT and RNase H cleavage of the tandem transcript can be performed simultaneously in the same vessel. A pattern of multimeric tandem repeats can be seen on a denaturing PAGE gel during the reaction, which coalesces to a single band on the target RNA upon completion of RNase H reaction (Figure 3A, lanes 1 and 2b). Typical yields using this method range between 30 and 70 nmol RNA per 1 mL IVT. Yet, the method based on RNase H cleavage of tandem repeats does not come without certain problems of its own. The RNase H cleavage reaction often does not go to completion when run simultaneously with T7 transcription (Figure 3A, lane 2a).
The separation of tandem units can be finalized by annealing the cleavage guide to the transcript and adding more RNase H (Figure 3A, lane 2b, step 2.1.2). As heating of large volumes is slow and leads to Mg2+-catalyzed hydrolysis of RNA, a conventional microwave oven was used, which heats the sample to &#62;95 &#176;C in 10-15 s. Adverse effects on the produced samples have not been observed so far. Some constructs show a minor second band that could not be eliminated by the optimization of the reaction conditions (Figure 3A, lane 4). Usually these are rather clearly visible as a shoulder in the HPLC chromatogram, if a well-optimized elution gradient is used, and can be removed (step 2.2.5). The following discussion is aimed to highlight critical steps in the protocol, specifically with respect to obtaining high-quality data that allow an interpretation of conformational dynamics.
RNase contamination 
Extracellular RNases are ubiquitous, highly stable, and pose the biggest threat for long-term stability of NMR samples. Therefore, it is crucial to work in an RNase-free environment and keep all reagents and plasticware RNase-free. The use of filter tips and maybe even facemasks is recommended. This is specifically important after HPLC purification. NMR samples contaminated with RNases typically exhibit narrow peaks visible in 1H-1D spectra after days or weeks due to single-nucleotide degradation products. Such a sample is not suitable for R1&#961;&#160;measurements.
NMR sample 
Owing to its highly charged nature, RNA can be used in high concentrations without precipitation when compared to most proteins. The use of Shigemi NMR tubes (see the Table of Materials) is advantageous as they allow centering of the highly concentrated sample in the center of the coil while still providing ideal shimming and locking conditions due to the susceptibility-matched glass bottom and plunger. This way, B1-inhomogeneity is reduced, giving rise to narrower lines. The typical sample volume in an NMR tube is 250 &#181;L, and typical concentration is 1-2 mM. Samples below 500 &#181;M are not recommended for RD experiments as the experiment would take too long and a good shim. Similarly, sample volume below 200 &#181;L is not recommended because a good shim and field stability (lock) is required. When inserting the plunger, it is crucial to avoid the formation of bubbles in the sample (step 2.4.5). If not fixed properly, the plunger can slide down into the sample, reducing the detectable volume. Furthermore, rapid changes in temperature can lead to the formation of new bubbles in the sample. Therefore, care should be taken when transporting the sample and when changing the probe temperature in the NMR spectrometer. Check the sample for bubbles when measuring again after a longer period.
RNA folding 
Dynamic RNA molecules can exist in multiple conformations when not folded properly. Even though melting temperatures of secondary structures can be only slightly above room temperature, a thorough heating-and-snap-cooling procedure is recommended before measurement. Highly concentrated hairpin samples folding under kinetic control (heating-and-snap-cooling) can form homodimers over time, which necessitates rigorous control of RNA folding before each NMR measurement. If the measured RNA is not a hairpin structure but an RNA duplex, slow folding under thermodynamic control should be applied.
In this case, the cooling process after heating should be in the range of hours, while the RNA is used at its final volume and concentration in the NMR sample. An initial count of expected imino and aromatic resonances can provide insight about the homogeneity of the sample. If the sample does not look like expected, it should be re-folded. Mg2+ (added as chloride salt) can help with folding RNA structures45. In practice, the folding control serves as a comparison to a sample that has been used to at least partially assign the NMR resonances and to solve the secondary structure experimentally.
Spin lock power and heating considerations 
In case of running the 1H R1&#961; RD experiments as 2D overview experiments, SL power should be no lower than 1.2 kHz. The radiofrequency transmitter frequency should be placed in the middle of the ppm region of the peaks of interest (e.g., 7.5 ppm for aromatic protons). The bandwidth of 1.2 kHz will then be large enough to spin-lock these protons without any major off-resonance effects. Such effects can be identified in the RD profile. If they occur, R2+REX values increase instead of decrease with increasing SL power values, especially for low SL power. Check if the calculated SL power values correspond to the power delivered to the sample. In practice, calculated SL power can be used if the 1H 90&#176; hard pulse was calibrated carefully on newer spectrometers; however, this can be checked by calibrating SL power for each desired bandwidth.
The range of SL power, which can be used in 1H R1&#961; RD experiments is very broad, leading to varying sample heating (1.2 kHz to 15 kHz for HSQC for&#160;HCP-based sequences and 50 Hz to 15 kHz for SELOPE experiments). Unequal sample heating can be detected as a slight change in chemical shift when comparing 1Ds obtained for low power SLs vs. high power SLs. This effect is usually not considered in heat compensations in R1&#961; experiments on heteronuclei. Heat compensation in those experiments is usually set up to correct for different heating due to the different spin lock durations specified in the vd list of each spin lock power series. Especially for the SELOPE experiment, a second heat compensation should be used across all applied SL strengths as described in20.
vd list considerations 
As mentioned earlier, the vd list should contain a time point long enough to obtain a significant decay of intensity (ideally down to 30% of initial signal, or as low as possible if it is not possible to reach a 70% decay within the specifications of the probe). Although the vd list was optimized for a low SL power (1.2 kHz), this vd list should also be tested at the highest SL power to be used (e.g., 15 kHz). This is due to the fact, that for peaks with significant REX contribution, the decay will be much slower at high SL power. So a sufficient decay should also be verified at high SL power. The same must be considered for decays at high offsets in off-resonance experiments. The ideal maximum time point of the vd list could be significantly different for the different regions of the dispersion experiment. In that case, more points could be included in the vd list, and the longer vd list points for higher SL power or higher offsets during analysis, based on the low SINO they will lead to, could be discarded. In general, 5-8 vd list points should be considered to be able to spot potential artefacts leading to non-exponential decays such as J-coupling (see below).
1D-HCP selectivity considerations 
Special care must be taken when running the HCP-based 1D version if there is another peak overlapping with the peak of interest in the 1H dimension of the 2D HSQC-based experiment. HCP-based transfers are very, but never 100% selective, and it can therefore happen that another peak contributes to the intensity and decay behavior of the peak of interest in the 1D. An indication for this would be a difference in on-resonance R1&#961; values obtained using the 1D and 2D versions of the labeled experiment.
ROE considerations: 
For off-resonance curves of atoms with slow-intermediate exchange, ROE artefacts can be identified based on a comparison of the obtained &#916;&#969; with a NOESY or ROESY spectrum. If a cross peak can be identified at a chemical shift difference corresponding to &#916;&#969;, then the observed excited state might in fact be a ROE artifact (e.g., ROEs were found between aromatic protons, which are all in the same chemical shift range and therefore covered by those off-resonance curves20). From experience, this always also led to poor fits with large errors, possibly due to the ROE not following the same pattern as REX with increasing SL power. The situation becomes more difficult for intermediate-fast exchange. While the on-resonance curve is (from comparison with 13C data obtained on the neighboring nucleus) still representative of the exchange process between the GS and ES, the off-resonance curve is influenced by multiple ROE artefacts.
In that case, the SL power to detect the exchange process is larger (&#62;1.5 kHz) and therefore spans a larger number of protons as off-resonance curves span over chemical shift differences of various ROE candidates (for H8 these would be: amino protons at ca. &#177;1000 Hz, H5/H1&#39;s at ca. -1200&#160;Hz, imino protons at ca. 3500 Hz). So far, no method has been found to suppress these ROE artefacts (other than using partially deuterated nucleotides46), and off-resonance data should not be recorded for fast-intermediate exchange, as no reliable information on the actual&#160;&#916;&#969;&#160;can be extracted with this method, if NOE/ROE contribution cannot be excluded via NOESY spectra.
J-Coupling (Hartmann-Hahn) considerations 
Although on-resonance curves for homonuclear J-coupled protons, such as H6, were successfully recorded10,20, special care must be taken for off-resonance measurements, especially for low SL power as Hartmann-Hahn matching conditions can span a wide range of the investigated offsets. Hartmann-Hahn artefacts can be identified as oscillations on the exponential decay or increasing R2+REX values with increasing SL strengths in on-resonance RD plots20.

