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

Summary

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.

Abstract

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. 

Introduction

Mass spectrometry (MS)-based sequencing methods, including top-down MS and tandem MS^1,2,3,4, 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 sequencing^5,6. 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....

Protocol

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.
   
   RNA #1: 5´-HO-CGCAUCUGACUGACCAAAA-OH-3´

Discussion

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 spectrometer^9,10. 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.......

Materials

Name	Company	Catalog Number	Comments
5' DNA Adenylation kit	New England Biolabs	E2610S	50uM concentration
6550 Q-TOF mass spectrometer	Agilent Technologies	5991-2116EN	Coupled to a 1290 Infinity LC system
A(5´)pp(5´)Cp-TEG-biotin-3´	ChemGenes	91718	HPLC purified
ATPγS	Sigma-Aldrich	11162306001	Lithium salt
Bicine	Sigma-Aldrich	B8660	BioXtra, ≥99% (titration)
Biotin maleimide	Vector Laboratories	SP-1501	Long arm
C18 column	Waters	186003532	50 mm × 2.1 mm Xbridge C18 column with a particle size of 1.7 μm
Centrifugal Vacuum Concentrator	Labconco	Refrig 115v/60hz 7310022	Labconco CentriVap
ChemBioDraw	PerkinElmer	ChemDraw Prime	Generate a chemical structure and property data of structures & fragments
CMC (N-cyclohexyl-Nʹ-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate)	Sigma-Aldrich	2491-17-0	95% Purifiy
Cyanine3 maleimide (Cy3)	Lumiprobe	11080	Water insoluble
DEPC-treated water	Thermo Fisher Scientific	AM9906	Autoclaved, certified nuclease-free
Diisopropylamine (DIPA)	Thermo Fisher Scientific	108-18-9	99% Alfa Aesar
DMSO	Sigma-Aldrich	276855	Anhydrous dimethyl sulfoxide, 99.9%
EDTA	Sigma-Aldrich	E6758	Anhydrous, crystalline, BioReagent, suitable for cell culture
Formic acid	Merck	64-18-6	98-100%, ACS reag, Ph Eur
Hexafluoro-2-propanol (HFIP)	Thermo Fisher Scientific	920-66-1	99% Acros Organics
LC-MS sample vials	Thermo Fisher Scientific	C4000-11	Plastic screw thread vials
LC-MS vial caps	Thermo Fisher Scientific	C5000-54A	Autosampler vial screw thread caps
Na2CO3 buffer	Sigma-Aldrich	88975	BioUltra, >0.1 M Na2CO3, >0.2 M NaHCO3
Oligo Clean & Concentrator	Zymo Research	D4060	Spin column
OriginLab	OriginLab	OriginPro	Data analysis and graphing software
pCp-biotin	TriLink BioTechnologies	NU-1706-BIO	20 ul (1 mM)
RNA #1--#6	Integrated DNA Technologies	Custom RNA oligos	19nt-21nt single-stranded RNAs, used without further purification
Rocking platform shaker	VWR	Orbital Shaker Standard 1000	Speed Range 40 to 300 rpm
Streptavidin magnetic beads	Thermo Fisher Scientific	88816	Binding approx. 55ug biotinylated rabbit lgG per mg of beads
Sulfonated Cyanine3 maleimide	Lumiprobe	11380	Water soluble
T4 DNA ligase 1	New England Biolabs	M0202S	400 units/uL
T4 polynucleotide kinase	Sigma-Aldrich	T4PNK-RO	From phage T4 am N81 pse T1 infected Escherichia coli BB
Tris-HCl buffer	Sigma-Aldrich	T6455	Tris-HCl Buffer, pH 10, 10×, Antigen Retriever
Urea	Sigma-Aldrich	81871	Urea for synthesis. CAS No. 57-13-6, EC Number 200-315-5.