WO1998045700A2 - Method for the mass spectrometric sequencing of biopolymers - Google Patents

Method for the mass spectrometric sequencing of biopolymers Download PDF

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WO1998045700A2
WO1998045700A2 PCT/DE1998/001016 DE9801016W WO9845700A2 WO 1998045700 A2 WO1998045700 A2 WO 1998045700A2 DE 9801016 W DE9801016 W DE 9801016W WO 9845700 A2 WO9845700 A2 WO 9845700A2
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rna
sequencing
dna
peptides
nucleic acids
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PCT/DE1998/001016
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French (fr)
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WO1998045700A3 (en
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Joachim W. Engels
Karlheinz WÖRNER
Konrad Faulstich
Hannelore Brill
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Engels Joachim W
Woerner Karlheinz
Konrad Faulstich
Hannelore Brill
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • RNA can be sequenced by exonuclease digestion, separation and detection of the fragments generated using mass spectrometry.
  • the method can be particularly useful for determining the structural structure of RNA (or also DNA) which is longer than 20 bases or modified nucleic acids, since the resolution of mass spectrometers is a limiting factor for the sequence analysis of longer nucleic acid fragments. It can also be valuable in determining the secondary structure of nucleic acids.
  • the nucleic acid fragments are different
  • REPLACEMENT BLA ⁇ Masses of nucleotides emerging during the digestion of exonucleases (primarily 5 ' ⁇ 3' exonuclease from calf spleen and 3 ' ⁇ 5' exonuclease from snake venom from crotalus du ⁇ ssus), determined by mass spectrometry, but U and C are recognized by different peak intensities in the mass spectrum. The differences the peak intensities are caused by different rates in the hydrolysis of the phosphodiester bonds by the enzyme.
  • fragments which contain a C at the ⁇ '-end are degraded less rapidly by 5 ' ⁇ 3'-phosphod ⁇ esterase and are therefore present in much greater concentrations than fragments containing 5'-U, for example.
  • 5'- A fragments made Adenosm is also easily distinguishable from the other nucleotides by its mass. It is possible to subject several nucleic acids simultaneously to this enzyme kinetics in order to increase the sequencing speed.
  • base-specific exo- / endonucleases can also be used for sequence analysis and rapid detection and determination of organisms, eg viruses, whose RNA or DNA is finger fingered (digestion of RNA or DNA with exo- / endonucleases that do not cut base-specifically on each nucleotide) or foot panning (digestion of RNA or DNA that interacts with foreign nucleic acid molecules and is subjected to hydrolysis with exo / endonucleases)
  • Separation and detection of the fragments generated by mass spectrometry can be used, for example, to prevent epidemics or to determine organisms and biological weapons. Finger and footprint are more precise than previous methods.
  • Example 1 Sequencing an 8mer with 5 ' ⁇ 3' phosphodiesterase and (see Fig. 1)
  • Linear continuous MALDI-TOF mass spectrometry was carried out with a Fisons VG TOF spec mass spectrometer (8mer, 9mere RNA and DNA, 1 6mer, 22mer, 1 20mer) and DE-MALDI-TOF measurements with a PerSeptive Biosystems Voyager mass spectrometer (1 6mer) contain a UV nitrogen laser with an emission frequency of 337nm The laser pulse width is 4 ns. The spectra were recorded in the negative mode with the exception of the 1 ⁇ mers and the 32 mers. These were measured in the positive mode.
  • Calf spleen The enzyme attacks the oligonucleotide at the 5 'end and leaves 3' nucleotides. 3 ' ⁇ 5' phosphodiesterase from crotalus du ⁇ ssus The enzyme attacks the oligonucleotide at the 3 'end and leaves 5' nucleotides RNase CL3 from chicken liver.
  • RNA cleaves RNA preferentially at Cp / N bonds and produces fragments with 3 'terminal cytidine phosphate Ap / N and Gp / N bonds are hydrolyzed much more slowly, Up / N bonds very rarely RNase CL3 / buffer solution (denaturing) 2 ⁇ ⁇ RNase CL3 (0.2U / ⁇ l) + 6 ⁇ ⁇ 8 M urea in water result in 8 ⁇ ⁇ 50 mU / ⁇ l enzyme solution 3 ' ⁇ 5'-phosphod ⁇ esterase / buffer solution 2 ⁇ ⁇ (4 mU / ⁇ l) 3' ⁇ 5'-phosphod ⁇ esterase + 1 8 ⁇ ⁇ 0 1 M ammonium citrate, pH 5.5 result in 20 ul 0 2 mU / ul enzyme solution sequences of the RNA pieces examined were 8mer as follows: 5'-HO-CAUGUGAC-OH-3 ', 9mer (RNA). 5'-HO-GCAUGUGAC-OH-3 ',
  • REPLACEMENT B ⁇ RULE 26 shown).
  • the samples were mixed with the matrix in a ratio of 1: 1, pipetted onto the sample plate of the spectrometer and air-dried for about 20 minutes. The drying and crystallization time can be shortened by careful drying. The enzymatic digestion stops when the samples are mixed with the matrix.
  • RNA 8mer (0.1 OD) 9.0 ⁇ l RNase CL3 / buffer solution (400 mU; denaturing) 8.0 ⁇ l
  • RNA 16mer (0.1 OD) 15.2 ul 5 ' ⁇ 3'-phosphodiesterase (24mU) 6.0 ul
  • REPLACEMENT BUTT (RULE 26) a) with 5 ' ⁇ 3'phosphodiesterase (from calf spleen)
  • RNA 22mer (0.1 OD) 9.0 ul RNA 8mer (0.1 OD) 9.0 ul
  • Example 6 Sequencing of a 16mer oligoribonucleotide (fingerprint) with RNase CL3 (sequence from Table 2; see FIG. 9)
  • RNA 16mer (0.1 OD) 9.0 ⁇ l RNase CL3 / buffer solution (100 mU; denaturing) 15.2 ⁇ l
  • the method uses not only the determination of the mass for data interpretation, but also the peak intensities. This means that spectrometers with a low resolution, which are also easy to use, can be used and the method is inexpensive - No exact mass determination is necessary. Therefore very long polymers can be sequenced.
  • the method can be used to predict the secondary structure of biopolymers.
  • the method can be used for the sequencing of modified biopolymers.
  • the method can be used to determine organisms (fingerprint / footprint).
  • the method can be automated and parallelized.

Abstract

The invention relates to a new method for sequencing biopolymers by mass spectrometry. The sequencing of biopolymers is either lengthy or requires exact determination of the mass of the fragments, which is difficult, especially in the case of long polymers. The speed of hydrolysis of phosphodiester, -peptide or -glycoside bonds with exo/endonucleases, -peptidases, -glycosidases or other hydrolytically acting substances is used in the inventive method for the sequence analysis of nucleic acids or other biopolymers. Separation and detection of the fragments produced take place by mass spectrometry by determining the mass and the different peak intensities. The main advantage of the method is that an exact determination of the mass is no longer necessary, and also that low-resolution mass spectrometers can be used. Furthermore, analysis and separation of fragments is extremely rapid, as there is no electrophoresis and the sequence of modified nucleic acids can be determined. These principles can also be applied to sequencing or secondary structure determination of other biopolymers, like, for example, peptides and oligosaccharides.

Description

Verfahren zur massenspektrometrischen Sequenzierung von Biopolymeren Process for the mass spectrometric sequencing of biopolymers
Beschreibungdescription
Die zunehmenden Aktivitäten in der Forschung von Nukleinsäuren, besonders von Ribonukleinsäuren sowie von Peptiden und O gosacchaπden in den letzten Jahren, erfordern eine schnelle Standard-Sequenziermethode, die auch Modifikationen detektieren kann. Bisherige Methoden für die Sequenzierung von RNA beruhen auf zweidimensionalen chromatographischen Methoden1 3, Maxam-Gilbert-Sequenzie rung4 7 oder Reverse Transkπptase Sanger Sequenzierung8 9 Neuere Entwicklungen benutzen Massenspektrometrie zur Pπmarstrukturbestimmung10 Eine Reihe von Arbeiten in den letzten Jahren haben sich mit dem physikalischen Abbau von O gonukleotiden beschäftigt, wie z B Tandem Massenspektrometrie mit Elektrospray Ionisierung, ESI (CID, collision induced dissociation)1 1 1 3, enzymati- sche Reaktionen unter Verwendung von Exonukleasen10 14 oder Oligonukleotid- Aufbau mit Polymerasen15 16 und physikalische spontane Fragmentierung wie "nozzle skimmer dissociation" (NS) von ESI generierten Nukleinsaure Ionen1 7 18 oder spontane Dissoziation der Nukleinsäuren nach Infrarot-Laser Beschüß einer in einer Matrix kristallisierten Probe18 24 Diese Methoden versagen aber bei derThe increasing activities in the research of nucleic acids, especially of ribonucleic acids as well as of peptides and O gosacchaπden in recent years require a fast standard sequencing method that can also detect modifications. Previous methods for the sequencing of RNA are based on two-dimensional chromatographic methods 1 3 , Maxam-Gilbert sequencing 4 7 or reverse transceptase singer sequencing 8 9 Recent developments use mass spectrometry for determining the structural structure 10 A number of works in recent years have dealt with the physical Degradation of oonucleotides, such as tandem mass spectrometry with electrospray ionization, ESI (CID, collision induced dissociation) 1 1 1 3 , enzymatic reactions using exonucleases 10 14 or oligonucleotide construction with polymerases 15 16 and physical spontaneous fragmentation such as "nozzle skimmer dissociation" (NS) nucleic acid ions 1 7 18 generated by ESI or spontaneous dissociation of the nucleic acids after infrared laser bombardment of a sample crystallized in a matrix 18 24 However, these methods fail with the
Sequenzierung von RNA, da die Nukleotide Uπdin (U , 306, 1 7) und Cytidin (C, 305, 1 8) fast gleiche Massen besitzen. Deshalb wurde argumentiert, daß die Sequenzierung von RNA durch Exonuklease Abbau (Verdau) und Detektion der erhaltenen Fragmente mit Massenspektrometrie nicht möglich sei25 Sequencing of RNA, since the nucleotides Uπdin (U, 306, 1 7) and cytidine (C, 305, 1 8) have almost the same mass. It was therefore argued that the sequencing of RNA by exonuclease degradation (digestion) and detection of the fragments obtained was not possible using mass spectrometry 25
Wir beschreiben hier eine Methode, mit der man RNA durch Exonukleaseverdau, Trennung und Detektion der erzeugten Fragmente mit Massenspektrometrie, sequenzieren kann Die Methode kann besonders nützlich sein für die Pπmar- strukturbestimmug von RNA (oder auch DNA) , die langer als 20 Basen ist oder modifizierten Nukleinsäuren, da die Auflosung von Massenspektrometern ein limitierender Faktor für die Sequenzanalyse längerer Nukleinsaurefragmente darstellt. Sie kann auch wertvoll bei der Bestimmung der Sekundarstruktur von Nukleinsäuren sein. Die Nukleinsaurefragmente werden durch unterschiedlicheWe describe here a method by which RNA can be sequenced by exonuclease digestion, separation and detection of the fragments generated using mass spectrometry. The method can be particularly useful for determining the structural structure of RNA (or also DNA) which is longer than 20 bases or modified nucleic acids, since the resolution of mass spectrometers is a limiting factor for the sequence analysis of longer nucleic acid fragments. It can also be valuable in determining the secondary structure of nucleic acids. The nucleic acid fragments are different
