Name: 2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications (Video) | JoVE Journal
Uploaded: 2020-07-10T13:00:00.000+00:00
Duration: 5 min 41 s
Description: Learn direct LC-MS-based RNA sequencing without cDNA intermediates for epitranscriptomic research. This method enables simultaneous detection of multiple RNA modifications with single-base precision and high accuracy.

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The authors acknowledge the R21 grant from National Institutes of Health&#160;(1R21HG009576) to S. Z. and W. L. and New York Institute of Technology (NYIT) Institutional Support for Research and Creativity grants to S. Z., which supported this work. The authors would like to thank PhD student Xuanting Wang (Columbia University) for assisting in figure-making, and thank Prof. Michael Hadjiargyrou (NYIT), Prof. Jingyue Ju (Columbia University), Drs. James Russo, Shiv Kumar, Xiaoxu Li, Steffen Jockusch, and other members of the Ju lab (Columbia University), Dr. Yongdong Wang (Cerno Bioscience), Meina Aziz (NYIT), and Wenhao Ni (NYIT) for helpful discussions and suggestions for our manuscript.

1. Design RNA oligonucleotides
<ol>
	<li>Design synthetic RNA oligonucleotides with different lengths (19 nt, 20 nt and 21 nt), including one (RNA #6) with both canonical and modified nucleotides. &#968; is employed as a model for non-mass-altering modifications, which is challenging for MS sequencing because it has an identical mass to U. m5C is chosen as a model for mass-altering modifications to demonstrate the robustness of the approach. 
	 
	RNA #1: 5&#180;-HO-CGCAUCUGACUGACCAAAA-OH-3&#180; 
	RNA #2: 5&#180;-HO-AUAGCCCAGUCAGUCUACGC-OH-3&#180; 
	RNA #3: 5&#180;-HO-AAACCGUUACCAUUACUGAG-OH-3&#180; 
	RNA #4: 5&#180;-HO-GCGUACAUCUUCCCCUUUAU-OH-3&#180; 
	RNA #5: 5&#180;-HO-GCGGAUUUAGCUCAGUUGGGA-OH-3&#180; 
	RNA #6: 5&#180;-HO-AAACCGU&#968;ACCAUUAm5CUGAG-OH-3&#180; 
	&#160;</li>
	<li>Dissolve each synthetic RNA in nuclease-free diethyl pyrocarbonate (DEPC)-treated water (expressed as DEPC-treated H2O unless otherwise indicated) to obtain a 100 mM RNA stock solution. Stock solutions are stored long-term at -20 &#176;C.</li>
	<li>To avoid possible RNA sample degradation, use RNase-free experimental supplies including DEPC-treated water, microcentrifuge tubes, and pipette tips. Frequently wipe down surfaces of lab supplies using&#160;RNase elimination wipes.</li>
</ol>
2. Label the 3&#180;-end of RNAs with biotin
<ol>
	<li>Two-step reaction protocol (adenylation and ligation)
	<ol>
		<li>Add 1 &#181;L of 10x adenylation reaction buffer containing 50 mM sodium acetate, pH 6.0, 10 mM MgCl2, 5 mM dichlorodiphenyltrichloroethane (DTT), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 &#181;L of 1 mM ATP, 1 &#181;L of 100 &#181;M biotinylated cytidine bisphosphate (pCp-biotin), 1 &#181;L of 50 &#181;M Mth RNA ligase, and 6 &#181;L of DEPC-treated H2O (a total volume of 10 &#181;L) into an RNase-free thin-walled 0.2 mL PCR tube. 
		NOTE: Store the reagents at -20 &#176;C before the two-step reaction. Thaw the reagents at room temperature and mix well by vortexing and centrifuging before adding to the reaction.</li>
		<li>Incubate the reaction in a PCR machine at 65 &#176;C for 1 h and inactivate the reaction at 85 &#176;C for 5 min.</li>
		<li>Conduct the ligation step in an RNase-free, thin walled 0.2 mL PCR tube containing 10 &#181;L of reaction solution from the previous step by adding 3 &#181;L of 10x T4 RNA ligase reaction buffer containing 50 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.8, 10 mM MgCl2, 1 mM DTT, 1.5 &#181;L of the 100 mM sample stock of the RNA to be sequenced, 3 &#181;L of anhydrous dimethyl sulfoxide (DMSO) to reach 10% (v/v), 1 &#181;L of T4 RNA ligase (10 units/&#181;L), and 11.5 &#181;L of DEPC-treated H2O (for a total volume of 30 mL). Incubate the reaction overnight at 16 &#176;C in a PCR machine. 
		NOTE: Combine reaction components at room temperature due to the high freezing point of DMSO (18.45 &#176;C).</li>
		<li>Incubate the reaction overnight at 16 &#176;C.</li>
		<li>Quench and purify the reaction by column purification to remove enzymes and free pCp-biotin using Oligo Clean &#38; Concentrator (Zymo Research, Irvine, CA, USA). Oligo Binding Buffer, DNA Wash Buffer, spin columns and collection tubes are provided in the kit. Add 20 mL of DEPC-treated H2O to the reaction solution to reach a 50 mL sample volume prior to adding the Binding Buffer.</li>
		<li>Add 100 mL of binding buffer to each reaction solution. Add 400 &#181;L of ethanol, mix by pipetting, and transfer the mixture to the column. Centrifuge at 10,000 x g for 30 s. Discard the flow-through.</li>
		<li>Add 750 &#181;L of DNA Wash Buffer to the column. Centrifuge at 10,000 x g and maximum speed for 30 s and 1 minute, respectively.</li>
		<li>Transfer the column to a 1.5 mL microcentrifuge tube. Add 15 &#181;L of DEPC-treated H2O to the column and centrifuge at 10,000 x g for 30 s to elute the RNA product. 
		NOTE: Samples can be stored at -20 &#176;C at this stage until the next step is performed.</li>
	</ol>
	</li>
	<li>One-step reaction protocol
	<ol>
		<li>Perform a one-step labeling reaction by combining 2 &#181;L of 150 &#956;M adenosine-5&#180;-5&#180;-diphosphate-{5&#180;-(cytidine-2&#180;-O-methyl-3&#180;-phosphate-TEG}C-biotin (AppCp-biotin), 3 &#181;L of 10x ligase reaction buffer, 1.5 &#181;L of the 100 mM sample stock of the RNA to be sequenced, 3 &#181;L of anhydrous DMSO to reach 10% (v/v), 1 &#181;L of T4 RNA ligase (10 units/&#181;L), and 19.5 &#181;L of DEPC-treated H2O (for a total volume of 30 mL) in a 1.5 mL RNase-free microcentrifuge tube.</li>
