WO2009095000A2 - Procédé de séquençage d'une molécule d'arn par spectrométrie de masse - Google Patents

Procédé de séquençage d'une molécule d'arn par spectrométrie de masse Download PDF

Info

Publication number
WO2009095000A2
WO2009095000A2 PCT/DE2009/000125 DE2009000125W WO2009095000A2 WO 2009095000 A2 WO2009095000 A2 WO 2009095000A2 DE 2009000125 W DE2009000125 W DE 2009000125W WO 2009095000 A2 WO2009095000 A2 WO 2009095000A2
Authority
WO
WIPO (PCT)
Prior art keywords
rna molecule
rna
acid
mass spectrometry
sequencing
Prior art date
Application number
PCT/DE2009/000125
Other languages
German (de)
English (en)
Other versions
WO2009095000A3 (fr
Inventor
Ute Bahr
Michael Karas
Original Assignee
Johann Wolfgang Goethe-Universität
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johann Wolfgang Goethe-Universität filed Critical Johann Wolfgang Goethe-Universität
Publication of WO2009095000A2 publication Critical patent/WO2009095000A2/fr
Publication of WO2009095000A3 publication Critical patent/WO2009095000A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6872—Methods for sequencing involving mass spectrometry

Definitions

  • the invention relates to a method for sequencing an RNA molecule, in particular an RNA oligonucleotide by means of mass spectrometry, and the use of such a method.
  • RNA molecules The mass spectroscopic analysis of RNA molecules is described in the prior art, in particular using enzymatic degradation preceding actual mass spectrometry. This enzymatic degradation is done using exonucleases or endonucleases and has been described by several authors
  • time of termination of the enzymatic digestion must be chosen precisely because it depends strongly on the base composition of the RNA molecule to be analyzed. Normally, therefore, samples are taken from the digestion solution at different times, since it is very rare, due to the enzyme kinetics, for all to be sequenced in a solution taken at a certain time
  • J5 fragments are included. Thus, several mass spectrometric measurements must be performed to determine a sequence.
  • Hahner et al. (Nucleic Acid Res 25, 1997, 1957 to 1964) describes a method for mass spectroscopic analysis of an RNA molecule by means of alkaline hydrolysis and subsequent mass spectrometry.
  • this method has several disadvantages.
  • the method according to Hahner et al. allows only the sequencing of the RNA molecule to be examined from one side of the RNA molecule. Complete sequencing is not possible since signals below about 1000 Da can no longer be detected. Both alkaline hydrolysis and enzymatic digestion of the analyte
  • RNA molecules take place in buffer solutions containing hydroxides or detergents.
  • the direct mass spectrometric analysis of these solutions leads to broad peaks with a low mass resolution.
  • the mass accuracy suffers, so that it is difficult to unambiguously identify the two nucleotides uridine and cytidine, which differ only by 1 Da. 0
  • the object of the present invention is to provide a method by means of which the sequencing of an RNA molecule can be achieved using mass spectrometry without the disadvantages described above occurring.
  • an RNA molecule is provided, for example in the form of an RNA oligonucleotide.
  • RNA molecule is subjected to acid hydrolysis so that exactly one phosphodiester bond of the RNA molecule is cleaved.
  • RNA molecule which can be sequenced by the method according to the invention has adenine, cytosine, guanine and / or uracil as bases.
  • the RNA molecule to be sequenced may also have further bases and / or modified bases (eg bases modified by methylation or deamination), modified sugars and / or modifications to the phosphate backbone (such as phosphorodiamidate morpholino residues) to the extent that these other bases, modified bases and / or chemical groups differ in their mass sufficiently from one another and sufficiently from the bases or chemical groups otherwise present in the RNA molecule, so that they can be distinguished and identified by mass spectrometry.
  • RNA oligonucleotides with a length of up to 30 nucleotides, preferably up to 21 or 22 nucleotides, or with a length of 3 to 30 nucleotides, preferably with a length of 3 nucleotides to 22 nucleotides or, preferably, from 6 nucleotides to 22 nucleotides are sequenced by the method according to the invention.
  • the RNA molecule to be sequenced is provided in a solution.
  • this is an aqueous solution.
  • the concentration of the RNA molecule in the solution or the aqueous solution is in an advantageous embodiment of the method according to the invention 15 .mu.mol / 1 to 25 .mu.mol / 1, wherein a concentration of 20 .mu.mol / 1 is preferred.
  • 1 pmol to 1000 pmol, preferably 100 pmol to 500 pmol, particularly preferably 200 pmol of RNA are used.
  • the acidic hydrolysis of the RNA molecule is advantageously carried out with a strong acid.
  • Suitable strong acids are, for example, formic acid, trifluoroacetic acid and hydrochloric acid, with formic acid and trifluoroacetic acid being preferred, since they are commercially available in high purity and contamination of the sample with salts or other impurities can not occur when they are used.
  • the pH of the solution is between 0 and 2.
  • Preferred for the acidic hydrolysis is a pH of ⁇ 2, more preferably a pH of 1.5 to 1.8.
  • the acidic hydrolysis can be carried out at different temperatures and for a different duration depending on the chosen temperature. In general, temperatures of about 4 0 C to about 60 ° C are possible, the respective incubation time of the sample must be adjusted so that the acid hydrolysis to the cleavage of only one