RNAs perform a multitude of regulatory1, catalytic2, and structural3 functions in the cell, many of which are correlated to a flexible molecular structure and intricate changes of those structures4,5,6,7. Low-populated states remain invisible to most methods of structure determination or do not allow the study of these hidden states at high atomic resolution. Solution-state nuclear magnetic resonance (NMR) spectroscopy combines both aspects by providing access to individual atomic nuclei as well as offering a large toolbox of experiments targeting dynamics through all time regimes8. RD NMR experiments provide access to conformational exchange in the intermediate timescale, wherein changes in base pairing patterns and local structural rearrangements can be expected5,9,10,11,12,13,14. RD experiments are performed as long R2 measurements in the form of a Carr-Purcell-Meiboom-Gill pulse train15 or as relaxation measurements in the rotating frame, called R1&#961; RD experiments16.
Although both can be used to extract population of and exchange rate and chemical shift difference to the minor state, R1&#961; RD experiments also give the sign of the chemical shift difference of the excited state. This allows an inference on secondary structure, which strongly correlates to chemical shift in RNA structures17. The chemical shift is a good indicator of helicity in the case of aromatic protons and carbons on the nucleobases, of base pairing partners for imino protons, and of sugar puckers on the C4&#39; and C1&#39; atoms18,19. It should be noted that recently a chemical exchange saturation transfer (CEST) experiment using higher spin lock (SL) power, thereby shifting the applicability of the CEST experiment to faster exchange timescales, was published as an alternative to the R1&#961; RD experiment for systems with one excited state.
Although 13C and 15N isotopes have often been used to access structural exchange, recent work from this laboratory used aromatic and imino protons as probes for conformational exchange9,10. The use of 1H as the observed nucleus brings several advantages, for example, access to exchange on faster and slower timescales, higher sensitivity, and shorter measurement times. This is further facilitated by the SELective Optimized Proton Experiment (SELOPE) approach, providing access to aromatic protons through decrowding of the one-dimensional (1D) spectrum using homonuclear scalar couplings, instead of a heteronuclear magnetization transfer, and eliminating the need for isotope labels20. This protocol addresses the measurement in 1H&#160;R1&#961;&#160;RD experiments of uniformly 13C/15N-labeled and unlabeled samples. Therefore, this paper presents a sample preparation method that was found to be the most versatile for different sample preparation needs21 and discusses alternatives in the last section of this article (Figure 1).
At this point, the reader should note that other sample preparation techniques are acceptable for 1H R1&#961; RD experiments, and that other methods of structural and functional analysis can be performed with the samples synthesized with the presented technique. 1H R1&#961; RD experiments require high RNA concentrations (ideally &#62;1 mM) as well as high homogeneity, both in RNA length and structural conformation to ensure reliable characterization of molecular dynamics. In vitro transcription (IVT) is the method of choice for many researchers to produce 13C/15N-labeled RNA samples due to the availability of labeled nucleoside triphosphates (NTPs) and facile incorporation in the enzymatic reaction22. However, the widely used T7 RNA polymerase (T7RNAP)23,24,25 suffers from low 5&#39; homogeneity in case of certain initiation sequences26,27 and often also 3&#39; homogeneity during transcription runoff28. Purification of the target RNA species becomes more expensive and laborious due to the need of large&#160;quantities of ~200 nmol. The method used here has been presented previously where advantages were discussed at large21. In brief, it solves described issues by transcribing a larger tandem transcript that is then site-specifically cleaved by Escherichia coli RNase H, guided by a chimeric oligonucleotide29,30 (see Figure 2 for details).
Incorporation of a spacer sequence at the 5&#39; and 3&#39; ends of the tandem transcript allows the use of a high-yield initiation sequence and removal of terminal overhangs close to the linearization site of the plasmid template, respectively (Figure&#160;2B). The method was shown to improve yields significantly, while reducing cost and labor, with the caveat of a more complex template synthesis and the need for an additional enzyme and oligonucleotide. The high specificity of RNase H cleavage facilitates purification due to the lack of RNA species in a similar size range. The present protocol uses an ion exchange high-performance liquid chromatography (HPLC) step that has been published by this laboratory recently31, although other methods are possible alternatives. 1H R1&#961;&#160;RD can, in general, be acquired on labeled or unlabeled samples with two respective pulse sequences, the &#8220;labeled&#8221; 1H R1&#961; heteronuclear single quantum correlation (HSQC)-based experiment with a 13C indirect dimension10&#160;and the &#8220;unlabeled&#8221; 1H&#160;R1&#961; SELOPE-based experiment with a 1H indirect dimension20.
These two-dimensional (2D) experiments can serve as a first check, regardless of whether dynamics on the R1&#961; timescale are present in the sample. An overview of RD for all resolved peaks in the spectra can be obtained, and peaks of interest for a more thorough RD analysis can be identified. This means that even unlabeled samples can be checked before a decision to produce a more expensive, labeled sample is made. Once a peak with conformational exchange contribution is selected to be studied more thoroughly, it is best to switch to the 1D versions of the above experiments (if the peak can still be resolved) to carry out so-called off-resonance experiments. For the labeled version, the HSQC transfer to 13C is replaced with a selective heteronuclear cross-polarization (HCP) step as used in 13C R1&#961; experiments32,33,34,35,&#160;while in the case of the SELOPE experiment, the experiment is simply run as a 1D, which is especially useful for H8 and H2 signals that are lying on the diagonal in the 2D anyway. One criterion as to which sequence to use, provided that both, a labeled as well as unlabeled sample are available, is how well isolated the peak of interest is in the two experiments.
In general, the SELOPE experiment is recommended for RNA samples of up to 50 nucleotides. For larger RNAs, the overlap will be bigger; however, structurally interesting nucleotides often appear in chemical shift regions that are less overlapped and still might be accessible in even larger RNAs. Another argument would be that in unlabeled samples, no J-coupling occurs between 1H and 12C. However, as the minimum spin lock power is defined by the minimum power used to decouple those two spins (~1 kHz) in the labeled experiment, the unlabeled experiment allows the use of a broader range of spin lock (SL) strengths and therefore, access to a broader timescale of exchange. These off-resonance experiments provide additional information to kex, such as population of the excited state (alternative conformer), pES, as well as very valuable chemical shift information in the form of&#160;&#916;&#969; (the chemical shift difference of the ground state and the excited state).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62470/62470fig1v2.jpg" /> 
Figure 1: Workflow of the presented protocol. Preparation before the actual large-scale sample production, consisting of template preparation and confirmation of successful in vitro transcription and RNase H cleavage. Large scale production including HPLC purification, filling of NMR tube, and confirmation of RNA folding. In case of isotope-labeled synthesis, an unlabeled purification should be performed for gradient optimization on the same day. NMR characterization of conformational dynamics with R1&#961; experiments. Each step can be performed independently, e.g., the 1H R1&#961; RD analysis can be applied to any suitable RNA sample produced with another method. Abbreviations: IVT = in vitro transcription; HPLC = high-performance liquid chromatography; NMR = nuclear magnetic resonance; RD = relaxation dispersion. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62470/62470fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The aim of this protocol is to provide practical details and critical parameters for the study of conformational dynamics with 1H R1&#961; relaxation dispersion in RNA hairpin molecules. After providing a detailed protocol of the design, synthesis, and ion exchange HPLC purification of a target RNA that can be performed using all, some, or none NTPs as 13C/15N-labeled versions, the workflow of finalizing the NMR sample and confirming the conformational exchange with NMR spectroscopy has been described. Finally, the details for the setup of 1H R1&#961; RD experiments on a Bruker NMR spectrometer are described (Figure 1). The protocol gives each step to set up the 1D version for labeled samples and additional comments and a table to adjust for the setting up of the SELOPE version (Table 2). After the protocol, critical steps and alternative routes to sample preparation and 1H R1&#961; RD setup are discussed.

<table><tbody><tr><td>40% Acrylamide/Bis Solution</td><td>&amp;nbsp;Bio-Rad</td><td>&amp;nbsp;161-0144</td><td></td></tr><tr><td>5-alpha Competent&amp;nbsp;&lt;em&gt;E. coli&lt;/em&gt;</td><td>NEB</td><td>C2987I</td><td></td></tr><tr><td>Acetic Acid</td><td>&amp;nbsp;Sigma-Aldrich</td><td>49199</td><td></td></tr><tr><td>Acetonitrile</td><td>&amp;nbsp;Sigma-Aldrich</td><td>34851</td><td></td></tr><tr><td>AFC-3000, HPLC Fraction collector&amp;nbsp;</td><td>Thermo Scientific</td><td>5702.1</td><td></td></tr><tr><td>Agarose</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;A9414</td><td></td></tr><tr><td>Amersham ImageQuant 800 UV</td><td>GE Healthcare</td><td>29399482</td><td>Replacing LAS-4000 or equivalent</td></tr><tr><td>Amicon ultra centrifugal filter unit</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;UFC900324</td><td></td></tr><tr><td>Ammonium persulfate</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;A3678</td><td></td></tr><tr><td>Ampicillin</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;A9518</td><td></td></tr><tr><td>ATP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;A2383</td><td></td></tr><tr><td>ATP-13C10/15N5</td><td>&amp;nbsp;Sigma-Aldrich</td><td>645702</td><td></td></tr><tr><td>BamHI restriction enzyme</td><td>NEB</td><td>&amp;nbsp;R0136L</td><td></td></tr><tr><td>Bottle top filter&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>514-1019</td><td></td></tr><tr><td>Bromophenol Blue</td><td>&amp;nbsp;Sigma-Aldrich</td><td>1081220005</td><td></td></tr><tr><td>Cleavage guide</td><td>IDT</td><td>N/A</td><td>or equivalent</td></tr><tr><td>CTP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;C1506</td><td></td></tr><tr><td>CTP-13C10/15N5</td><td>&amp;nbsp;Sigma-Aldrich</td><td>645699</td><td></td></tr><tr><td>D2O</td><td>&amp;nbsp;Sigma-Aldrich</td><td>151882</td><td></td></tr><tr><td>Dionex Ultimate 3000 UHPLC system&amp;nbsp;</td><td>Thermo Scientific</td><td>N/A</td><td></td></tr><tr><td>DL-Dithiotreitol</td><td>&amp;nbsp;Sigma-Aldrich</td><td>43815</td><td></td></tr><tr><td>DMSO</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;D8418</td><td></td></tr><tr><td>DNAPac PA200 22x250 Semi-Prep column&amp;nbsp;</td><td>Thermo Scientific</td><td>SP6734</td><td></td></tr><tr><td>DNAPac PA200 22x50 guard column&amp;nbsp;</td><td>Thermo Scientific</td><td>SP6731</td><td></td></tr><tr><td>E.coli RNase H</td><td>&amp;nbsp;NEB</td><td>&amp;nbsp;M0297L</td><td>&amp;nbsp;or made in-house uniprot ref. P0A7Y4&amp;nbsp;</td></tr><tr><td>EDTA</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;E6758</td><td></td></tr><tr><td>Eppendorf centrifuge, rotor: A-4-44</td><td>Eppendorf&amp;nbsp;</td><td>5804R</td><td></td></tr><tr><td>Ethanol 95%</td><td>&amp;nbsp;Fisher scientific</td><td>11574139</td><td></td></tr><tr><td>Ethanol 95% denatured</td><td>&amp;nbsp;VWR</td><td>85829.29</td><td></td></tr><tr><td>Formamide</td><td>&amp;nbsp;Sigma-Aldrich</td><td>47671</td><td></td></tr><tr><td>GelRed</td><td>&amp;nbsp;VWR</td><td>41003</td><td></td></tr><tr><td>GeneRuler 1kbp Plus</td><td>&amp;nbsp;Fisher Scientific</td><td>&amp;nbsp;SM1333</td><td>Optional</td></tr><tr><td>GMP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;G8377</td><td></td></tr><tr><td>GMP-13C10/15N5</td><td>&amp;nbsp;Sigma-Aldrich</td><td>650684</td><td></td></tr><tr><td>GTP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;G8877</td><td></td></tr><tr><td>GTP-13C10/15N5</td><td>&amp;nbsp;Sigma-Aldrich</td><td>645680</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;H1758</td><td></td></tr><tr><td>Inorganic pyrophosphatase</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;I1643-100UN</td><td>&amp;nbsp;or made in-house uniprot ref. P0A7A9</td></tr><tr><td>Invitrogen UltraPure 10X TBE-buffer</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;T4415</td><td></td></tr><tr><td>Julabo TW8 Water bath&amp;nbsp;</td><td>VWR</td><td>461-3117</td><td></td></tr><tr><td>kuroGEL Midi 13 Horizontal gel electrophoresis</td><td>VWR</td><td>700-0056</td><td>or comparable</td></tr><tr><td>LB broth (Lennox)</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;L3022</td><td></td></tr><tr><td>LB broth with agar (Lennox)</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;L2897</td><td></td></tr><tr><td>Low Range ssRNA Ladder&amp;nbsp;</td><td>NEB</td><td>&amp;nbsp;N0364S</td><td>Optional</td></tr><tr><td>LPG-3400RS Pump&amp;nbsp;</td><td>Thermo Scientific</td><td>5040.0036</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>&amp;nbsp;Sigma-Aldrich</td><td>63068</td><td></td></tr><tr><td>microRNA Marker&amp;nbsp;</td><td>NEB</td><td>N2102S</td><td></td></tr><tr><td>Microwave oven</td><td>&amp;nbsp;Samsung</td><td>&amp;nbsp;MS23F301EAW</td><td></td></tr><tr><td>Mini-PROTEAN electrophoresis equipment</td><td>&amp;nbsp;Bio-Rad</td><td>1658004</td><td></td></tr><tr><td>NucleoBond Xtra Maxi</td><td>&amp;nbsp;Machinery-Nagel</td><td>&amp;nbsp;740414.10M</td><td></td></tr><tr><td>pUC19 plasmid containing tandem insert</td><td>&amp;nbsp;Genscript</td><td>&amp;nbsp;N/A</td><td>or equivalent</td></tr><tr><td>RNaseZAP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;R2020</td><td></td></tr><tr><td>Shigemi tube 5mm&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Z529427</td><td></td></tr><tr><td>Single-use syringe, Luer lock tip</td><td>VWR</td><td>613-2008</td><td></td></tr><tr><td>Sodium acetate</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;S2889</td><td></td></tr><tr><td>Sodium chloride</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;730-1470</td><td></td></tr><tr><td>Sodium perchlorate</td><td>&amp;nbsp;Sigma-Aldrich</td><td>71853</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;S3264</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;S3139</td><td></td></tr><tr><td>Spermidine trihydrochloride</td><td>&amp;nbsp;Sigma-Aldrich</td><td>85578</td><td></td></tr><tr><td>SYBR Gold</td><td>&amp;nbsp;ThermoFisher</td><td>&amp;nbsp;S11494</td><td></td></tr><tr><td>Syringe filters</td><td>VWR</td><td>514-0061</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>&amp;nbsp;Sigma-Aldrich</td><td>10881767001</td><td>&amp;nbsp;or made in-house uniprot ref. P00573</td></tr><tr><td>TCC-3000RS Column thermostat&amp;nbsp;</td><td>Thermo Scientific</td><td>5730</td><td></td></tr><tr><td>Tetramethylethylenediamine</td><td>&amp;nbsp;Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris Base</td><td>&amp;nbsp;Fisher Scientific</td><td>10103203</td><td></td></tr><tr><td>UMP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>U6375</td><td></td></tr><tr><td>UMP-13C9/15N2&amp;nbsp;</td><td>Sigma-Aldrich</td><td>651370</td><td></td></tr><tr><td>Urea</td><td>&amp;nbsp;Sigma-Aldrich</td><td>U5378</td><td></td></tr><tr><td>UTP</td><td>&amp;nbsp;Sigma-Aldrich</td><td>U6625</td><td></td></tr><tr><td>UTP-13C10/15N5</td><td>&amp;nbsp;Sigma-Aldrich</td><td>645672</td><td></td></tr><tr><td>VWD-3100 Detector&amp;nbsp;</td><td>Thermo Scientific</td><td>5074.0005</td><td></td></tr></tbody></table>

practical aspects of sample preparation and setup of 1h r1&#961; relaxation dispersion experiments of rna

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

The protocol for RNA production facilitates purification through the generation of high-purity transcripts. Figure 3A shows the results of several cleavage reactions of tandem transcripts, providing both successful and unsuccessful reactions. Lane 1 shows the optimal case of a fully cleaved transcript with only faint traces of side products. Lane 2a shows incomplete cleavage, which can be resolved by re-annealing and the addition of more RNase H (Lane 2b, step 2.1.2). The RNA constructs of lanes 1, 2a, and 2b are the same. The sample in lane 3 shows unsuccessful cleavage. Troubleshooting this reaction would involve a check of the cleavage guide sequence, purity of DNA template, and annealing temperatures. Potentially, RNase H cleavage will have to be performed after T7 IVT as shown for sample 2.
The sample in lane 4 shows a significant amount of cleavage side products, which are difficult to remove via ion-exchange HPLC. Troubleshooting such a sample can involve (a) lowering temperature, amount of RNase H, or reaction time, (b) reducing elution gradient and injection volume and attempting to separate the target fractions from the side products. Further information on how to increase the resolution in ion exchange HPLC purification has been discussed by Karlsson et al.31. HPLC separates the target RNA from longer or shorter nucleic acids and protein or small-molecule contaminants. Figure 3B shows the optimal result for the ion-exchange HPLC purification. The elution gradient should be chosen such that the target RNA species elutes at least one column volume (in this example: 35 mL) after the next smaller species and one column volume before the next larger species.
Smaller species in this method include single nucleotides, abortive products (8-12 nt), 3&#39; and 5&#39; spacer sequences (5-14 nt), and cleavage guide (12 nt chimeric nucleic acid), whereas longer sequences are potentially uncleaved tandem repeats and the plasmid. When a well-separated elution peak is achieved, purification can be scaled up to the equivalent of ~20 mL of IVT reaction per injection. The correct fold of an RNA sample is crucial for RD experiments and has to be confirmed before every measurement. Figure 4 shows an A-labeled 22-mer RNA before the folding protocol in step 2.4 (blue) was applied, and the same sample after the correct folding has been achieved (red). A Mc-Fold secondary structure prediction (Figure 4C) proposes the presented hairpin structure with 4 base pairs resulting in 5 imino signals.
Both spectra in Figure 4A confirm these predicted signals, albeit with slightly different relative intensities, which indicates that some misfolded structure (here, a dimer) can be problematic to assess with only 1H 1D spectra. An aromatic 1H,13C-HSQC spectrum (Figure 4B), however, shows only 3 of the aromatic signals for the sample before the folding protocol (blue), but all 4 signals for the sample that has been folded according to step 2.4 (red). The sample shown in blue likely formed a homodimer (structure proposed in Figure 4D) that would result in the same imino signals as the hairpin. The signal of A13H2 seems exchange-broadened. These results help to highlight the importance of folding confirmation with both imino and aromatic fingerprint experiments before each RD experiment. The 1H R1&#961; pulse sequences described in this protocol allow the detection of dynamics in the intermediate exchange regime. Initially an on-resonance curve is recorded, and if dynamics are present for a specific residue, a dispersion is visible within the obtained R2+REX values, while this curve is flat for residues without exchange.
Figure 5&#160;shows representative on-resonance curves obtained for two different H8 atoms in a synthetic RNA hairpin (Figure 5A), wherein G6H8 experiences exchange (Figure 5C), while A4H8 does not (Figure 5B). As the exchange is relatively slow in this sample (kEX = 292 &#177; 40 Hz), the advantage of the SELOPE experiment to achieve low SL strengths was exploited, and the two on-resonance curves were recorded using the 1D version of the pulse sequence. The same pulse sequence was then used to obtain off-resonance data for the residue showing dispersion in the on-resonance profile. Figure 5D shows the obtained R1&#961; values vs. offset wherein a slight asymmetry of the curve already indicates the sign of&#160;&#916;&#969;.
This becomes even more apparent in the R2+REX plot where the R1 contribution is removed (Figure 5E). The right column of the same figure shows representative on-resonance curves obtained for two different H8 atoms in a slightly different synthetic RNA hairpin with faster exchange, wherein G6H8 experiences exchange (Figure 5G), whereas A4H8 does not (Figure 5F). The faster exchange rate (kEX = 43,502 &#177; 38,478 Hz) allowed the RD recording of all aromatic protons at once using the SELOPE 2D version to obtain both, on- and off-resonance data (G6H8 data displayed in Figure 5H,I).