ERSATZBLAπ (REGEL 26) Massen, der beim Verdau von Exonukleasen (vornehmlich 5'→3'-Exonuklease aus Kalbsmilz und 3'→5'-Exonuklease aus Schlangengift von crotalus duπssus) austretenden Nukleotide, massenspektrometrisch bestimmt U und C werden aber durch unterschiedliche Peakintensitäten im Massenspektrum erkannt Die Unterschiede in den Peakintensitäten werden durch unterschiedliche Geschwindigkeiten bei der Hydrolyse der Phosphodieseterbindungen durch das Enzym hervorgerufen. Dadurch werden Fragmente, die am δ'-Ende ein C enthalten durch 5'→3'-Phosphodιesterase weniger schnell abgebaut und liegen deshalb in viel größeren Konzentrationen vor als z B 5'-U enthaltende Fragmente Die gleiche Beobachtung wird auch für 5'-A-Fragmente gemacht Adenosm ist jedoch auch durch seine Masse von den anderen Nukleotiden leicht zu unterscheiden Es ist möglich mehrere Nukleinsäuren gleichzeitig dieser Enzymkinetik zu unterwerfen, um die Sequenziergeschwindigkeit zu erhohen Auch der Einsatz von basen- spezifischen Exo-/Endonukleasen kann zur Sequenzanalyse und zur schnellen Erkennung und Bestimmung von Organismen, z B Viren herangezogen werden, deren RNA oder DNA einem "Fingerpπnting" (Verdau von RNA oder DNA mit Exo- /Endonukleasen, die nicht an jedem Nukleotid basenspezifisch schneiden) oder "Footpπnting " (Verdau von RNA oder DNA, die mit Nukleinsaure fremden Molekülen wechselwirken und der Hydrolyse mit Exo-/Endonukleasen ausgesetzt werden) unterworfen wird Die Trennung und Detektion der erzeugten Fragmente durch Massenspektrometrie kann so z B zur Prävention von Seuchen oder zur Bestimmung von Organismen und biologischen Waffen eingesetzt werden Finger und Footprint sind genauer als bisherige Methoden Es werden nach der Hydrolyse einer Phosphodiesterbindung beide Spaltfragmente detektiert, wahrend herkomm- liehe Verfahren nur das markierte Fragment detektieren können Eine Sekundar- strukturvorhersage von Nukleinsäuren ist schließlich dadurch möglich, daß die Enzyme oft bevorzugt an einzelstrangigen, linearen Bereichen schneiden Somit können Domänen, Sekundär- und Tertiarstrukturen wie z B 'Hairpins' oder 'internal loops' an ihren doppelstrangigen Bereichen erkannt werdenREPLACEMENT BLAπ (RULE 26) Masses of nucleotides emerging during the digestion of exonucleases (primarily 5 '→ 3' exonuclease from calf spleen and 3 '→ 5' exonuclease from snake venom from crotalus duπssus), determined by mass spectrometry, but U and C are recognized by different peak intensities in the mass spectrum. The differences the peak intensities are caused by different rates in the hydrolysis of the phosphodiester bonds by the enzyme. As a result, fragments which contain a C at the δ'-end are degraded less rapidly by 5 '→ 3'-phosphodιesterase and are therefore present in much greater concentrations than fragments containing 5'-U, for example. The same observation is also made for 5'- A fragments made Adenosm is also easily distinguishable from the other nucleotides by its mass. It is possible to subject several nucleic acids simultaneously to this enzyme kinetics in order to increase the sequencing speed. The use of base-specific exo- / endonucleases can also be used for sequence analysis and rapid detection and determination of organisms, eg viruses, whose RNA or DNA is finger fingered (digestion of RNA or DNA with exo- / endonucleases that do not cut base-specifically on each nucleotide) or foot panning (digestion of RNA or DNA that interacts with foreign nucleic acid molecules and is subjected to hydrolysis with exo / endonucleases) Separation and detection of the fragments generated by mass spectrometry can be used, for example, to prevent epidemics or to determine organisms and biological weapons. Finger and footprint are more precise than previous methods. After hydrolysis of a phosphodiester bond, both cleavage fragments are detected using conventional methods only the labeled fragment can be detected Finally, a secondary structure prediction of nucleic acids is possible by the fact that the enzymes often prefer to cut on single-stranded, linear regions. Thus, domains, secondary and tertiary structures such as 'hairpins' or 'internal loops' on their double-stranded ones Areas are recognized
Diese Prinzipien lassen sich auch auf die Sequenzierung bzw Sekundarstrukturbe- stimmung anderer Biopolymere anwenden, wie z B Peptide und Oligosacchaπde Die Methode ist sowohl mit MALDI als auch mit DE-MALDI26 27 durchfuhrbarThese principles can also be applied to the sequencing or secondary structure determination of other biopolymers, such as peptides and oligosaccharides. The method can be carried out with both MALDI and DE-MALDI 26 27
ERSATZBLÄ1T (REGEL 26) BeispieleERSATZBLÄ1T (RULE 26) Examples
Beispiel 1 : Sequenzierung eines 8mers mit 5'→3' Phosphodiesterase und (siehe Fig. 1 )Example 1: Sequencing an 8mer with 5 '→ 3' phosphodiesterase and (see Fig. 1)
Sequenzierung eines 8mers mit mit RNase CL3 (siehe Fig. 2)Sequencing an 8-mer with RNase CL3 (see FIG. 2)
Tabelle 1 : Massen und Sequenzen (5'→3'Rιchtung) der Sequenzierfragmente eines δmers, die durch enzymatischen Verdau erzeugt wurdenTable 1: Masses and sequences (5 '→ 3' direction) of the sequencing fragments of a δmer, which were generated by enzymatic digestion
a) mit 5'→3' Phosphodiesterase (aus Kalbsmilz)a) with 5 '→ 3' phosphodiesterase (from calf spleen)
Figure imgf000005_0001
Figure imgf000005_0001
b) mit RNase CL3 (aus Hühnerleber)b) with RNase CL3 (from chicken liver)