  • the reaction can be stopped after about 2 minutes. If the acidic hydrolysis is carried out at a temperature of 4 ° C., the necessary incubation time can be 16 hours. A person skilled in the art will be able to determine the parameters of temperature and incubation length by means of routine experiments.
  • the reaction solution or part of the reaction solution can be used directly for mass spectroscopic sequence determination, without the need for a further purification step.
  • sequence determination of the RNA molecule is carried out by means of MALDI (matrix assisted laser desorption / ionization) mass spectrometry.
  • sequencing on the basis of a single spectrum in both 5 'to 3', and 3 'to 5' direction take place.
  • sequencing is in both directions to allow complete sequencing of the RNA molecule.
  • sequencing in both directions serves as an internal control of sequencing in the other direction.
  • mass analyzers are all combinable with MALDI devices into consideration, such as. Linear time of flight mass spectrometers (TOF), reflector TOFs, orthogonal TOFs, FTICR-MS or Orbitrap MS. All of these mass analyzers are known to the person skilled in the art.
  • TOF Linear time of flight mass spectrometers
  • reflector TOFs reflector TOFs
  • orthogonal TOFs FTICR-MS
  • Orbitrap MS Orbitrap MS. All of these mass analyzers are known to the person skilled in the art.
  • RNA fragments obtained from the acidic hydrolysis in conjunction with a matrix.
  • Suitable matrix materials are all matrix compounds which can be used for MALDI, for example 3-hydroxy-picolinic acid (HPA), 6-aza-2-thiothymine (ATT) and 2,4,6-trihydroxyacetophenone. 6-aza-2-thiothymine (ATT) has proven to be particularly advantageous for this sequencing of RNA molecules, in particular of RNA oligonucleotides.
  • Performing the MALDI mass spectrometry leads to a specific intensity value for each detectable mass (m / z) 5.
  • mass peaks result, at which the measured signal intensity reaches a local maximum.
  • the mass difference is determined for every two such mass peaks that are adjacent to each other. From this mass difference can be concluded that the base at a certain position 10 of the RNA molecule or on the nucleotide.
  • the terminal sequence can be determined by MS / MS fragmentation in order to improve the identifiability of the terminal one to two nucleotides of the RNA molecule to be sequenced.
  • the problem underlying the invention is also solved by the use of a method described above for sequencing an RNA molecule, in particular for sequencing an RNA oligonucleotide by mass spectrometry.
  • the description of the use according to the invention is based on the description contained herein.
  • RNA molecules in particular for siRNA molecules, antisense molecules and miRNA molecules.
  • Fig. 1 shows the reaction scheme of acid hydrolysis of an RNA molecule. Both obtained molecular fragments can be detected by mass spectrometry as protonated or deprotonated (0 molecules;
  • the terminal sequence can be amplified by MS / MS.
  • Fragmentation can be determined.
  • the fragment ion of interest can be isolated in the mass analyzer and fragmented by collisions with inert gas atoms.
  • the fragment ion spectra are shown here using the example of two dimers with inverse sequence (FIG. 4a: UGp, FIG. 4b: GUp) and a 3mer (FIG. 4c: GGUp).
  • the fragmentation leads to so-called y- and c-fragments (see FIG. 4d), whereby the y-
  • Ions always have a higher intensity than the c ions, yi or y 2 ions are formed by cleavage of the 3 '-terminal nucleotide, C 1 - and c 2 -ions by cleavage of the 5' -terminal nucleotide.
  • the inventive method allows the complete sequencing of an RNA molecule in the form of an RNA oligonucleotide of 21 nucleotides.
  • a sequencing in 5 '-3' direction (conductor 2)
  • a sequencing in 3 '-5' direction (conductor 1).
  • RNA oligonucleotide of the sequence UAU CAC UUG AUC UCG UAC ATT (SEQ ID NO: 1) is treated in an aqueous solution in a concentration of 20 .mu.mol / 1 with formic acid as a strong acid, that the pH of the solution ⁇ 2 is.
  • the acidic hydrolysis takes place at a temperature of 60 ° C. for 2 minutes and is terminated by rapid cooling of the sample.
  • RNA oligonucleotide As preliminary experiments have shown, the hydrolysis of exactly one phosphodiester bond per RNA oligonucleotide takes place at this temperature and incubation time on average, so that all possible RNA oligonucleotide fragments which can be generated by hydrolysis of a phosphodiester bond are contained in the solution in the same frequency.
  • MS analysis 0.5 ⁇ l of the RNA solution is mixed with 2 ⁇ l of 6-aza-2-thiothymine (ATT) on a sample plate and dried. The obtained spectrum is shown in FIG. The sequence is determined by starting from the intact molecular peak between two mass peaks adjacent in the spectrum, the mass difference is formed.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de séquençage d'une molécule d'ARN, en particulier d'un oligonucléotide d'ARN, par spectrométrie de masse. Le procédé est caractérisé en ce qu'il présente les étapes suivantes : a) préparation d'une molécule d'ARN; b) scission de la molécule d'ARN par hydrolyse acide; et c) détermination de la séquence de la molécule d'ARN par spectrométrie de masse.
PCT/DE2009/000125 2008-01-31 2009-01-30 Procédé de séquençage d'une molécule d'arn par spectrométrie de masse WO2009095000A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200810007112 DE102008007112B3 (de) 2008-01-31 2008-01-31 Verfahren zur Sequenzierung eines RNA-Moleküls mittels Massenspektrometrie
DE102008007112.9 2008-01-31

Publications (2)

Publication Number Publication Date
WO2009095000A2 true WO2009095000A2 (fr) 2009-08-06
WO2009095000A3 WO2009095000A3 (fr) 2009-11-05

Family

ID=40822374

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2009/000125 WO2009095000A2 (fr) 2008-01-31 2009-01-30 Procédé de séquençage d'une molécule d'arn par spectrométrie de masse

Country Status (2)

Country Link
DE (1) DE102008007112B3 (fr)
WO (1) WO2009095000A2 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19714558A1 (de) * 1997-04-09 1998-10-15 Joachim W Prof Dr Engels Ein neues Verfahren zur Sequenzierung von Biopolymeren mit Massenspektrometrie
WO1999057318A2 (fr) * 1998-05-07 1999-11-11 Sequenom, Inc. Analyse de molecules par spectrometrie de masse a desorption/ionisation par laser, assistee par matrice absorbant l'infrarouge

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19714558A1 (de) * 1997-04-09 1998-10-15 Joachim W Prof Dr Engels Ein neues Verfahren zur Sequenzierung von Biopolymeren mit Massenspektrometrie
WO1999057318A2 (fr) * 1998-05-07 1999-11-11 Sequenom, Inc. Analyse de molecules par spectrometrie de masse a desorption/ionisation par laser, assistee par matrice absorbant l'infrarouge