		<li>Incubate the reaction overnight at 16 &#176;C in a PCR machine.</li>
		<li>Perform column purification as described above in steps 2.1.5-2.1.8. 
		NOTE: Prepare a separate/exclusive reaction tube for each RNA sample (150 pmol scale of RNA). Labeling of the 5&#180;-end of the RNA(s) with sulfo-Cyanine3 (Cy3) or Cy3 may be needed (e.g., for bidirectional sequencing verification). The method is different than that of 3&#180;-biotinylation and is described in a previous publication9.</li>
	</ol>
	</li>
</ol>
3. Capture biotinylated RNA sample on streptavidin beads
<ol>
	<li>Activate 200 &#181;L of streptavidin C1 magnet beads by adding 200 &#181;L of 1x B&#38;W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl) in a 1.5 mL RNase-free microcentrifuge tube. Vortex this solution and place it on a magnet stand for 2 min. Then discard the supernatant by carefully pipetting out the solution.</li>
	<li>Wash the beads twice with 200 &#181;L of Solution A (DEPC-treated 0.1 M NaOH and DEPC-treated 0.05 M NaCl) and once in 200 &#181;L of Solution B (DEPC-treated 0.1 M NaCl). For each wash step, vortex the solution and place it on a magnet stand for 2 min, followed by discarding of the supernatant. Then add 100 &#181;L of 2x B&#38;W buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl).</li>
	<li>Add 1x B&#38;W buffer to the biotinylated RNA sample until the volume is 100 &#181;L. Then add this solution to the washed beads stored in 100 &#181;L of 2x B&#38;W buffer. Incubate for 30 min at room temperature on a rocking platform shaker at 100 rpm. Place the tube on a magnet stand for 2 min and discard the supernatant.</li>
	<li>Wash the coated beads 3 times in 1x B&#38;W buffer and measure the final concentration of supernatant in each wash step by Nanodrop for recovery analysis, to confirm that the target RNA molecules remain on the beads.</li>
	<li>Incubate the beads in 10 mM EDTA, pH 8.2 with 95% formamide at 65 &#176;C for 5 min in a PCR machine. Keep the tube on the magnet stand for 2 min and collect the supernatant (containing the biotinylated RNAs released from the streptavidin beads) by pipet. 
	NOTE: This physical separation step prior to acid degradation is only used for sequencing of RNA#1 in Figure 1c, and is not mandatory for the 2D-HELS MS Seq since the hydrophobic biotin label can cause the 3&#180;-labeled ladder fragments to have a significantly delayed tR during LC-MS measurement, which can clearly distinguish the labeled 3&#180;-ladder fragments from the unlabeled 5&#180;-ladder fragments in the 2D mass-tR plot.</li>
</ol>
4. Acid hydrolysis of RNA to generate MS ladders for sequencing
<ol>
	<li>Divide each RNA sample into three equal aliquots. For instance, divide an RNA sample with a volume of 15 &#181;L RNA sample into three aliquots of 5 &#181;L.</li>
	<li>Add an equal volume of formic acid to achieve 50% (v/v) formic acid in the reaction mixture8,9.</li>
	<li>Incubate the reaction at 40 &#176;C in a PCR machine, with one reaction running for 2 min, one for 5 min, and one for 15 min, respectively.</li>
	<li>Quench the acid degradation by immediately freezing the sample on dry ice after each reaction finishes.</li>
	<li>Use a centrifugal vacuum concentrator to dry the sample. The sample is typically completely dried within 30 min, and formic acid is removed together with H2O during the drying process because formic acid has a boiling point (100.8 &#176;C) similar to that of H2O (100 &#176;C).</li>
	<li>Suspend and combine a total of three dried samples in 20 &#181;L of DEPC-treated H2O for LC-MS measurement. 
	NOTE: Samples can be stored at -20 &#176;C at this stage while waiting for LC-MS measurement.</li>
</ol>
5. Convert &#968; to CMC-&#968; adduct
<ol>
	<li>Add 80 &#181;L of DEPC-treated H2O into a 1.5 mL RNase-free microcentrifuge tube containing 0.0141 g of N-cyclohexyl-N&#697;-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMC) and 0.07 g of urea. Add 10 &#181;L of the 100 &#181;M sample stock of the RNA to be sequenced, 8 &#181;L of 1 M bicine buffer (pH 8.3), and 1.28 &#181;L of 0.5 M EDTA. Add DEPC-treated H2O to reach a total volume of 160 &#181;L. Final concentrations are 0.17 M CMC, 7 M urea, and 4 mM EDTA in 50 mM bicine (pH 8.3)11. 
	NOTE: This protocol is applicable to either a single synthetic RNA sequence or RNA mixtures.</li>
	<li>Divide the 160 &#181;L reaction solution into four equal aliquots in RNase-free, thin walled 0.2 mL PCR tubes and incubate at 37 &#176;C for 20 min in a PCR machine. 
	NOTE: 50 &#181;L per tube is the maximum reaction volume that can be used in a PCR machine.</li>
	<li>Quench each reaction with 10 &#181;L of 1.5 M sodium acetate and 0.5 mM EDTA (pH 5.6).</li>
	<li>Perform column purification with four parallel spin columns to remove excessive reactants according to the procedure as described in steps 2.1.5-2.1.8. Dissolve the purified product in 15 &#181;L of DEPC-treated H2O in each 1.5 mL RNase-free microcentrifuge tube.</li>
	<li>Transfer the purified product to four RNase-free, thin walled 0.2 mL PCR tubes. Add 20 &#181;L of 0.1 M Na2CO3&#160;buffer (pH 10.4) into each 15 &#181;L of purified product and add DEPC-treated H2O to make a final volume of 40 &#181;L for each reaction tube (in total four tubes). Incubate the reaction at 37 &#176;C for 2 h in a PCR machine.</li>
	<li>Quench and purify the reaction by column purification with four parallel spin columns as described in step 2.1.5. Elute the CMC-&#968; converted product to a 1.5 mL RNase-free microcentrifuge tube each with 15 &#181;L of DEPC-treated H2O.</li>
	<li>Combine the purified CMC-&#968; converted sample from four collection tubes into one tube. Perform formic acid degradation 50% (v/v) according to the procedures as described in steps 4.1-4.6 to generate MS ladders for sequencing.</li>
</ol>
6. LC-MS measurement
<ol>
	<li>Prepare mobile phases for LC-MS measurement. Mobile phase A is 25 mM hexafluoro-2-propanol with 10 mM diisopropylamine in LC-MS grade water; mobile phase B is methanol.</li>
	<li>Transfer the sample to LC-MS sample vial for analysis. Each sample injection volume is 20 &#181;L containing 100-400 pmol of RNA.</li>
	<li>Use the following LC conditions: column temperature of 35 &#176;C, flow rate of 0.3 mL/min; a linear gradient from 2&#8211;20% mobile phase B over 15 min followed by a 2 min wash step with 90% mobile phase B. 
	NOTE: For more hydrophobic end-labels such as Cy3 and sulfo-Cy3 as mentioned in Section 2, a higher percentage of organic solvent may be necessary for sample elution (i.e., a similar gradient can be used but with an increased percentage range of mobile phase B). For instance, 2&#8211;38% mobile phase B over 30 min with a 2 min wash step with 90% mobile phase B.</li>
	<li>Separate and analyze samples on an Agilent Q-TOF (Quadrupole Time-of-Flight) mass spectrometer coupled to an LC system equipped with an autosampler and an MS HPLC (High Performance Liquid Chromatography) system. The LC column is a 50 mm x 2.1 mm C18 column with a particle size of 1.7 &#956;m. Use the following MS settings: negative ion mode; range, 350 m/z to 3200 m/z; scan rate, 2 spectra/s; drying gas flow, 17 L/min; drying gas temperature, 250 &#176;C; nebulizer pressure, 30 psig; capillary voltage, 3500 V; and fragmentor voltage, 365 V. Please note that these parameters are specific to the type or model of mass spectrometer being used.</li>
	<li>Acquire data with Agilent MassHunter acquisition software. Use Agilent molecular feature extraction (MFE) workflow to extract compound information including mass, retention time, volume (the MFE abundance for the respective ion species), and quality score, etc. Use the following MFE settings: &#8220;centroid data format, small molecules (chromatographic), peak with height &#8805; 100, up to a maximum of 1000, quality score &#8805; 50&#8221;. 
	NOTE: Optimize MFE settings to extract as many potential compounds as possible, up to a maximum of 1000, with quality scores of &#8805; 50.</li>
</ol>
7. Automate RNA sequence generation by a computational algorithm
NOTE: This procedure is shown only for RNA #1 in Figure 1c.
<ol>
	<li>Sort MFE extracted compounds in order of decreasing volume (peak intensity) and tR. Perform data pre-selection via 1) setting tR from 4 to 10 min to select the RNA fragments labeled by the biotin, since the tRs of the biotin-labeled mass ladder components are shifted to this tR window (4 min to 10 min), and 2) using an order-of-magnitude higher of input compounds than the number of ladder fragments for algorithm computation to reduce data amount based on volume. For instance, for a 20 nt RNA, 20 labeled mass-tR ladder components will be required for sequencing of the 20 nt RNA, and thus, 200 compounds from MFE data file will be selected based on volume. Please note that the tR window may be different when a different type or model of mass spectrometer is used.</li>
	<li>Perform data processing and sequence generation of RNA #1 using a revised version of a published algorithm8. The source codes of the revised algorithm are described previously (https://academic.oup.com/nar/article/47/20/e125/5558343#supplementary-data)9.</li>
	<li>In addition to automating sequence generation using the algorithm, manually calculate the mass differences between two adjacent ladder components for base calling. All bases in the RNA can be called manually and matched with the theoretical ones in the RNA nucleotide and modification database8; thus, the complete sequence of the RNA strand can be accurately read out manually, which is used to confirm the accuracy of the algorithm-reported sequence read. More structures of RNA modifications can be found in RNA modification databases12, and their corresponding theoretical masses are obtained by ChemBioDraw. In Tables S1&#8211;S2, the ppm (parts-per-million) mass difference is shown when comparing the observed mass to its theoretical mass for a specific ladder component, and a value less than 10 ppm is considered a good match for each base calling.</li>
</ol>
8. Sequencing RNA mixtures
<ol>
	<li>Label a mixture of five RNA strands (RNA #1 to #5) at their 3&#180;-ends with A(5&#180;)pp(5&#180;)Cp-TEG-biotin using a one-step protocol described in step 2.2. In a total volume of 150 &#181;L reaction solution, add 15 &#181;L of 10x T4 RNA ligase reaction buffer, 1.5 &#181;L of each RNA strand (100 &#181;M stock of RNA #1 to #5, respectively, for a total volume of 7.5 &#181;L), 10 &#181;L of 150 &#181;M A(5&#180;)pp(5&#180;)Cp-TEG-biotin, 15 &#181;L of anhydrous DMSO, 5 &#181;L of T4 RNA ligase (10 units/&#181;L), and 97.5 &#181;L of DEPC-treated H2O. Equally distribute the reaction solution into five aliquots. Each RNase-free microcentrifuge tube contains 30 &#181;L of reaction solution.</li>
	<li>Incubate the reaction overnight at 16 &#176;C in a PCR machine.</li>
	<li>Perform column purification according to the procedure as described in steps 2.1.5-2.1.8&#160;with five parallel spin columns. Elute a mixture sample of 3&#180;-biotinylated 5 RNA strands (mixture of RNA #1 to #5) to a 1.5 mL RNase-free microcentrifuge tube each with 15 &#181;L of DEPC-treated H2O.</li>
	<li>Combine the purified mixture samples from the five collection tubes into one tube. Perform formic acid degradation according to the procedure described in Section 4.</li>
	<li>Measure samples by LC-MS as described in Section 6, and analyze the data using the data analysis software with optimized MFE settings to extract data containing mass, tR, and volume as described in step 6.5. The typical processing and base-calling algorithm is not applied due to the significantly increased data complexity resulting from the mixture. All bases in the RNA of the mixed sample are called manually in a method similar to Section 7.3 and match well with the theoretical ones in the RNA nucleotide and modification database8, thus the complete sequences of all five RNA strands in the mixed sample are accurately read out. In Tables S7&#8211;S11, all information is listed including observed mass, tR, volume, quality score and ppm mass difference.</li>
</ol>

<ol><li>Addepalli, B., Venus, S., Thakur, P., Limbach, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+ribonuclease+activity+of+cusativin+from+Cucumis+sativus+for+mapping+nucleoside+modifications+in+RNA%5BTitle%5D)+AND+%22Analytical+and+Bioanalytical+Chemistry%22%5BJournal%5D)">Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.</a> Analytical and Bioanalytical Chemistry. 409 (24), 5645-5654 (2017).</li>
<li>Gao, H., Liu, Y., Rumley, M., Yuan, H., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequence+confirmation+of+chemically+modified+RNAs+using+exonuclease+digestion+and+matrix-assisted+laser+desorption%2Fionization+time-of-flight+mass+spectrometry%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 23 (21), 3423-3430 (2009).</li>
<li>McLuckey, S. A., Van Berkel, G. J., Glish, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tandem+mass+spectrometry+of+small%2C+multiply+charged+oligonucleotides%5BTitle%5D)+AND+%22Journal+of+The+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">Tandem mass spectrometry of small, multiply charged oligonucleotides.</a> Journal of The American Society for Mass Spectrometry. 3 (1), 60-70 (1992).</li>
<li>Fountain, K. J., Gilar, M., Gebler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+native+and+chemically+modified+oligonucleotides+by+tandem+ion-pair+reversed-phase+high-performance+liquid+chromatography%2Felectrospray+ionization+mass+spectrometry%5BTitle%5D)+AND+%22Rapid+Communications+in+Mass+Spectrometry%22%5BJournal%5D)">Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 17 (7), 646-653 (2003).</li>
<li>Taucher, M., Breuker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+modified+RNA+by+top-down+mass+spectrometry%5BTitle%5D)+AND+%22Angewandte+Chemie+International+Edition+in+English%22%5BJournal%5D)">Characterization of modified RNA by top-down mass spectrometry.</a> Angewandte Chemie International Edition in English. 51 (45), 11289-11292 (2012).</li>
<li>Kellner, S., Burhenne, J., Helm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+RNA+modifications%5BTitle%5D)+AND+%22RNA+Biology%22%5BJournal%5D)">Detection of RNA modifications.</a> RNA Biology. 7 (2), 237-247 (2010).</li>
<li>Thomas, B., Akoulitchev, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+of+RNA%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Mass spectrometry of RNA.</a> Trends in Biochemical Sciences. 31 (3), 173-181 (2006).</li>
<li>Bjorkbom, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bidirectional+direct+sequencing+of+noncanonical+RNA+by+two-dimensional+analysis+of+mass+chromatograms%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Bidirectional direct sequencing of noncanonical RNA by two-dimensional analysis of mass chromatograms.</a> Journal of the American Chemical Society. 137 (45), 14430-14438 (2015).</li>
<li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+general+LC-MS-based+RNA+sequencing+method+for+direct+analysis+of+multiple-base+modifications+in+RNA+mixtures%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.</a> Nucleic Acids Research. 47 (20), 125(2019).</li>
<li>Zhang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+sequencing+of+tRNA+by+2D-HELS-AA+MS+Seq+reveals+its+different+isoforms+and+dynamic+base+modifications%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">Direct sequencing of tRNA by 2D-HELS-AA MS Seq reveals its different isoforms and dynamic base modifications.</a> ACS Chemical Biology. 15 (6), 1464-1472 (2020).</li>
<li>Bakin, A., Ofengand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four+newly+located+pseudouridylate+residues+in+Escherichia+coli+23S+ribosomal+RNA+are+all+at+the+peptidyltransferase+center%3A+analysis+by+the+application+of+a+new+sequencing+technique%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique.</a> Biochemistry. 32 (37), 9754-9762 (1993).</li>
<li>Cantara, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+Modification+Database%2C+RNAMDB%3A+2011+update%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">The RNA Modification Database, RNAMDB: 2011 update.</a> Nucleic Acids Research. 39 (Database issue), D195-D201 (2011).</li>
</ol>

The authors have filed a provisional patent related to the technology discussed in this manuscript.

Unlike tandem-based MS fragmentation, highly controlled acidic hydrolysis is used in the 2D-HELS MS Seq approach to fragment the RNA before analysis with a mass spectrometer9,10. As a result, each acid-degraded fragment can be detected by the instrument, forming the equivalent of a sequencing ladder. Under optimal conditions, this method creates an &#8220;ideal&#8221; sequence ladder from RNA via, on average, one-per-molecule site-specific RNA cleavage e...

Mass spectrometry (MS)-based sequencing methods, including top-down MS and tandem MS1,2,3,4, have been developed for direct sequencing of RNA. However, in situ fragmentation techniques for effectively generating high-quality RNA ladders in mass spectrometers currently can not be applied to de novo sequencing5,6. Furthermore, it is not very trivial to analyze the traditional one-dimensional (1D) MS data for de novo sequencing of even one purified RNA sequence, and it would be even more challenging for MS sequencing of mixed RNA samples7,8. Therefore, a two-dimensional (2D) liquid chromatography (LC)-MS-based RNA sequencing method has been developed, incorporating production of 2D mass-retention time (tR) ladders to replace 1D mass ladders, making it much easier to identify ladder components needed for de novo sequencing of RNAs8. However, the 2D LC-MS-based RNA sequencing method is mainly limited to purified synthetic short RNA, as it cannot read a complete sequence solely based on one single ladder, but must rely on two co-existing adjacent ladders (5&#180;- and 3&#180;-ladders)8. More specifically, this approach requires bidirectional paired-end reads for reading terminal nucleobases in the low-mass region8. The added complexity of the paired-end reading results in this method being untenable for sequencing of RNA mixtures because confusion is raised on which ladder fragment belongs to which ladder for the unknown samples.&#160;
To overcome the abovementioned barriers in MS-based RNA sequencing approaches and to broaden such applications in direct RNA sequencing, two issues must be addressed: 1) how to generate a high-quality mass ladder that can be used to read a complete sequence, from the first nucleotide to the last in an RNA strand,&#160;and 2) how to effectively identify each RNA/mass ladder in a complex MS dataset. Together with well-controlled acid degradation, we have developed a new sequencing method by introducing a hydrophobic end labeling strategy (HELS) into the MS-based sequencing technique, and successfully addressed these two issues by adding a hydrophobic tag at either 5&#180;- and/or 3&#180;-end of the RNAs to be sequenced9. This method creates an &#8220;ideal&#8221; sequence ladder from RNA&#8212;each ladder fragment derives from site-specific RNA cleavage exclusively at each phosphodiester bond, and the mass difference between two adjacent ladder fragments is the exact mass of either the nucleotide or nucleotide modification at that position 8,9,10. This is possible because we include a highly controlled acidic hydrolysis step, which fragments the RNA, on average, once per molecule, before it is injected into the instrument. As a result, each degradation fragment product is detected on the mass spectrometer and all fragments together form a sequencing ladder8,9,10. This new strategy enables complete reading of an RNA sequence from one single ladder of an RNA strand without paired-end reading from the other ladder of the RNA, and additionally allows MS sequencing of RNA mixtures with multiple different strands that contain combinatorial nucleotide modifications9. By adding a tag at the 5&#180;- and/or 3&#180;-end of the RNA, the labeled ladder fragments display a significant delay of tR, which can help to distinguish the two mass ladders from each other and also from the noisy low-mass region. The mass-tR shift caused by adding the hydrophobic tag facilitates mass ladder identification and simplifies data analysis for sequence generation. Furthermore, the addition of the hydrophobic tag can help to identify the terminal base in the strand by preventing its corresponding ladder fragment from being in the noisy low-mass-tR region due to the mass and hydrophobicity increase caused by the tag, thus allowing identification of the complete sequence of an RNA from a single ladder; no paired-end reads are required. As a result, we have previously demonstrated the successful sequencing of a complex mixture of up to 12 RNA distinct strands without the use of any advanced sequencing algorithm9, which opens the door for de novo MS sequencing of RNA containing both canonical and modified nucleotides and makes it more feasible for the sequencing of mixed and more complex RNA samples. In fact, using 2D-HELS MS Seq, we have even successfully sequenced a mixed population of tRNA samples10 and are actively expanding its application to other complex RNA samples.&#160;
To facilitate 2D-HELS MS Seq to directly sequence a broader range of RNA samples, here we will focus on the technical aspects of this sequencing approach and will cover all of the essential steps needed when applying the technique towards direct sequencing of RNA samples. Specific examples will be used to illustrate the sequencing technique, including synthetic single RNA sequences, mixtures of multiple distinct RNA sequences, and modified RNAs containing both canonical and modified nucleotides such as pseudouridine (&#968;) and 5-methylcytosine (m5C). Since RNAs all contain phosphodiester bonds, any type of RNA can be acid-hydrolyzed to generate an ideal sequence ladder for 2D-HELS MS Seq under optimal conditions8,9. However, detection of all ladder fragments of a given RNA is instrument dependent. On a standard high-resolution LC-MS (40K), the minimal loading amount for sequencing a purified short RNA sample (&#60;35 nt) is 100 pmol per run. However, more material is required (up to 400 pmol per RNA sample) when additional experiments must be conducted (e.g., to distinguish isomeric base modifications that share identical masses). The protocol used in sequencing the model synthetic modified RNAs will also be applicable to sequencing broader RNA samples, including biological RNA samples with unknown base modifications. However, an even larger sample amount, such as 1000 pmol for sequencing tRNA (~76 nt) using a standard LC-MS instrument, is required to sequence the complete tRNA with all the modifications, and an advanced algorithm must be developed for its de novo sequencing10.

<table><tbody><tr><td>5&#039; DNA Adenylation kit</td><td>New England Biolabs</td><td>E2610S</td><td>50uM concentration</td></tr><tr><td>6550 Q-TOF mass spectrometer</td><td>Agilent Technologies</td><td>5991-2116EN</td><td>Coupled to a 1290 Infinity LC system</td></tr><tr><td>A(5&amp;acute;)pp(5&amp;acute;)Cp-TEG-biotin-3&amp;acute;</td><td>ChemGenes</td><td>91718</td><td>HPLC purified</td></tr><tr><td>ATP&amp;gamma;S</td><td>Sigma-Aldrich</td><td>11162306001</td><td>Lithium salt</td></tr><tr><td>Bicine</td><td>Sigma-Aldrich</td><td>B8660</td><td>BioXtra, &amp;ge;99% (titration)</td></tr><tr><td>Biotin maleimide</td><td>Vector Laboratories</td><td>SP-1501</td><td>Long arm</td></tr><tr><td>C18 column</td><td>Waters</td><td>186003532</td><td>50 mm &amp;times; 2.1 mm Xbridge C18 column with a particle size of 1.7 &amp;mu;m</td></tr><tr><td>Centrifugal Vacuum Concentrator</td><td>Labconco</td><td>Refrig 115v/60hz 7310022</td><td>Labconco CentriVap</td></tr><tr><td>ChemBioDraw</td><td>PerkinElmer</td><td>ChemDraw Prime</td><td>Generate a chemical structure and property data of structures &amp;amp; fragments</td></tr><tr><td>CMC (N-cyclohexyl-N?-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate)</td><td>Sigma-Aldrich</td><td>2491-17-0</td><td>95% Purifiy</td></tr><tr><td>Cyanine3 maleimide (Cy3)</td><td>Lumiprobe</td><td>11080</td><td>Water insoluble</td></tr><tr><td>DEPC-treated water</td><td>Thermo Fisher Scientific</td><td>AM9906</td><td>Autoclaved, certified nuclease-free</td></tr><tr><td>Diisopropylamine (DIPA)</td><td>Thermo Fisher Scientific</td><td>108-18-9</td><td>99% Alfa Aesar</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>276855</td><td>Anhydrous dimethyl sulfoxide, 99.9%</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6758</td><td>Anhydrous, crystalline, BioReagent, suitable for cell culture</td></tr><tr><td>Formic acid</td><td>Merck</td><td>64-18-6</td><td>98-100%, ACS reag, Ph Eur</td></tr><tr><td>Hexafluoro-2-propanol (HFIP)</td><td>Thermo Fisher Scientific</td><td>920-66-1</td><td>99% Acros Organics</td></tr><tr><td>LC-MS sample vials</td><td>Thermo Fisher Scientific</td><td>C4000-11</td><td>Plastic screw thread vials</td></tr><tr><td>LC-MS vial caps</td><td>Thermo Fisher Scientific</td><td>C5000-54A</td><td>Autosampler vial screw thread caps</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt; buffer</td><td>Sigma-Aldrich</td><td>88975</td><td>BioUltra, &gt;0.1 M Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;, &gt;0.2 M NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Oligo Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4060</td><td>Spin column</td></tr><tr><td>OriginLab</td><td>OriginLab</td><td>OriginPro</td><td>Data analysis and graphing software</td></tr><tr><td>pCp-biotin</td><td>TriLink BioTechnologies</td><td>NU-1706-BIO</td><td>20 ul (1 mM)</td></tr><tr><td>RNA #1--#6</td><td>Integrated DNA Technologies</td><td>Custom RNA oligos</td><td>19nt-21nt single-stranded RNAs, used without further purification</td></tr><tr><td>Rocking platform shaker</td><td>VWR</td><td>Orbital Shaker Standard 1000</td><td>Speed Range 40 to 300 rpm</td></tr><tr><td>Streptavidin magnetic beads</td><td>Thermo Fisher Scientific</td><td>88816</td><td>Binding approx. 55ug biotinylated rabbit lgG per mg of beads</td></tr><tr><td>Sulfonated Cyanine3 maleimide</td><td>Lumiprobe</td><td>11380</td><td>Water soluble</td></tr><tr><td>T4 DNA ligase 1</td><td>New England Biolabs</td><td>M0202S</td><td>400 units/uL</td></tr><tr><td>T4 polynucleotide kinase</td><td>Sigma-Aldrich</td><td>T4PNK-RO</td><td>From phage T4 am N81 pse T1 infected Escherichia coli BB</td></tr><tr><td>Tris-HCl buffer</td><td>Sigma-Aldrich</td><td>T6455</td><td>Tris-HCl Buffer, pH 10, 10&amp;times;, Antigen Retriever</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>81871</td><td>Urea for synthesis. CAS No. 57-13-6, EC Number 200-315-5.</td></tr></tbody></table>

2d-hels ms seq: a general lc-ms-based method for direct and de novo sequencing of rna mixtures with different nucleotide modifications

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;

Introducing a biotin tag to the 3´-end of RNA to produce easily-identifiable mass-tR ladders. The workflow of the 2D-HELS MS Seq approach is demonstrated in Figure 1a. The hydrophobic biotin label introduced to the 3´-end of the RNA (see Section 2) increases the masses and tRs of the 3´-labeled ladder components when compared to those of their unlabeled counterparts. Thus, the 3´-ladder curve is shifted to greater y-axis values (due to the increase in t...

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 ...

2d-hels-ms-seq-general-lc-ms-based-method-for-direct-de-novo

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

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

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