Figure imgf000005_0002
Experimentelles:
Figure imgf000005_0002
Experimental:
Für alle Beispiele der massenspektrometπschen RNA Sequenzierungen gilt, falls nicht anders angegeben. Linear kontinuierliche MALDI-TOF Massenspektrometrie wurde mit einem Fisons VG TOF spec Massenspektrometer (8mer, 9mere RNA und DNA, 1 6mer, 22mer, 1 20mer) und DE-MALDI-TOF Messungen mit einem PerSeptive Biosystems Voyager Massenspektrometer ( 1 6mer) durchgeführt, die einen UV Stickstofflaser mit einer Emissionsfrequenz von 337nm enthalten Die Laser Pulsbreite ist 4 ns. Die Spektren wurden im negativ Modus aufgenommen mit Ausnahme des 1 δmers und des 32 mers Diese wurden im positiv Modus gemessen 2,4,6-Tπhydroxyacetophenon/Ammonιumcιtrat wurde in allen Sequenzierexperimenten als Matrix verwendet Herstellung der Matrix Losung 1 (2,4,6-Tπhydroxyacetophenon gesattigt in Ethanol Wasser, 1 1 ) und Losung 2 (0, 1 M Ammoniumcitratin Wasser ~ pH 5,5) werden im Verhältnis 2 1 gemischt Enzyme wurden von Boehπnger Mannheim bezogen 5'→3' Phosphodiesterase ausUnless otherwise stated, the following applies to all examples of mass spectrometric RNA sequencing. Linear continuous MALDI-TOF mass spectrometry was carried out with a Fisons VG TOF spec mass spectrometer (8mer, 9mere RNA and DNA, 1 6mer, 22mer, 1 20mer) and DE-MALDI-TOF measurements with a PerSeptive Biosystems Voyager mass spectrometer (1 6mer) contain a UV nitrogen laser with an emission frequency of 337nm The laser pulse width is 4 ns. The spectra were recorded in the negative mode with the exception of the 1 δmers and the 32 mers. These were measured in the positive mode. 2,4,6-Tπhydroxyacetophenon / Ammonιumcιtrat was used as a matrix in all sequencing experiments Preparation of the matrix solution 1 (2,4,6- Tπhydroxyacetophenone saturated in ethanol water, 1 1) and solution 2 (0, 1 M ammonium citrate in water ~ pH 5.5) are mixed in a ratio of 2 1. Enzymes were obtained from Boehπnger Mannheim 5 '→ 3' phosphodiesterase
Kalbsmilz Das Enzym greift das Oligonukleotid am 5'-Ende an und hinterlasst 3' Nukleotide. 3'→5' Phosphodiesterase aus crotalus duπssus Das Enzym greift das Oligonukleotid am 3'-Ende an und hinterlasst 5' Nukleotide RNase CL3 aus Huhnerleber Das Enzym spaltet RNA bevorzugt an Cp/N-Bindungen und produziert Fragmente mit 3' endstandigem Cytidinphosphat Ap/N- und Gp/N-Bmdungen werden viel langsamer hydrolysiert, Up/N-Bindungen sehr selten RNase CL3/Pufferlosung (denaturierend) 2 μ\ RNase CL3 (0,2U/μl) + 6 μ\ 8 M Harnstoff in Wasser resultieren in 8 μ\ 50 mU/μl Enzymlosung 3'→5'-Phosphodιester- ase/Pufferlosung 2 μ\ (4 mU/μl) 3'→5'-Phosphodιesterase + 1 8 μ\ 0 1 M Ammoniumcitrat, pH 5,5 resultieren in 20 μl 0 2 mU/μl Enzymlosung Sequenzen der untersuchten RNA-Stucke waren wie folgt 8mer: 5'-HO-CAUGUGAC-OH-3', 9mer (RNA). 5'-HO-GCAUGUGAC-OH-3', 9mer (DNA) 5'-HO-GTCACATGC-OH-3', 1 6mer. 5'-HO-GCGUACAUCUUCCCCU-OH-3',Calf spleen The enzyme attacks the oligonucleotide at the 5 'end and leaves 3' nucleotides. 3 '→ 5' phosphodiesterase from crotalus duπssus The enzyme attacks the oligonucleotide at the 3 'end and leaves 5' nucleotides RNase CL3 from chicken liver. The enzyme cleaves RNA preferentially at Cp / N bonds and produces fragments with 3 'terminal cytidine phosphate Ap / N and Gp / N bonds are hydrolyzed much more slowly, Up / N bonds very rarely RNase CL3 / buffer solution (denaturing) 2 μ \ RNase CL3 (0.2U / μl) + 6 μ \ 8 M urea in water result in 8 μ \ 50 mU / μl enzyme solution 3 '→ 5'-phosphodιesterase / buffer solution 2 μ \ (4 mU / μl) 3' → 5'-phosphodιesterase + 1 8 μ \ 0 1 M ammonium citrate, pH 5.5 result in 20 ul 0 2 mU / ul enzyme solution sequences of the RNA pieces examined were 8mer as follows: 5'-HO-CAUGUGAC-OH-3 ', 9mer (RNA). 5'-HO-GCAUGUGAC-OH-3 ', 9mer (DNA) 5'-HO-GTCACATGC-OH-3', 16mer. 5'-HO-GCGUACAUCUUCCCCU-OH-3 ',
22mer. 5'-HO-GCUCUUUUCU * UUUUUCUUUUCC-OH-3\ (U * = , 3C markiertes22mer. 5'-HO-GCUCUUUUCU * UUUUUCUUUUCC-OH-3 \ (U * = , 3 C labeled
Uπdin an allen fünf Kohlenstoffatomen des Zuckerbausteins),Uπdin on all five carbon atoms of the sugar building block),
1 20mer (5s-rιbosomale RNA) 5'-pUGCCUGGCGGCCGUAGCGCGGUGGUCCCAC1 20mer (5s-rιbosomal RNA) 5'-pUGCCUGGCGGCCGUAGCGCGGUGGUCCCAC
CUGACCCCAUGCCGAACUCAGAAGUGAAACGCCGUAGCGCCGAUGGUAGUG UGGGGUCUCCCCAUGCGAGAGUAGGGAACUGCCAGGCAU-OH-3'CUGACCCCAUGCCGAACUCAGAAGUGAAACGCCGUAGCGCCGAUGGUAGUG UGGGGUCUCCCCAUGCGAGAGUAGGGAACUGCCAGGCAU-OH-3 '
In allen Experimenten wurden Proben von je 1 μl nach einer Inkubationszeit von 1 , 3, 6, 1 0, 20 und 60 Minuten genommen (es sind meist nicht alle SpektrenIn all experiments, samples of 1 μl were taken after an incubation time of 1, 3, 6, 10, 20 and 60 minutes (mostly not all spectra are
ERSATZB π REGEL 26 gezeigt). Die Proben wurden mit der Matrix im Verhältnis 1 : 1 gemischt, auf die Probenplatte des Spektrometers pipettiert und ca. 20 Minuten an der Luft getrocknet. Die Trocknungs- und Kristallisationszeit kann durch vorsichtiges Anfönen verkürzt werden. Der enzymatische Verdau stoppt, wenn die Proben mit der Matrix vermischt werden.REPLACEMENT B π RULE 26 shown). The samples were mixed with the matrix in a ratio of 1: 1, pipetted onto the sample plate of the spectrometer and air-dried for about 20 minutes. The drying and crystallization time can be shortened by careful drying. The enzymatic digestion stops when the samples are mixed with the matrix.
RNA 8mer (0, 1 OD) 9,0 μlRNA 8mer (0.1 OD) 9.0 ul
5'→3'-Phosphodiesterase (24mU) 6,0 μl5 '→ 3'-phosphodiesterase (24mU) 6.0 ul
Σ 1 5,0 μlΣ 1 5.0 μl
Inkubationstemperatur: 22 ° CIncubation temperature: 22 ° C
Inkubationszeit: 6 MinutenIncubation time: 6 minutes
RNA 8mer (0, 1 OD) 9,0 μl RNase CL3/Pufferlösung (400 mU; denaturierend) 8,0 μlRNA 8mer (0.1 OD) 9.0 μl RNase CL3 / buffer solution (400 mU; denaturing) 8.0 μl
1 7 ,0 μl1 7.0 ul
Inkubationstemperatur: 50 °C Inkubationszeit: 1 0 MinutenIncubation temperature: 50 ° C Incubation time: 10 minutes
Beispiel 2: Sequenzierung eines 1 6mers mit 5'-»3' Phosphodiesterase (siehe Fig. 3)Example 2: Sequencing of a 1 6mer with 5 '- »3' phosphodiesterase (see Fig. 3)
Sequenzierung eines 1 δmers mit 3'→5' Phosphodiesterase (siehe Fig. 4)Sequencing a 1 δmer with 3 '→ 5' phosphodiesterase (see Fig. 4)
Tabelle 2: Massen und Sequenzen (5'→3'Richtung) der Sequenzierfragmente eines 1 6 mers (aufgenommen mit DE-MALDI) Table 2: Masses and sequences (5 '→ 3' direction) of the sequencing fragments of a 1 6 mer (recorded with DE-MALDI)
mit 5'-*3' Phosphodiesterase (aus Kalbsmilz)with 5 '- * 3' phosphodiesterase (from calf spleen)
Figure imgf000008_0001
Figure imgf000008_0001
b) mit 3'→5' Phosphodiesterase (aus crotalus durissus)b) with 3 '→ 5' phosphodiesterase (from crotalus durissus)
Figure imgf000009_0001
Figure imgf000009_0001
Experimentelles:Experimental:
RNA 16mer (0,1 OD) 15,2μl 5'→3'-Phosphodiesterase (24mU) 6,0 μlRNA 16mer (0.1 OD) 15.2 ul 5 '→ 3'-phosphodiesterase (24mU) 6.0 ul
21,2 l21.2 l
Inkubationstemperatur: 22°CIncubation temperature: 22 ° C
RNA 16mer (0,1 OD) 15,2μlRNA 16mer (0.1 OD) 15.2 µl
3'→5'-Phosphodiesterase/Pufferlösung (0,6 mU) 3,0 μl3 '→ 5'-phosphodiesterase / buffer solution (0.6 mU) 3.0 ul
18,2μl18.2μl
Inkubationstemperatur: 40°CIncubation temperature: 40 ° C
ERSATZBUOT (REGEL 26) Beispiel 3: Sequenzierung eines 22mers mit 5'→3' Phosphodiesterase (siehe Fig. 5)REPLACEMENT BOOT (RULE 26) Example 3: Sequencing a 22mer with 5 '→ 3' phosphodiesterase (see Fig. 5)
Sequenzierung eines 22mers mit 3'→5' Phosphodiesterase (siehe Fig. 6)Sequencing a 22mer with 3 '→ 5' phosphodiesterase (see Fig. 6)
Tabelle 3: Massen und Sequenzen (5'→3' Richtung) der Sequenzierfragmente eines 22mersTable 3: Masses and sequences (5 '→ 3' direction) of the sequencing fragments of a 22mer
ERSATZBUTT (REGEL 26) a) mit 5'→3'Phosphodiesterase (aus Kalbsmilz)REPLACEMENT BUTT (RULE 26) a) with 5 '→ 3'phosphodiesterase (from calf spleen)
Figure imgf000011_0002
Figure imgf000011_0001
Figure imgf000011_0002
Figure imgf000011_0001
b) mit 3'→5' Phosphodiesterase (aus crotalus durissus)b) with 3 '→ 5' phosphodiesterase (from crotalus durissus)
10 rt u10 rt u
1515
a n r> na n r> n
2020th
25
Figure imgf000012_0001
25th
Figure imgf000012_0001
Experimentelles:Experimental:
RNA 22mer (0,1 OD) 9,0 μlRNA 22mer (0.1 OD) 9.0 ul
5'-*3'-Phosphodiesterase (24mU) 6,0 μl5 '- * 3'-phosphodiesterase (24mU) 6.0 ul
15,0μl15.0μl
Inkubationstemperatur: 22°CIncubation temperature: 22 ° C
RNA 22mer (0,1 OD) 9,0 μlRNA 22mer (0.1 OD) 9.0 ul
3'→5'-Phosphodiesterase/Pufferlösung (0,6 mU) 3,0 μl3 '→ 5'-phosphodiesterase / buffer solution (0.6 mU) 3.0 ul
Σ 12,0μlΣ 12.0μl
Inkubationstemperatur: 40°CIncubation temperature: 40 ° C
Beispiel 4: Simultane Sequenzierung von zwei Oligoribonukleotiden (8mer undExample 4: Simultaneous sequencing of two oligoribonucleotides (8mer and
22mer; siehe Fig.7)22mer; see Fig. 7)
Bezüglich Massen und Sequenzen der Sequenzierfragmente siehe Tabellen 1a und 3a.For the masses and sequences of the sequencing fragments, see Tables 1a and 3a.
Experimentelles:Experimental:
RNA 22mer (0,1 OD) 9,0 μl RNA 8mer(0,1 OD) 9,0 μlRNA 22mer (0.1 OD) 9.0 ul RNA 8mer (0.1 OD) 9.0 ul
5'-→3'-Phosphodiesterase (20mU) 5,0 μl5'- → 3'-phosphodiesterase (20mU) 5.0 ul
23,0 μl23.0 ul
Inkubationstemperatur: 22°CIncubation temperature: 22 ° C
Inkubationszeit: 20 Minuten Beispiel 5: Fingerprint einer 5s-ribosomalen RNA (120mer) mit RNase CL3 (siehe Fig.8)Incubation time: 20 minutes Example 5: Fingerprint of a 5s-ribosomal RNA (120mer) with RNase CL3 (see Fig. 8)
Experimentelles:Experimental:
RNA 120mer (1 OD) 10,0 μlRNA 120mer (1 OD) 10.0 ul
RNase CL3/Pufferlösung (400 mU; denaturierend) 8,0 μlRNase CL3 / buffer solution (400 mU; denaturing) 8.0 μl
Figure imgf000014_0001
Figure imgf000014_0001
Inkubationstemperatur: 50°CIncubation temperature: 50 ° C
Inkubationszeit: 15 MinutenIncubation time: 15 minutes
Beispiel 6: Sequenzierung eines 16mer Oligoribonukleotids (Fingerprint) mit RNase CL3 (Sequenz aus Tabelle 2; siehe Fig.9)Example 6: Sequencing of a 16mer oligoribonucleotide (fingerprint) with RNase CL3 (sequence from Table 2; see FIG. 9)
Experimentelles:Experimental:
RNA 16mer (0,1 OD) 9,0 μl RNase CL3/Pufferlösung (100 mU; denaturierend) 15,2μlRNA 16mer (0.1 OD) 9.0 μl RNase CL3 / buffer solution (100 mU; denaturing) 15.2 μl
24,2 μl24.2 ul
Inkubationstemperatur: 50°C Inkubationszeit: 1 Minute Incubation temperature: 50 ° C Incubation time: 1 minute
Beispiel 7 : Simultansequenzierung zweier 9mere (DNA und RNA; siehe Fig. 10)Example 7: Simultaneous sequencing of two 9mers (DNA and RNA; see Fig. 10)
Tabelle 4: Fragmente und Massen des Verdaus eines RNA 9mers (5'-H0- GCAUGUGAC-OH-3') mit 3'→5'-Phosphodiesterase (aus crotalus durissus)Table 4: Fragments and masses of the digestion of an RNA 9mer (5'-H0-GCAUGUGAC-OH-3 ') with 3' → 5'-phosphodiesterase (from crotalus durissus)
Figure imgf000015_0001
Figure imgf000015_0001
Tabelle 5: Fragmente und Massen des Verdaus eines DNA 9mers (5'-HO- d(GTCACATGC)-OH-3') mit 3'→5'-Phosphodiesterase (aus crotalus durissus)Table 5: Fragments and masses of the digestion of a DNA 9mer (5'-HO- (GTCACATGC) -OH-3 ') with 3' → 5'-phosphodiesterase (from crotalus durissus)
Figure imgf000015_0002
Tabelle 6: DNA-Abgangsgruppen:
Figure imgf000015_0002
Table 6: DNA leaving groups:
Figure imgf000016_0001
Figure imgf000016_0001
Tabelle 7 : RNA-Abgangsgruppen.Table 7: RNA leaving groups.
Figure imgf000016_0002
Figure imgf000016_0002
Experimentelles:Experimental:
RNA (0, 1 OD) 1 2,5 μlRNA (0.1 OD) 1 2.5 ul
DNA (0, 1 OD) 2,3 μlDNA (0.1 OD) 2.3 ul
3'→5'-Phosphodιesterase/Pufferlosung (2mU) 1 0,0 μl3 '→ 5'-phosphodιesterase / buffer solution (2mU) 1 0.0 μl
24,8 μl24.8 ul
Inkubationstemperatur: 40 ° CIncubation temperature: 40 ° C
Im folgenden werden die Vorteile der Methode nochmals aufgeführtThe advantages of the method are listed again below
- Das Verfahren arbeitet elektrophoresefrei und ist damit sehr schnell- The process works without electrophoresis and is therefore very fast
- Es wird kein zusätzlicher Marker zur Detektion benotigt auch keine Radioaktivität. Damit entfallen alle Markierungsschritte- No additional marker is required for detection and no radioactivity. This eliminates all marking steps
Das Verfahren nutzt nicht nur die Bestimmung der Masse zur Dateninterpreta- tion, sondern auch die Peakintensitäten. Damit sind auch Spektrometer mit geringer Auflosung, die auch leicht bedienbar sind, einsetzbar und das Verfahren wird kostengünstig - Es ist keine genaue Massenbestimmung nötig. Deshalb können auch sehr lange Polymere sequenziert werden.The method uses not only the determination of the mass for data interpretation, but also the peak intensities. This means that spectrometers with a low resolution, which are also easy to use, can be used and the method is inexpensive - No exact mass determination is necessary. Therefore very long polymers can be sequenced.
- Die Methode kann zur Sekundärstrukturvorhersage von Biopolymeren heran- gezogen werden.- The method can be used to predict the secondary structure of biopolymers.
- Das Verfahren kann zur Sequenzierung von modifizierten Bioploymeren dienen.- The method can be used for the sequencing of modified biopolymers.
- Das Verfahren kann zur Bestimmung von Organismen dienen (Finger- print/Footprint).- The method can be used to determine organisms (fingerprint / footprint).
- Die Methode ist automatisierbar und parallelisierbar.- The method can be automated and parallelized.
ERSATZBUTT (REGEL 26) LiteraturREPLACEMENT BUTT (RULE 26) literature
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(3) Silberklang, M., Gillum, A. M. and RajBhandary, U. L., in Methods in Enzymology. Wu, R. and Grossmann, L. (eds), Academic Press Inc., London and New York, (1979) 59, 58(3) Silberklang, M., Gillum, A.M. and RajBhandary, U.L., in Methods in Enzymology. Wu, R. and Grossmann, L. (eds), Academic Press Inc., London and New York, (1979) 59, 58
(4) Maxam, A. M. and Gilbert, W., Proc. Natl. Acad. Sei. USA, (1977) 74, 560-564(4) Maxam, A.M. and Gilbert, W., Proc. Natl. Acad. Be. USA, (1977) 74, 560-564
(5) Stahl, D. A., Krupp, G. and Stackebrandt, E., in Nucleic Acids Sequencing, a practical aproach, Howe, C. J. and Ward, E. S. (eds), IRL Press at Oxford University Press, Oxford, New York, Tokyo, (1989) 137(5) Stahl, DA, Krupp, G. and Stackebrandt, E., in Nucleic Acids Sequencing, a practical aproach, Howe, CJ and Ward, ES (eds), IRL Press at Oxford University Press, Oxford, New York, Tokyo , (1989) 137
(6) Waldmann, R., Gross, H. J. and Krupp, G., Nucleic Acids Res., (1937) 15, 7209.(6) Waldmann, R., Gross, H.J. and Krupp, G., Nucleic Acids Res., (1937) 15, 7209.
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180, 121 (10) Pieles, U., Zürcher, W., Schär, M., Moser, H. E., Nucleic Acids Res., (1993) 21, 3191- 3196180, 121 (10) Pieles, U., Zürcher, W., Schär, M., Moser, H.E., Nucleic Acids Res., (1993) 21, 3191-3196
(II) McLuckey, S. A., Habibi-Goudarzi, S., J. Am. Chem. Soc, (1993) 115, 12085-12095(II) McLuckey, S.A., Habibi-Goudarzi, S., J. Am. Chem. Soc, (1993) 115, 12085-12095
(12) Wolter, M. A., Engels, J. W., Eur. Mass Spectrom., (1995) 1, 583-590,(12) Wolter, M.A., Engels, J.W., Eur. Mass Spectrom., (1995) 1, 583-590,
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(14) Limbach, P. A., McCIoskey, J. A., Crain, P. F., Nucleic Acids Res. Symp. Ser., (1994) 31, 127-128(14) Limbach, P.A., McCIoskey, J.A., Crain, P.F., Nucleic Acids Res. Symp. Ser., (1994) 31, 127-128
(15) Roskey, M. T., Juhasz, P., Smirnov, I. P., Takach, E. J., Martin, S. A. and Haff, L. A., Proc. Natl. Acad. Sei. USA, (1996) 93, 4724-4729(15) Roskey, M.T., Juhasz, P., Smirnov, I.P., Takach, E.J., Martin, S.A. and Haff, L.A., Proc. Natl. Acad. Be. USA, (1996) 93, 4724-4729
(16) Köster, H., Tang, K., Fu, D.-J., Braun, A., van den Boom, D., Smith, C. L., Cotter, R. J. and Cantor, C.R., Nature Biotechnology, (1996) 14, 1123- 1128(16) Köster, H., Tang, K., Fu, D.-J., Braun, A., van den Boom, D., Smith, CL, Cotter, RJ and Cantor, CR, Nature Biotechnology, (1996 ) 14, 1123-1128
(17) Loo, J. A., Udseth, H., R., Smith, R., D., Rapid Commun. Mass Spectrom. (1988) 2, 207-210(17) Loo, J.A., Udseth, H., R., Smith, R., D., Rapid Commun. Mass Spectrom. (1988) 2, 207-210
(18) Little, D. P., Chorush, R. A., Speir, J. P., Senko, M. W., Kelleher, N. L.,(18) Little, D.P., Chorush, R.A., Speir, J.P., Senko, M.W., Kelleher, N.L.,
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(20) Little, D. P., Speir, J. P. , Senko, M. W., O'Connor, P. B., McLafferty, F. W., Anal. Chem., (1994) 66, 2809-2815 (21 ) Nordhoff, E., Karas, M.,Cramer, R., Hahner, S., Hillenkamp, F.,Kirpekar, F.,(20) Little, D.P., Speir, J.P., Senko, M.W., O'Connor, P.B., McLafferty, F.W., Anal. Chem., (1994) 66, 2809-2815 (21) Nordhoff, E., Karas, M., Cramer, R., Hahner, S., Hillenkamp, F., Kirpekar, F.,
Lezius, A., Muth, J., Meier, C, Engels, J. W., J. Mass Spectrom., (1995) 30, 99-112 (22) Little, D. P., McLafferty, F. W., J. Am. Chem. Soc, (1995) 117, 6783- 6784 (23) Wu, K. J., Shaler, T. A. and Becker, H., Anal. Chem., (1994) 66, 1637-Lezius, A., Muth, J., Meier, C, Engels, J.W., J. Mass Spectrom., (1995) 30, 99-112 (22) Little, D.P., McLafferty, F.W., J. Am. Chem. Soc, (1995) 117, 6783-6784 (23) Wu, K.J., Shaler, T.A. and Becker, H., Anal. Chem., (1994) 66, 1637-
1645 (24) Nordhoff, E., Cramer, R., Karas, M., Hillenkamp, F., Kirpekar, F., Kristiansen, K. and Roepstorff, P., Nucleic Acids Res., (1993) 21, 3347- 3357 (25) Kirpekar, F., Nordhoff, E., Kristiansen, K., Roepstorff, ?., Lezius, A.,1645 (24) Nordhoff, E., Cramer, R., Karas, M., Hillenkamp, F., Kirpekar, F., Kristiansen, K. and Roepstorff, P., Nucleic Acids Res., (1993) 21, 3347 - 3357 (25) Kirpekar, F., Nordhoff, E., Kristiansen, K., Roepstorff,?., Lezius, A.,
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(27) Juhasz, P., Roskey, M. T., Smirnov, I. P., Haff, L. A., Vestal, M. L. and Martin, S. A., Anal. Chem., (1996) 68, 941-946(27) Juhasz, P., Roskey, M.T., Smirnov, I.P., Haff, L.A., Vestal, M.L. and Martin, S.A., Anal. Chem., (1996) 68, 941-946
ERSATZBUTT (REGEL 26) REPLACEMENT BUTT (RULE 26)

Claims

Patentansprüche claims
ir beanspruchen-I claim
Ein Verfahren zur Sequenzierung von Biopolymeren mit Massenspektrometrie, vornehmlich Ribonukleinsäuren (RNA) oder Nukleinsäuren (DNA) oder Peptiden oder O gosacchaπden durch Verdau des zu untersuchenden RNA- oder DNA- oder Peptid- oder Oligosacchaπd- Stranges mit spezifischen Exo- /Endonukleasen, -peptidasen, -carboxyesterasen, -amidasen oder -glycosidasen oder anderen sequenz- oder basenspezifisch spaltendenA method for the sequencing of biopolymers with mass spectrometry, primarily ribonucleic acids (RNA) or nucleic acids (DNA) or peptides or O gosacchaπden by digesting the RNA or DNA or peptide or oligosaccharid strand to be examined with specific exo- / endonucleases, peptidases , carboxyesterases, amidases or glycosidases or other sequence- or base-specific cleaving
Verbindungen, wobei die Trennung und Detektion der erzeugten Fragmente einer Kinetik folgend durch Massenspektrometrie, vornehmlich MALDI , erfolgt und unterschiedliche Peakintensitäten, hervorgerufen durch enzymatische oder chemische Hydrolyse der entsprechenden einzelnen Bindungen, in den Massenspektren zur Interpretation der Sequenzdaten herangezogen werdenCompounds, the separation and detection of the generated fragments following a kinetic by mass spectrometry, primarily MALDI, and different peak intensities, caused by enzymatic or chemical hydrolysis of the corresponding individual bonds, are used in the mass spectra to interpret the sequence data
Die Anwendung des Verfahrens nach Anspruch 1 zur Sequenzierung von modifizierter und unmodifizierter DNA, RNA, Peptide und Oligosacchaπde Modifikationen können bei Nukleosiden und Nukleotiden -verglichen mit Adenosm, Guanosin, Uπdin, Cytidin und Thymidin bzw deren Phosphate undThe use of the method according to claim 1 for the sequencing of modified and unmodified DNA, RNA, peptides and oligosaccharide modifications can be carried out on nucleosides and nucleotides - compared with adenosm, guanosine, urinin, cytidine and thymidine or their phosphates and
Oligomere- Veränderungen an der Base, am Zucker oder an der Phosphatgruppe bzw. des Phosphatruckgrates betreffen, bei Peptiden Abweichungen der als '20 natürliche Aminosäuren' bekannten Monomere von Ppetiden und bei Oligosacchaπden den bekannten 'naturlichen' SacchaπdenOligomeric changes to the base, to the sugar or to the phosphate group or the phosphate backbone concern, in the case of peptides, deviations from the monomers of ppetids known as '20 natural amino acids' and in the case of oligosaccharides the known 'natural' saccharides
Die Verwendung von Enzymen oder anderen sequenz- oder basenspezifisch spaltenden Verbindungen, die bei der Hydrolyse von Bindungen unterschiedliche Geschwindigkiten aufweisen und zur Sequenzierung von Nukleinsäuren (RNA/DNA), Peptiden oder O gosacchaπden in einem Verfahren nach Anspruch 1 und 2 eingesetzt werdenThe use of enzymes or other sequence- or base-specific cleaving compounds which have different speeds in the hydrolysis of bonds and are used for the sequencing of nucleic acids (RNA / DNA), peptides or O gosacchaπden in a method according to claim 1 and 2
Ein Verfahren zur simultanen Sequenzierung von mehreren Ribonukleinsäuren (RNA), Nukleinsäuren (DNA) , Peptiden und O gosacchaπden nach Anspruch 1 , d a d u r c h g e k e n n z e i c h n e t, daß der Verdau des zu untersuchenden RNA-, DNA-, Peptid- oder Oligosacchaπd-A method for the simultaneous sequencing of several ribonucleic acids (RNA), nucleic acids (DNA), peptides and Ogosacchaπden according to claim 1, d a d u r c h g e k e n n z e i c h n e t that the digestion of the RNA, DNA, peptide or oligosaccharide to be examined
Stranges mit spezifischen Exo-/Endonukleasen, -peptidasen, -amidasen, - carboxyesterasen oder Exo- und Endoglycosidasen oder anderen sequenz- oder basenspezifisch spaltenden Verbindungen gleichzeitig erfolgt, wobei dieStrands with specific exo / endonucleases, peptidases, amidases, carboxyesterases or exo- and endoglycosidases or other sequence- or base-specific cleaving compounds takes place simultaneously, the
ERSATZBUTT (REGEL 26) Trennung und Detektion der erzeugten Fragmente einer Kinetik folgend durch Massenspektrometrie, vornehmlich MALDI, erfolgt und unterschiedliche Peakintensitäten, hervorgerufen durch enzymatische oder chemische Hydrolyse der entsprechenden einzelnen Bindungen, in den Massenspektren zur Interpretation der Sequenzdaten herangezogen werdenREPLACEMENT BUTT (RULE 26) Separation and detection of the generated fragments following kinetics by mass spectrometry, primarily MALDI, and different peak intensities, caused by enzymatic or chemical hydrolysis of the corresponding individual bonds, are used in the mass spectra to interpret the sequence data
Ein Verfahren zur Sequenzierung von RNA, DNA, Peptiden und Oligosacchaπden durch Verdau des zu untersuchenden Stranges mit spezifischen Endonukleasen, -peptidasen, -carboxyesterasen, -amidasen, Endoglycosidasen und anderen sequenz- oder basenspezifisch Endo spaltenden Verbindungen, wobei die Trennung und Detektion der erzeugten Fragmente durch Massenspektrometrie, vornehmlich MALDI, erfolgtA method for the sequencing of RNA, DNA, peptides and oligosaccharides by digestion of the strand to be examined with specific endonucleases, peptidases, carboxyesterases, amidases, endoglycosidases and other sequence- or base-specific endo-cleaving compounds, the separation and detection of the fragments generated by mass spectrometry, primarily MALDI
Ein Verfahren zur Analyse und Bestimmung von Organismen nach Anspruch 1 d a d u rc h g e ke n n z e i c h n e t, daß deren RNA oder DNA einem "Fingerpπnting" oder "Footpπnting" unterworfen werden und die Trennung und Detektion der erzeugten Fragmente durch Massenspektrometrie, vornehmlich MALDI, erfolgtA method for the analysis and determination of organisms according to claim 1, that the RNA or DNA are subjected to "finger panning" or "foot panning" and that the fragments generated are separated and detected by mass spectrometry, primarily MALDI
Ein Verfahren zur Sekundarstrukturbestimmung von NukleinsäurenA method for the secondary structure determination of nucleic acids
(DNA/RNA), Peptiden und Oligosacchaπden durch Verdau der Biopolymeren mit Exo-/Endonukleasen, -peptidasen, -carboxyesterasen, -amidasen und -glycosidasen oder anderen sequenz- oder basenspezifisch spaltenden Verbindungen und Trennung und Detektion der erzeugten Fragmente mit Massenspektrometrie, vornehmlich MALDI, nach Anspruch 1 d a d u r c h g e k e n n z e i c h n e t, daß(DNA / RNA), peptides and oligosacchaπden by digestion of the biopolymers with exo- / endonucleases, -peptidases, -carboxyesterases, -amidases and -glycosidases or other sequence- or base-specific cleaving compounds and separation and detection of the generated fragments with mass spectrometry, mainly MALDI , according to claim 1, characterized in that
Sekundarstrukturbereiche (z B loops, doppelstrangige Bereiche) nur langsam oder gar nicht bzw sehr viel schneller von diesen Enzymen angegriffen werdenSecondary structure areas (eg loops, double-stranded areas) are attacked only slowly or not at all or much faster by these enzymes
Kombination der Verfahren nach Anspruch 1-7 zur Analyse von (Ribo-) Nukleinsäuren, Peptiden und Oligosacchaπden Combination of the method according to claims 1-7 for the analysis of (ribo) nucleic acids, peptides and oligosacchases
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