General identifiers for positive and negative results 
Positive results in the tandem IVT and RNase H cleavage can be identified as follows: 1) The target band is the strongest band in the denaturing PAGE gel. 2) There are no or only weak bands around the main band. 3) There are no or only weak higher molecular weight species. 4) The HPLC chromatogram shows a well separated peak of the target RNA. 5) When the main peak is sampled, only one band appears on a denaturing PAGE gel.
Negative results in the tandem IVT and RNase H cleavage present as follows: 1) No or just a weak main band is visible on a denaturing PAGE gel. 2) A pattern of high molecular weight species from RNA tandem repeats is visible. 3) Although the main band is present, bands of similar intensity are above or below the main band within &#177; 3 nt.
A well-folded sample can be identified as follows: 1) The number of observed imino protons matches the number of imino protons expected from a secondary structure simulation (e.g., Mc-Fold39, Figure 4A). 2) The syn G-U wobble base pair in a UUCG loop (if present) is visible at ~9.5 ppm, sometimes only visible at lower temperature. Further fingerprinting of the UUCG loop has been described by F&#252;rtig and colleagues40. 3) The aromatic fingerprint agrees with a previously assigned sample that has been confirmed to fold correctly (Figure 4C).
A misfolded or degraded sample can be identified as follows: 1) There are more imino signals than a secondary structure simulation predicts (NOTE: fewer imino signals do not necessarily imply misfolding, as closing base pairs are often not visible, and conformational exchange broadens lines). 2) Absence of imino signals. 3) Narrow signals of high intensity in the aromatic region, indicating single nucleotide degradation products. 4) Divergence between imino or aromatic signals to a reference sample of confirmed folding (Figure 4C).
An atom showing no exchange in the detectable timescale can be identified as follows: 1) from a flat RD profile (due to the missing REX contribution varying with the applied SL power) (Figure 5B and Figure 5F). 2) Care has to be taken for the case of slow-intermediate exchange when kEX and &#916;&#969; are of the same magnitude. In that case, the on-resonance contribution can be very small as can be seen in Figure 5C (in this case the fitted parameters are kEX = 292 &#177; 40 Hz and &#916;&#969; = 112 &#177; 4 Hz). If in doubt, a low SL off-resonance curve can be recorded for verification.
An atom showing exchange in the intermediate time scale can be identified 1) from a non-flat relaxation dispersion profile in an on-resonance RD experiment (Figure 5B and Figure 5F); 2) a broader linewidth in the HSQC or SELOPE experiment can also be an indicator for exchange.
Well-selected SL power values for off-resonance curves (Figure 5E,F): 1) have a considerable kEX contribution in the on-resonance curve (selected SL power values are indicated in Figure 5C and Figure 5G). 2) As off-resonance curves are measured for at least 3 SL power values, the selected SL power values should be spread out over the region of the on-resonance curve with kEX contribution. 3) Lead to non-flat R2+REX curves after the Laguerre fit (e.g., Figure 5D: SL strengths 25, 50, and 75 Hz; Figure 5E). &#160;
Poorly selected SL power values for off-resonance curves (Figure 5E,F) lead to flat R2+REX curves after the Laguerre fit. An example is shown in Figure 5E, wherein the 100 Hz off-resonance curve is very flat and therefore does not provide significant information on &#916;&#969;.
Indications for rotating-frame nuclear Overhauser effect (ROE) artefacts: 1) &#916;&#969; obtained from off resonance curves match chemical shifts of protons in spatial vicinity / protons, which show a cross peak with the peak of interest in the nuclear Overhauser effect spectroscopy (NOESY) spectrum. (e.g., Figure 5I shows broad off-resonance curves as expected for fast-intermediate exchange, but the curves also have sharper features, e.g., at -3000 Hz and +1500 Hz. These are very likely due to an ROE artifact rather than a chemical shift for this H8 in a different conformer). 2) Laguerre fit does work, but does not work well (gives high errors or physically impossible values) for an on-resonance and at least 3 off-resonance curves, even though exponentials were obtained from experiments with high SINO (&#62;20) (e.g., kEX = 43,502 &#177; 38,478 Hz). Often each SL fits individually well, but fitting them together gives a much higher error; the opposite behavior is expected for a true excited state.