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FAULSTICH K ET AL: "A sequencing method for RNA oligonucleotides based on mass spectrometry." ANALYTICAL CHEMISTRY 1 NOV 1997, Bd. 69, Nr. 21, 1. November 1997 (1997-11-01), Seiten 4349-4353, XP002540491 ISSN: 0003-2700 *
HAHNER STEPHANIE ET AL: "Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of endonuclease digests of RNA" NUCLEIC ACIDS RESEARCH, Bd. 25, Nr. 10, 1997, Seiten 1957-1964, XP002540493 ISSN: 0305-1048 *
NORDHOFF E ET AL: "Direct mass spectrometric sequencing of low-picomole amounts of oligodeoxynucleotides with up to 21 bases by matrix-assisted laser desorption/ionization mass spectrometry" JOURNAL OF MASS SPECTROMETRY 1995 GB, Bd. 30, Nr. 1, 1995, Seiten 99-112, XP002540492 ISSN: 1076-5174 *
TOLSON D A ET AL: "Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF" NUCLEIC ACIDS RESEARCH, Bd. 26, Nr. 2, 15. Januar 1998 (1998-01-15), Seiten 446-451, XP002540494 ISSN: 0305-1048 *

Also Published As

Publication number Publication date
DE102008007112B3 (de) 2009-08-06
WO2009095000A3 (fr) 2009-11-05

Similar Documents

Publication Publication Date Title
Nordhoff et al. Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ionization mass spectrometry
EP0925373A1 (fr) Procede pour la detection d'acides nucleiques par determination de la masse
DE19802905C2 (de) Verfahren zur bevorzugten Herstellung nur eines Stranges selektierten Genmaterials für massenspektrometrische Messungen
DE60219132T2 (de) Mutationsanalyse mittels PCR und Massenspektrometrie
DE102009013653B4 (de) Protein-Sequenzierung mit MALDI-Massenspektrometrie
US6723564B2 (en) IR MALDI mass spectrometry of nucleic acids using liquid matrices
Wu et al. Gas-phase fragmentation of oligonucleotide ions
Cohen et al. Emerging technologies for sequencing antisense oligonucleotides: capillary electrophoresis and mass spectrometry
DE69838519T2 (de) Massenmarkierte Hybridisierungssonden
DE19710166C1 (de) Zwei-Schritt-Verfahren der DNA-Amplifikation für MALDI-TOF-Messungen
Muddiman et al. Sequencing and characterization of larger oligonucleotides by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry
Kirpekar et al. RNA fragmentation studied in a matrix‐assisted laser desorption/ionisation tandem quadrupole/orthogonal time‐of‐flight mass spectrometer
EP0612994A2 (fr) Matrice pour la spectrométrie de masse avec désorption par laser assistée par matrice
DE19853398C1 (de) Verfahren zur Identifikation von Cytosin-Methylierungsmustern in genomischer DNA
Distler et al. 5-Methoxysalicylic acid and spermine: A new matrix for the matrix-assisted laser desorption/ionization mass spectrometry analysis of oligonucleotides
Schultz et al. Rapid electron capture dissociation of mass-selectively accumulated oligodeoxynucleotide dications
DE102005061425A1 (de) Rückgesteuerte Fragmentierung in Ionenfallen-Massenspektrometern
WO2009095000A2 (fr) Procédé de séquençage d'une molécule d'arn par spectrométrie de masse
Kellersberger et al. Atmospheric pressure MALDI-FTMS of normal and chemically modified RNA
Galhena et al. Influence of cluster size and ion activation method on the dissociation of cesium iodide clusters
Gao et al. Sequence confirmation of chemically modified RNAs using exonuclease digestion and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Gao et al. Sequence verification of oligonucleotides containing multiple arylamine modifications by enzymatic digestion and liquid chromatography mass spectrometry (LC/MS)
Luo et al. Charge effects for differentiation of oligodeoxynucleotide isomers containing 8-oxo-dG residues
Polo et al. Analysis of oligonucleotides by electrospray ionization mass spectrometry
DE19714558A1 (de) Ein neues Verfahren zur Sequenzierung von Biopolymeren mit Massenspektrometrie

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09705849

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 09705849

Country of ref document: EP

Kind code of ref document: A2