It is important to note that this work was a collaborative effort between three institutions, two research groups, and one core facility. The distribution and workload were carried out as follows: Protein (Xrn-1) expression was carried out at the University of Denver (Denver, CO). Oligonucleotide synthesis, quantification, and experimentation (mainly enzymatic degradation) were conducted at the University of Colorado Denver (Denver, CO). Optimization was also carried out there. MALDI-TOF spotting, acquisition, and analysis were carried out at the Analytical Resources Core Facility at Colorado State University. (Fort Collins, CO). SS would like to acknowledge a UROP Award (CU Denver) and Eureca grants (CU Denver) for support. E. G. C. acknowledges support from NIGMS, via R00GM115757. MJER acknowledges support from NIGMS, via 1R15GM132816. K. B. acknowledges resource ID: SCR_021758. The work was also supported by a Teacher-Scholar Award (MJER), TH-21-028, from the Henry Dreyfus Foundation.

RNase-free ultra pure water (Table 1) was used for the present study.
1. Concentration determination of RNA solution
<ol>
	<li>Prepare the RNA sample following the steps below.
	<ol>
		<li>Use a microcentrifuge tube (0.6 mL) to prepare a solution of RNA by diluting 1 &#181;L of stock solution (obtained via solid-phase synthesis)19 into 159 &#181;L of RNase-free H2O. Mix the solution by pipetting the mixture up and down repeatedly (10x). 
		NOTE: Since a wide range of cuvettes are commercially available, the needed volumes may differ. Cuvettes with a 1 cm pathlength and reduced volume (150 &#181;L) were used in this work.</li>
		<li>Rinse the UV-vis cuvette with methanol (2x), followed by water (ultrapure H2O, 3x, see Table of Materials). Use a flow of nitrogen gas to dry the cuvette thoroughly.</li>
		<li>Use a pipette to transfer the solution (step 1.1.1) into the cuvette, ensuring no air bubbles. Cap the cuvette and place it to the side until needed.</li>
	</ol>
	</li>
	<li>Determine the concentration of the prepared RNA solution using UV-vis Spectrophotometer.
	<ol>
		<li>Turn on the UV-Vis spectrophotometer by flipping the switch located in the back of the instrument. Allow the instrument to complete the startup procedure and click on the UV WinLab icon to open the instrument operation/control window.</li>
		<li>Turn on the Peltier Temperature Controller spectrophotometer using the switch located to the instrument&#39;s right.</li>
		<li>Under Base Methods, click on Scan - Lambda 365. Another screen will open and prompt the user to ensure that the cell holders are empty. Click on OK and allow the instrument to conduct its system checks. The system will then display a list of checks, ensure that all &#34;Pass&#34; and click on OK. 
		NOTE: It is recommended to follow steps 1.2.1-1.2.3 in the provided order; altering this sequence of events may lead to software malfunction.</li>
		<li>Adjust the scanning parameters by selecting Data Collection. In the new window under Scan Settings change the Start to 350 nm and the End to 215 nm.</li>
		<li>Activate the Peltier by selecting the + to the left of the Accessory. Click on Multiple Cell Peltier, change the Temperature &#176;C to 25, and click on Peltier On.</li>
		<li>Navigate to the Sample Info tab and enter the number of samples, names, and cell position as desired.</li>
		<li>Click on Autozero and insert the cuvette containing the background solution. Ensure to position this cuvette in the same slot where measurements will be carried out.</li>
		<li>Click on Start and insert the cuvette (step 1.1.3) in the desired cell. Ensure that the cuvette window is oriented parallel to the front face of the instrument. Click on OK to begin the first scan at 25 &#176;C.</li>
		<li>For measurements at higher temperatures, repeat steps 1.2.5-1.2.8 and change the temperature to the desired sample temperature (90 &#176;C in the present case).</li>
		<li>Click on File &#62; Export and choose the desired file location. Select XY Data to obtain a .txt file. 
		NOTE: An illustrative aid of step 1.2 can be found in Supplementary File 1 (S1-S7).</li>
	</ol>
	</li>
	<li>Perform data acquisition for determining the RNA concentration.
	<ol>
		<li>Find the file for each spectrum under the Results tab. To obtain the absorbance, hover over the line, navigate to 260 nm, and record the displayed absorbances. Alternatively, through the File &#62; Export cascade, export the data as a .txt file for further graphical analysis using other plotting software. 
		NOTE: If 0.1 &#60; A &#8805; 1, it will be necessary to either dilute or increase the concentration of the RNA sample prepared in step 1.1.1.</li>
		<li>Use the Beer-Lambert law to calculate the concentration of the solution. 
		NOTE: Beer&#39;s Law: A = &#949;cl, where: 
		A:&#160;Absorbance (at 260 nm in this case, from step 1.3.1) 
		&#949;: Extinction coefficient. For the sequence used herein, 208,000 L mol-1cm-1 is the value obtained from an OligoAnalyzer tool. 
		l: Path length, 1 cm 
		c: concentration of the sample</li>
		<li>Use these calculations to prepare the desired solution of RNA to be used for subsequent experiments. Solutions with [RNA] = 200 &#181;M were used in the present study.</li>
	</ol>
	</li>
</ol>
2. Hydrolysis of oligonucleotides of RNA by Xrn-1
<ol>
	<li>Perform 5&#39; phosphorylation of RNA following the steps below.
	<ol>
		<li>Use a 0.6 mL microcentrifuge tube to prepare the following solution (50 &#181;L total volume).
		<ol>
			<li>Add RNase-free H2O (33.5 &#181;L); volumes may vary depending on [RNA].</li>
			<li>Then, add solution A (5 L, Table 1).</li>
			<li>Add an aqueous solution of adenosine triphosphate (ATP, see Table of Materials) (6 L, 10 mM, 60 nmol).</li>
			<li>Add RNA aqueous solution (1 &#181;L, 200 &#181;M, 200 pmol).</li>
			<li>Add polynucleotide kinase (PNK enzyme, see Table of Materials) solution (4.5 L, 45 units). Mix gently by pipetting the mixture up and down (10x).</li>
		</ol>
		</li>
		<li>Incubate the reaction mixture at 37 &#176;C for 45 min by placing it in a water bath.</li>
		<li>Inactivate the enzyme by placing the reaction tube in a pre-heated heat block (65 &#176;C) and incubating the solution for 10 min.</li>
		<li>Allow the solution to cool to room temperature (RNA phosphorylation was characterized via MALDI-TOF analysis, see Representative Results) to yield a final 5&#39;-phosphorylated RNA solution (4 &#181;M, 50 &#181;L).</li>
	</ol>
	</li>
	<li>Perform RNA hydrolysis by Xrn-1 following the steps below.
	<ol>
		<li>Using the solution (50 &#181;L) from step 2.1.4, carry out the following steps.
		<ol>
			<li>Add solution B (5 L, Table 1).</li>
			<li>Add a solution of Xrn-1 (0.5 L, 1 ng, 30 fmol). Mix the solution by gently pipetting up and down (10x).</li>
			<li>Incubate the reaction tube at room temperature for 2 h.</li>
		</ol>
		</li>
		<li>Transfer the reaction mixture (step 2.2.1.3) into a 10 kDa pore-sized centrifugal device (see Table of Materials), and filter the enzymes by centrifuging (700 x g for 10 min at room temperature).</li>
		<li>Transfer the filtrate into a 0.6 mL centrifuge tube.</li>
		<li>Wash the residual RNA on the centrifugal tube (step 2.2.2.) by adding RNase-free H2O (20 L), followed by centrifugation (700 x g for 10 min at room temperature).</li>
		<li>Combine the filtrate with that obtained from step 2.2.3.</li>
		<li>Store the tube containing the resulting solution in a freezer (0 &#176;C) or ship for analysis.</li>
	</ol>
	</li>
</ol>
3. Desalting of RNA solution, MALDI-TOF plate spotting
<ol>
	<li>Desalt and concentrate the sample using commercially available cation exchange C18 pipette tips (see Table of Materials).
	<ol>
		<li>Position a 10 L C18 pipette tip (pipette tip loaded with a bed of C18 chromatography media fixed at its end) onto a 10 &#181;L pipette, and then continue with the following:
		<ol>
			<li>Wash the C18 tip with 50% aqueous acetonitrile (10 &#181;L) solution two times. Discard the used volumes into a separate waste tube each time.</li>
			<li>Equilibrate the C18 tip with an aqueous trifluoroacetic acid solution (0.1% TFA, 10 &#181;L) two times. Discard the used volumes into a separate waste tube each time.</li>
			<li>Manually remove the C18 tip from the pipette and secure it onto a 200 &#181;L pipette containing a P200 pipette tip (the C18 tip will now be attached to the P200 pipette tip).</li>
			<li>Immerse the C18 tip into the solution (step 2.2.6), and aspirate-release the solution through the C18 tip ten times. 
			NOTE: The RNA sample becomes bound to the cation exchange resin inside the tip.</li>
			<li>Detach the C18 tip from the P200 pipette and position it onto a 10 &#181;L pipette.</li>
			<li>Wash the C18 tip using an aqueous solution of 0.1% TFA (10 &#181;L) two times. Discard the used volumes into a separate waste tube each time.</li>
			<li>Wash the C18 tip with RNase-free water (10 &#181;L) two times. Discard the used volumes into a separate waste tube each time.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Spot onto the MALDI plate following the steps below.
	<ol>
		<li>Elute the RNA oligonucleotide from the C18 tip (step 3.1.1.7) by immersing the sample into the desired matrix (10 &#181;L, 1:1 solution of C and D, Table 1), followed by dispensing the solution back into the tube. Repeat this process ten times.</li>
		<li>Pipette the solution from 3.2.1 onto two separate spots on the plate (1 &#181;L each, 20 pmol). Allow spots to air dry (Figure 1A and Video 1). 
		NOTE: This process can be repeated on the same spot to increase the sample concentration on each spot.</li>
		<li>Pipette desired calibrant (1 L) on two separate spots physically close to the sample location and allow to air dry. 
		NOTE: The test standard used in the present study was obtained from commercial sources (see Table of Materials).</li>
		<li>Over-lay the [fully dried] calibrant with the desired matrix (1 L). Allow to air dry. 
		&#8203;NOTE: It is important to track the position where the samples and calibrants are spotted prior to insertion into the instrument. Make a note of this using the plate coordinate system, e.g., sample 1 is located at position (C,3)</li>
	</ol>
	</li>
</ol>
4. Data acquisition and processing
NOTE: In general, THAP requires higher laser power to achieve ionization. Additionally, oligos can be difficult to ionize without fragmenting. Thus, it is usually necessary to use as low laser power as possible and raise detector gains and/or lower laser frequency to maximize detection and minimize fragmentation.
<ol>
	<li>Perform data acquisition following the steps below. 
	NOTE: Molecular weight measurements were performed using a mass spectrometer (see Table of Materials) in positive ion, linear mode with an ion source voltage of 20 kV, detector gain of ~2.8 kV, and a laser frequency of 20 Hz. Scan ranges were set based on expected molecular weight(s). These were calculated and expressed as mass over charge (m/z), where the charge was 1. External calibration was performed using a protein calibration mixture (Bacterial Test Standard, see Table of Materials) on a spot adjacent to the sample. The raw data was then processed in the flexAnalysis software (see Table of materials).
	<ol>
		<li>Open &#34;Flexcontrol software&#34; for operation. Press the instrument&#39;s green in/out button, wait for the target stage to move, and vacuum to vent. Open the lid, place the fully dried target plate into the instrument. Ensure to align in the right orientation (Figure 1B).</li>
		<li>Gently lower the lid, and then press the green in/out button. Wait for the plate to be retracted and the instrument to pump back down. Once the status bar (lower right corner of the software window) reflects &#39;Ready&#39;, proceed with calibration.</li>
		<li>To calibrate the instruments using the software, carry out the following.
		<ol>
			<li>Select the desired method by clicking on the File &#62; Select Method or pressing the Select button adjacent to the loaded method. Click on the Coordinate for the calibrant spot. Navigate to the Calibration tab and confirm the correct calibrant is selected. Ensure Random Walk is turned off (in the Sample Carrier tab).</li>
			<li>Press Start and manually direct the laser along a crystal (clicking the mouse arrow at the desired location in the camera view). Adjust laser power, if necessary. Once satisfied with the settings, press Start to collect spectra and add them to the sum buffer.</li>
			<li>Add spectra to the sum until sufficient intensity is reached. Toggle to view only the sum buffer, subtract the baseline, and smooth. Then click on Automatic Assign. Check each assigned peak and note the ppm error. If satisfied with assignments and errors, click on Apply.</li>
			<li>Clear the sum buffer and navigate to the position of the first sample.</li>
			<li>Confirm the desired scan range (&#39;Detection&#39; tab). Adjust the bars in the &#34;Mass Range&#34; so that the green region is representative of expected mass(es).</li>
			<li>Click directly on the Magnification window so that the crosshairs are oriented at the desired crystalized fragment (larger crystals often yield the best results, Figure 2).</li>
			<li>Collect a few spectra along the large crystals, adjusting the laser power as necessary. The proper setting is 5%-10% above the power at which peaks appear.</li>
		</ol>
		</li>
		<li>To obtain spectra, perform the following.
		<ol>
			<li>Click on Start to observe the laser flash in tandem with increasing spectral intensity (shown in the window in the upper right). Move the laser along the length of the crystal by clicking the mouse arrow up and down the crystal (Video 2).</li>
			<li>Click on Add directly below Start.</li>
			<li>Repeat this several times until desired intensity/number of shots is reached (reflected on the y-axis of the spectral window)</li>
			<li>Adjust the laser power and/or speed and/or detector gains if the initial scans are unsatisfactory. Repeat steps 4.1.4.1-4.1.4.3 as needed.</li>
			<li>If satisfied with the spectrum, click on Save As to save the spectrum under a specified name. Then, if other samples are needed, click on Clear Sum and repeat steps under step 4.1.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform data processing. 
	NOTE: Step 4.2 describes one way to analyze the data using the flexAnalysis software.
	<ol>
		<li>Open the desired software. Then, open spectrum (a) by selecting File. From the dropdown menu, select Open.</li>
		<li>A new window will be displayed. Click on Browse to locate the files saved from step 4.1.4. Check the boxes for the desired files, and then click on Open on the left. If only a single spectrum requires analysis, simply drag, and drop the file into the software window.</li>
		<li>Highlight the loaded file(s) for bulk analysis. Navigate to the Process tab on the toolbar. In the dropdown, select Subtract Mass Spectrum Baseline. 
		NOTE: The algorithm used for the subtraction will be determined by the method selected in step 4.1.3.</li>
		<li>Navigate to the Process tab on the toolbar. In the dropdown, select Smooth Mass Spectrum. 
		NOTE: The algorithm used for the smoothing will be determined by the method selected in step 4.1.3.</li>
		<li>Click on the Mass List tab; in the dropdown, select Find. This command automatically labels peaks with their masses; a list of the masses will be displayed on the screen to the right.</li>
		<li>To add or delete peaks, proceed to the Mass List tab. In the dropdown, click on Edit.</li>
		<li>Hover over the x-axis of the spectrum; a vertical line represents the location of the spectrum where the cursor is on. 
		NOTE: An icon with a graph and cursor will appear when a new mass can be added by clicking. This icon will appear when hovering over peaks that can be deleted by clicking (see step 4.2.7 in Supplementary File 1).</li>
		<li>Repeat steps 4.1.3-4.1.7 for additional spectra. Export the mass list for all spectra by clicking and dragging to highlighting the files on the left and execute the following menu cascade File &#62; Export &#62; Mass List to Excel.</li>
		<li>To export spectra as shown on the screen, either take a screenshot or export it as a report. For the latter, navigate to the Report menu. In the dropdown, select Save as PDF. 
		NOTE: One can change how the spectra are displayed using the three tabs below the spectra and/or varying the highlighted files. A screen will pop up. Name the PDF document as desired, adjust the settings/layout, and click on Save to generate an image of the spectrum.</li>
		<li>The image generated will reflect the spectral view in the software program. To manipulate the view, perform the following.
		<ol>
			<li>Zooming in: Click on the Zoom In button in the toolbar.</li>
			<li>Hover over the x-axis and click until the desired range is achieved.</li>
			<li>Zooming out/undo: Click on the Zoom Out button and click anywhere in the spectrum to return to full view. 
			NOTE: An illustrative aid for steps 4.1 and 4.2 can be found in Supplementary File 1 (pp S8-S23 and S24-S30, respectively).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Langeberg, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+characterization+of+yeast+Xrn1%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Biochemical characterization of yeast Xrn1.</a> Biochemistry. 59 (15), 1493-1507 (2020).</li>
<li>Stevens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+characterization+of+a+Saccharomyces+cerevisiae+exoribonuclease+which+yields+5%27-mononucleotides+by+a+5%27+leads+to+3%27+mode+of+hydrolysis%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Purification and characterization of a Saccharomyces cerevisiae exoribonuclease which yields 5'-mononucleotides by a 5' leads to 3' mode of hydrolysis.</a> Journal of Biological Chemistry. 255 (7), 3080-3085 (1980).</li>
<li>Yan, L. L., Simms, C. L., McLoughlin, F., Vierstra, R. D., Zaher, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidation+and+alkylation+stresses+activate+ribosome-quality+control%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Oxidation and alkylation stresses activate ribosome-quality control.</a> Nature Communications. 10 (1), 5611(2019).</li>
<li>Fasnacht, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+23S+rRNA+modification+ho5C2501+benefits+Escherichia+coli+under+oxidative+stress%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Dynamic 23S rRNA modification ho5C2501 benefits Escherichia coli under oxidative stress.</a> Nucleic Acids Research. 50 (1), 473-489 (2022).</li>
<li>Estevez, M., Valesyan, S., Jora, M., Limbach, P. A., Addepalli, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidative+damage+to+RNA+is+altered+by+the+presence+of+interacting+proteins+or+modified+nucleosides%5BTitle%5D)+AND+%22Frontiers+in+Molecular+Biosciences%22%5BJournal%5D)">Oxidative damage to RNA is altered by the presence of interacting proteins or modified nucleosides.</a> Frontiers in Molecular Biosciences. 8, 697149(2021).</li>
<li>Tomar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+sequence+modulates+the+efficiency+of+NEIL1-catalyzed+excision+of+the+aflatoxin+B1-induced+formamidopyrimidine+guanine+adduct%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">DNA sequence modulates the efficiency of NEIL1-catalyzed excision of the aflatoxin B1-induced formamidopyrimidine guanine adduct.</a> Journal of the American Chemical Society. 34 (3), 901-911 (2021).</li>
<li>Gaffney, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+env-dependent+cell+killing+by+bifunctional+small-molecule%2Fpeptide+conjugates%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">HIV-1 env-dependent cell killing by bifunctional small-molecule/peptide conjugates.</a> ACS Chemical Biology. 16 (1), 193-204 (2021).</li>
<li>Sikorski, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+display+of+a+chemoattractant+agonist+on+cancer+cells+activates+the+formyl+peptide+receptor+1+on+immune+cells%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Selective display of a chemoattractant agonist on cancer cells activates the formyl peptide receptor 1 on immune cells.</a> ChemBioChem. , 202100521(2022).</li>
<li>Kubo, T., Nishimura, Y., Sato, Y., Yanagihara, K., Seyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sixteen+different+types+of+lipid-conjugated+siRNAs+containing+saturated+and+unsaturated+fatty+Acids+and+exhibiting+enhanced+RNAi+potency%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">Sixteen different types of lipid-conjugated siRNAs containing saturated and unsaturated fatty Acids and exhibiting enhanced RNAi potency.</a> ACS Chemical Biology. 16 (1), 150-164 (2021).</li>
</ol>

The authors have nothing to disclose.

The main challenge in this workflow arose between finalizing the experiments and carrying out the mass spectrometric analyses. Experiments were carried out and completed at the University of Colorado Denver and shipped (overnight) to the Colorado State University facilities. Data acquisition was carried out upon receipt, as per convenience. Several unexpected circumstances led to time delays in the process. In one instance, unexpected instrument malfunctions required the samples to be frozen (one time for 21 days) prior ...

MALDI-TOF1,2,3 is a widely used technique for the characterization and/or detection of molecules of varying sizes and characteristics. Some of its uses include diverse applications such as detecting tannins from natural resources4, imaging metabolites in food5, discovery or monitoring of cellular drug targets or markers6, and clinical diagnostics7, to name a few. Of relevance to the present work is the use of MALDI-TOF with DNA or RNA, with its use on oligonucleotides dating back to over three decades8, where several limitations were noted. This technique has now evolved to a reliable, commonly used means to characterize both biopolymers9 and identify/understand chemical and biochemical reactions, e.g., characterization of platinated sites in RNA10, identification of RNA fragments following strand cleavage11,12, or formation of protein-DNA cross-links13. Thus, it is valuable to illustrate and highlight important aspects of using this technique. The basics of MALDI-TOF have been described in video format as well14 and will not be further elaborated herein. Furthermore, its application in a DNA or protein context has been previously described and illustrated in the said format15,16,17.
The protocol for detecting RNA fragments formed after enzymatic hydrolysis is reported herein. The experimental model was chosen based on a recent finding published by our group18, where MALDI-TOF was used to determine the unique reactivity between the exoribonuclease Xrn-1 and oligonucleotides of RNA containing the oxidative lesion 8-oxoG. The 20-nucleotide long strands were obtained via solid-phase synthesis19, [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU], while Xrn-1 was expressed and purified following the previously described report20. In brief, Xrn-121 is a 5&#39;-3&#39; exoribonuclease with various key biological roles that degrade multiple types of RNA, including oxidized RNA22. It was found that the processivity of the enzyme stalls upon encountering 8-oxoG, which led to RNA fragments containing 5&#39;-phosphorylated ends [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]18.
Finally, it is important to note that mass spectrometry is a powerful method that, through various methodologies, can be adapted to other purposes23,24; thus, choosing the right ionization method as well as other experimental set up is of utmost importance.

<table><tbody><tr><td>0.6 mL MCT Graduated Violet</td><td>Fisher Scientific</td><td>05-408-127</td><td></td></tr><tr><td>6&amp;rsquo;-Trihydroxyacetophenone monohydrate 98%</td><td>Sigma Aldrich</td><td>480-66-0</td><td></td></tr><tr><td>Acetonitrile 99.9%, HPLC grade</td><td>Fisher Scientific</td><td>75-05-8</td><td></td></tr><tr><td>Adenosine triphosphate, 10 mM</td><td>New Englang Bioscience</td><td>P0756S</td><td></td></tr><tr><td>Ammonium citrate, dibasic 98%</td><td>Sigma Aldrich</td><td>3012-65-5</td><td></td></tr><tr><td>Ammonium Fluoride 98.0%, ACS grade</td><td>Alfa Aesar</td><td>12125-01-8</td><td></td></tr><tr><td>Bruker bacterial test standard</td><td>Bruker Daltonics</td><td>8255343</td><td></td></tr><tr><td>Commercial source of Xrn-1</td><td>New England BioLabs</td><td>M0338S</td><td></td></tr><tr><td>Diethyl pyrocarbonate, 97%</td><td>ACROS Organics</td><td>A0368487</td><td></td></tr><tr><td>Flex analysis software</td><td>Bruker daltonics</td><td></td><td>FlexAnalysis software version 3.4, Bruker Daltonics</td></tr><tr><td>Lambda 365 UV-vis spectrophotometer</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>MALDI plate: MSP 96 ground steel target</td><td>Bruker Daltonics</td><td>280799</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Bruker</td><td></td><td>Microflex LRFTOF mass spectrometer (Bruker Daltonics, Billerica, MA)</td></tr><tr><td>Mili-Q IQ 7000</td><td>Milipore Sigma</td><td></td><td>A Mili-Q system was used to purify all water used in this work</td></tr><tr><td>Nanosep Centrifugal Devices with OmegaTM Membrane 10 K, blue (24/pkg)</td><td>Pall Corporation</td><td>OD010C33</td><td>filter media, Omega (modified polyethersulfone) 10 K pore size</td></tr><tr><td>NEBuffer 3</td><td>New England Biolabs</td><td>B7003S</td><td>This is solution B</td></tr><tr><td>Oligo Analyzer tool</td><td>IDT-DNA</td><td></td><td>https://www.idtdna.com/calc/analyzer</td></tr><tr><td>Pipette tips P10</td><td>Fisher Scientific</td><td>02-707-441</td><td></td></tr><tr><td>Pipette tips P200</td><td>Fisher Scientific</td><td>02-707-419</td><td></td></tr><tr><td>RNase Away</td><td>Molecular BioProducts</td><td>7005-11</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England BioLabs</td><td>M0201S</td><td></td></tr><tr><td>T4 Polynucleotide Kinase Reaction Buffer</td><td>New England BioLabs</td><td>B0201S</td><td>This is solution A</td></tr><tr><td>Triflouroacetic Acid</td><td>Alfa Aesar</td><td>76-05-1</td><td></td></tr><tr><td>Xrn-1 exoribonuclease</td><td>Expressed in house</td><td>See ref. 20</td><td></td></tr><tr><td>ZipTip Pipette Tips for Sample preparation</td><td>Millipore</td><td>ZTC 18S 096</td><td>10 &amp;micro;L pipette tips loaded with a C18 standard 0.6 &amp;micro;L bed</td></tr></tbody></table>

identification of rna fragments resulting from enzymatic degradation using maldi-tof mass spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

The oligonucleotides used in this work were synthesized, characterized, and quantified prior to use. The concentration of all oligonucleotides was determined via UV-vis spectroscopy recorded at 90 &#176;C to avoid erroneous readings arising from the potential formation of the secondary structures. Figure 3 displays the spectra of the model oligonucleotides of RNA used in this work, taken at room temperature and after applying heat.
The overall procedure, ...

Watch this Scientific Journal Video about Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry at JoVE.com

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

identification-rna-fragments-resulting-from-enzymatic-degradation

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Cell Biology

Biochemistry

Unit 1

Analyzing Cells and Proteins

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3.2K Views.  University of Colorado, Denver. Massaspectrometrie is op grote schaal toegepast voor verschillende toepassingen. De methodologie in dit artikel biedt de ondubbelzinnige karakterisering van RNA-fragmenten verkregen uit een in vitro enzymatisch proces. Voordelen van deze techniek zijn onder meer dat het molecuul ijzerpiek wordt verkregen, dat de experimenten kunnen worden uitgevoerd met kleine hoeveelheden materiaal voor in vitro toepassingen en dat het protocol kan worden bereikt met minimale training.De beschreven me...

Video: Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Chemistry

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 ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Bioengineering

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 ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Genetics

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Developmental Biology

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Immunology and Infection

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (&bull;OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.1 When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA1 and RNA2 with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein1 and RNA-protein complexes. Equilibrium3 and kinetic4 transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.5
In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H2O2 (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O2 (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

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