Indications for &#8220;true&#8221; exchange &#916;&#969;: 1)&#160;&#916;&#969; obtained from off-resonance curves do not match chemical shifts of protons in spatial vicinity/protons, which show a cross peak with the peak of interest in the NOESY spectrum (e.g., Figure 5E). 2)&#160;Laguerre fit gives low errors for an on-resonance and at least 3 off-resonance curves (e.g., Figure 5E vs. Figure 5I, see caption for fit results).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62470/62470fig3v5.jpg" /> 
Figure 3: Sample production by T7 tandem IVT and RNase H cleavage reaction. (A) Denaturing PAGE of positive and negative results of tandem IVT and RNase H cleavage. Ladder height refers to RNA references, 12* refers to the chimeric cleavage guide. Lane 1: Successful generation of a 20 nt target RNA. Few shorter and longer products are present. Lane 2a: Incomplete cleavage of the tandem transcript. Although HPLC purification is possible, a lot of material would be wasted. Lane 2b: Continued RNase H cleavage of Lane 2 produces a clean sample ready for HPLC injection (identical to Lane 1). Lane 4: RNase H cleavage was largely unsuccessful, and no target band was produced. The full-length tandem transcript is still visible at 600 nt. Lane 5: A target band was produced, but a strong -1 band is present. Although HPLC can be performed, careful removal of the side product is necessary. (B) Example of a successful HPLC injection. The peak at 38 min contains pure RNA of the target length, while longer and shorter products are well-separated from the target RNA. Panel B has been modified from 21. Abbreviations: IVT = in vitro transcription; HPLC = high-performance liquid chromatography; nt = nucleotides; AU = arbitrary units. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62470/62470fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62470/62470fig4v2.jpg" /> 
Figure 4: Example of an RNA hairpin before (blue) and after (red) the folding step 2.4 (see protocol) in NMR. (A) Imino region of a 1H-1D spectrum of an A-labeled 22-mer RNA. Expected regions for base pair identity of imino signals are indicated in gray below. (B) 1H,13C-HSQC spectrum of the aromatic resonances of the RNA from panel A. The sample after folding (red) shows 4 signals as expected, while the sample before folding (blue) shows only 3 signals. (C) Mc-Fold prediction of the 22-mer RNA as a hairpin. Five imino signals are to be expected from this secondary structure, which can be found in both samples in panel A. (D) Proposed structure of a homodimer formed by the 22-mer RNA, resulting in the same 5 base pairs as the hairpin structure. Abbreviations: NMR = nuclear magnetic resonance; 1D = one-dimensional; HSQC = heteronuclear single quantum correlation; ppm = parts per million. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62470/62470fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62470/62470fig5v2.jpg" /> 
Figure. 5: 1H R1&#961; RD representative results for two different constructs based on an RNA hairpin. (A) The left column shows results obtained on the RNA with a C-G base pair above the bulged U, while the right column shows results obtained on a sample where the base pair was switched to G-C instead. (B) and (F) show flat dispersion profiles as obtained for A4H8 for the two constructs, indicating no conformational exchange. (C&#8211;E) show on-resonance, off-resonance, and fitted data obtained for G6 in the (G-C) construct. The Laguerre fit leads to the following result: R1 = 2.87 &#177; 0.01 Hz, R2 = 7.76 &#177; 0.03 Hz, kEX =292 &#177; 40 Hz, pES = 0.31 &#177; 0.03 %, &#916;&#969; = 112 &#177; 4 Hz. (G&#8211;I) show on-resonance, off-resonance, and fitted data obtained for G6 in the (G-C) construct. The Laguerre fit leads to the following result: R1 = 1.93 &#177; 0.02 Hz, R2 = 6.71 &#177; 0.86 Hz, kEX = 43,502 &#177; 38,478 Hz, pES = 27 &#177; 16 %, &#916;&#969; = 203 &#177; 166 Hz. This figure was modified from 20. Abbreviation: SL = spin lock. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62470/62470fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Practical Aspects of Sample Preparation and Setup of 1H R1Ï Relaxation Dispersion Experiments of RNA at JoVE.com

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

practical-aspects-sample-preparation-setup-1h-r1-relaxation

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

4.6K Views.  Karolinska Institutet. We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1ρ relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

Video: Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Genetics

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Chemistry

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biology

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

Practical Aspects of Sample Preparation and Setup of ¹H R_1ρ Relaxation Dispersion Experiments of RNA

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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Identification of Circular RNAs using RNA Sequencing

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

¹⁵N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

NMR ¹⁵N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins