JoVE Logo
Faculty Resource Center

Sign In





Representative Results






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

Published: July 10th, 2020



1Department of Biological and Chemical Sciences, New York Institute of Technology, 2Department of Chemical Engineering, Columbia University, 3Department of Chemistry, Hunter College, City University of New York, 4Department of Computer Science, New York Institute of Technology

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 (<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 direct sequencing 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 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 (pseudouridine (Ψ) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide 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 (<35 nt) when using a standard high-resolution LC-MS system, and can also be used for sequence verification of modified therapeutic RNA oligonucleotides. In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples. 

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 sequenc....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Design RNA oligonucleotides

  1. Design synthetic RNA oligonucleotides with different lengths (19 nt, 20 nt and 21 nt), including one (RNA #6) with both canonical and modified nucleotides. ψ 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.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 “ideal” sequence ladder from RNA via, on average, one-per-molecule site-specific RNA cleavage e.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The authors acknowledge the R21 grant from National Institutes of Health (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 suggestio....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
5' DNA Adenylation kitNew England BiolabsE2610S50uM concentration
6550 Q-TOF mass spectrometerAgilent Technologies5991-2116ENCoupled to a 1290 Infinity LC system
A(5´)pp(5´)Cp-TEG-biotin-3´ChemGenes91718HPLC purified
ATPγSSigma-Aldrich11162306001Lithium salt
BicineSigma-AldrichB8660BioXtra, ≥99% (titration)
Biotin maleimideVector LaboratoriesSP-1501Long arm
C18 columnWaters18600353250 mm × 2.1 mm Xbridge C18 column with a particle size of 1.7 μm
Centrifugal Vacuum ConcentratorLabconcoRefrig 115v/60hz 7310022Labconco CentriVap
ChemBioDrawPerkinElmerChemDraw PrimeGenerate a chemical structure and property data of structures & fragments
CMC (N-cyclohexyl-Nʹ-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate)Sigma-Aldrich2491-17-095% Purifiy
Cyanine3 maleimide (Cy3)Lumiprobe11080Water insoluble
DEPC-treated waterThermo Fisher ScientificAM9906Autoclaved, certified nuclease-free
Diisopropylamine (DIPA)Thermo Fisher Scientific108-18-999% Alfa Aesar
DMSOSigma-Aldrich276855Anhydrous dimethyl sulfoxide, 99.9%
EDTASigma-AldrichE6758Anhydrous, crystalline, BioReagent, suitable for cell culture
Formic acidMerck64-18-698-100%, ACS reag, Ph Eur
Hexafluoro-2-propanol (HFIP)Thermo Fisher Scientific920-66-199% Acros Organics
LC-MS sample vialsThermo Fisher ScientificC4000-11Plastic screw thread vials
LC-MS vial capsThermo Fisher ScientificC5000-54AAutosampler vial screw thread caps
Na2CO3 bufferSigma-Aldrich88975BioUltra, >0.1 M Na2CO3, >0.2 M NaHCO3
Oligo Clean & ConcentratorZymo ResearchD4060Spin column
OriginLabOriginLabOriginProData analysis and graphing software
pCp-biotinTriLink BioTechnologiesNU-1706-BIO20 ul (1 mM)
RNA #1--#6Integrated DNA TechnologiesCustom RNA oligos19nt-21nt single-stranded RNAs, used without further purification
Rocking platform shakerVWROrbital Shaker Standard 1000Speed Range 40 to 300 rpm
Streptavidin magnetic beadsThermo Fisher Scientific88816Binding approx. 55ug biotinylated rabbit lgG per mg of beads
Sulfonated Cyanine3 maleimideLumiprobe11380Water soluble
T4 DNA ligase 1New England BiolabsM0202S400 units/uL
T4 polynucleotide kinaseSigma-AldrichT4PNK-ROFrom phage T4 am N81 pse T1 infected Escherichia coli BB
Tris-HCl bufferSigma-AldrichT6455Tris-HCl Buffer, pH 10, 10×, Antigen Retriever
UreaSigma-Aldrich81871Urea for synthesis. CAS No. 57-13-6, EC Number 200-315-5.

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

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved