DE19714558A1 - A new method for sequencing biopolymers using mass spectrometry - Google Patents

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. A further advantage is that unmarked nucleic acids can be used and no radioactivity is need. The method can be conducted in parallel with a simultaneous sequence analysis of various nucleic acids, which increases sequencing speed. The method can also be used for detecting and determining organisms (fingerprint, footprint), whereby the finger and footprints are more precise than in previous methods: both cleavage fragments are detected after hydrolysis of a phophodiester bond, whereas hitherto known methods could detect only a marked fragment. Finally, the method can contribute to elucidation of the secondary structure of nucleic acids by mass spectrometry. These principles can also be applied to sequencing or secondary structure determination of other biopolymers, like, for example, peptides and oligosaccharides.

Description

The increasing activities in the research of nucleic acids, especially ribonucleic acids as well as peptides and oligosaccharides in recent years, require a fast standard sequencing method that can also detect modifications. Previous methods for sequencing RNA are based on two-dimensional chromatographic methods ^1-3 , Maxam-Gilbert sequencing ^4-7 or reverse transcriptase Sanger sequencing ^8.9 . More recent developments use mass spectrometry to determine the primary structure ¹⁰ . A number of works in recent years have dealt with the physical degradation of oligonucleotides, such as. B. Tandem mass spectrometry with electrospray ionization, ESI (CID, collision induced dissociation) ^11-13 , 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 generated by ESI ^17,18 or spontaneous dissociation of the nucleic acids after infrared laser bombardment of a sample ^{18-24 crystallized in a matrix} Cytidine (C, 305,18) have almost the same masses. It has therefore been argued that the sequencing of RNA by exonuclease degradation (digestion) and detection of the fragments obtained with mass spectrometry is not possible ²⁵ .

We describe here a method by which RNA can be digested by exonuclease, separation and detecting the generated fragments with mass spectrometry. the Method can be particularly useful for determining the primary structure of RNA (or also DNA) that is longer than 20 bases or modified nucleic acids, as the dissolution of Mass spectrometers are a limiting factor for longer sequence analysis Represents nucleic acid fragments. They can also be valuable in determining who Be secondary structure of nucleic acids. The nucleic acid fragments are through different masses that occur during the digestion of exonucleases (mainly 5 '→ 3'- Exonuclease from calf's spleen and 3 '→ 5' exonuclease from snake venom from crotalus durissus) emerging nucleotides, determined by mass spectrometry. U and C are however through different peak intensities recognized in the mass spectrum. The differences in the Peak intensities are due to different rates in the hydrolysis of the Phosphodieseter bonds caused by the enzyme. This will remove fragments that contain a C at the 5 'end and are less rapidly degraded by 5' → 3'-phosphodiesterase are therefore in much larger concentrations than z. B. 5'-U containing fragments. the the same observation is made for 5 'A fragments. However, adenosine is also through its mass can be easily distinguished from the other nucleotides. It is possible several To subject nucleic acids to these enzyme kinetics at the same time in order to achieve the Increase sequencing speed. Also the use of base-specific exo / endo Nucleases can be used for sequence analysis and for the rapid recognition and determination of Organisms, e.g. B. Viruses are used whose RNA or DNA a "fingerprinting" (Digestion of RNA or DNA with exo / endonucleases that do not attach to any nucleotide base-specific cut) or "footprinting" (digestion of RNA or DNA, which with Foreign nucleic acid molecules interact and hydrolyze with exo- / endonucleases exposed). The separation and detection of the fragments generated by mass spectrometry, for. B. for the prevention of epidemics or for determination used by organisms and biological weapons. Fingerprint and footprint are more accurate than previous methods: after hydrolysis, a phosphodiester bond is formed both cleavage fragments are detected, while conventional methods only detect the labeled fragment can detect. A secondary structure prediction of nucleic acids is finally possible because the enzymes often preferentially on single-stranded, linear regions cut. Thus, domains, secondary and tertiary structures such as e.g. B. "Hairpins" or "intrnal loops" can be recognized by their double-stranded areas.

These principles can also be applied to sequencing or secondary structure determination apply other biopolymers, such as B. peptides and oligosaccharides.

The method can be carried out with both MALDI and DE-MALDI ^26,27 .

Examples example 1 Sequencing an 8mer with 5 '→ 3' phosphodiesterase (see Fig. 1) Sequencing of an 8mer with with RNase CL3 (see Fig. 2)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

b) with RNase CL3 (from chicken liver)

Experimental

Unless 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 (8-mer, 9-mer RNA and DNA, 16-mer, 22-mer, 120-mer) and DE-MALDI-TOF measurements a PerSeptive Biosysteins Voyager mass spectrometer (16mer) containing a UV nitrogen laser with an emission frequency of 337 nm. The laser pulse width is 4 ns. The spectra were recorded in negative mode with the exception of the 16mers and 32mers. These were measured in positive mode. 2,4,6-trihydroxyacetophenone / ammonium citrate was used as a matrix in all sequencing experiments. Preparation of the matrix: Solution 1 (2,4,6-trihydroxyacetophenone 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 Boehringer Mannheim: 5 '→ 3' phosphodiesterase from calf's spleen: the enzyme attacks the oligonucleotide at the 5 'end and leaves 3' nucleotides behind. 3 '→ 5' phosphodiesterase from crotalus durissus: The enzyme attacks the oligonucleotide at the 3 'end and leaves 5' nucleotides behind. 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 are very rare. RNase CL3 / buffer solution (denaturing): 2 µl RNase CL3 (0.2 U / µl) + 6 µl 8 M urea in water result in 8 µl 50 mU / µl enzyme solution. 3 '→ 5' phosphodiesterase / buffer solution: 2 µl (4 mU / µl) 3 '→ 5'-phosphodiesterase + 18 µl 0.1 M ammonium citrate, pH 5.5 result in 20 µl 0.2 mU / µl enzyme solution. Sequences of the investigated RNA pieces were 8-mer as follows: 5'-HO-CAUGUGAC-OH-3 '; 9mer (RNA): 5'-HO-GCAUGUGAC-OH-3 '; 9mer (DNA): 5'-HO-GTCACATGC-OH-3 '; 16-mer: 5'-HO-GCGUACAUCUUCCCCU-OH-3 '; 22mer: 5'-HO-GCUCUUUUCU * UUUUUCUUUUCC-OH-3 '; (U * = ¹³ C labeled uridine on all five carbon atoms of the sugar building block); 120mer (5s ribosomal RNA): 5'- pUGCCUGGCGGCCGUAGCGCGGUGGUCCCACCUGACCCCAUGCCGAACUCAGAAGUGAAACGCCG UAGCGCCGAUGGUAGUGUGGGGUCUCCCCAUGCGAGAGUAGGGAACUGCC-3AGGCAU-OH '.

In all experiments, samples of 1 µl each were taken after an incubation time of 1, 3, 6, 10, 20 and 60 minutes (mostly not all spectra are 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 tinting. The enzymatic digestion stops when the samples are mixed with the matrix.

RNA 8mer (0.1 OD) 9.0 µl 5 '→ 3'-phosphodiesterase (24 mU) 6.0 µl Σ 15.0 µl

Incubation temperature: 22 ° C
Incubation time: 6 minutes

RNA 8mer (0.1 OD) 9.0 µl RNase CL3 / buffer solution (400 mU; denaturing) 8.0 µl Σ 17.0 µl

Incubation temperature: 50 ° C
Incubation time: 10 minutes

Example 2 Sequencing a 16mer with 5 '→ 3' phosphodiesterase (see Fig. 3) Sequencing a 16mer with 3 '→ 5' phosphodiesterase (see Fig. 4)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

b) with 3 '→ 5' phosphodiesterase (from crotalus durissus)

Experimental

RNA 16mer (0.1 OD) 15.2 µl 5 '→ 3'-phosphodiesterase (24 mU) 6.0 µl Σ 21.2 µl

Incubation temperature: 22 ° C

RNA 16mer (0.1 OD) 15.2 µl 3 '→ 5'-phosphodiesterase / buffer solution (0.6 mU) 3.0 µl Σ 18.2 µl

Incubation temperature: 40 ° C

Example 3 Sequencing a 22mer with 5 '→ 3' phosphodiesterase (see Fig. 5) Sequencing a 22mer with 3 '→ 5' phosphodiesterase (see Fig. 6)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

a) with 5 '→ 3' phosphodiesterase (from calf's spleen)

b) with 3 '→ 5' phosphodiesterase (from crotalus durissus)

Experimental

RNA 22mer (0.1 OD) 9.0 µl 5 '→ 3'-phosphodiesterase (24 mU) 6.0 µl Σ 15.0 µl

Incubation temperature: 22 ° C

RNA 22mer (0.1 OD) 9.0 µl 3 '→ 5'-phosphodiesterase / buffer solution (0.6 mU) 3.0 µl Σ 12.0 µl

Incubation temperature: 40 ° C

Example 4 Simultaneous sequencing of two oligoribonucleotides (8-mer and 22-mer; see Fig. 7) For the masses and sequences of the sequencing fragments, see Tables 1a and 3a Experimental

RNA 22mer (0.1 OD) 9.0 µl RNA 8mer (0.1 OD) 9.0 µl 5 '→ 3'-phosphodiesterase (20 mU) 5.0 µl Σ 23.0 µl

Incubation temperature: 22 ° C
Incubation time: 20 minutes

Example 5 Fingerprint of a 5s ribosomal RNA (120mer) with RNase CL3 (see Fig. 8) Experimental

RNA 120mer (1 OD) 10.0 µl RNase CL3 / buffer solution (400 mU; denaturing) 8.0 µl Σ 18.0 µl

Incubation temperature: 50 ° C
Incubation time: 15 minutes

Example 6 Sequencing of a 16mer oligoribonucleotide (fingerprint) with RNase CL3 (Sequence from Table 2; see Fig. 9) Experimental

RNA 16mer (0.1 OD) 9.0 µl RNase CL3 / buffer solution (100 mU; denaturing) 15.2 µl Σ 24.2 µl

Incubation temperature: 50 ° C
Incubation time: 1 minute

Example 7 Simultaneous sequencing of two 9mers (DNA and RNA; see Fig. 10)

Fragments and masses of the digestion of an RNA 9mer (5'-HO-GCAUGUGAC-OH- 3 ') with 3' → 5'-phosphodiesterase (from crotalus durissus)

Fragments and masses of the digestion of an RNA 9mer (5'-HO-GCAUGUGAC-OH- 3 ') with 3' → 5'-phosphodiesterase (from crotalus durissus)

Fragments and masses of the digestion of a DNA 9mer (5'-HO-d (GTCACATGC) - OH-3 ') with 3' → 5'-phosphodiester star (from crotalus durissus)

Fragments and masses of the digestion of a DNA 9mer (5'-HO-d (GTCACATGC) - OH-3 ') with 3' → 5'-phosphodiester star (from crotalus durissus)

DNA leaving groups

DNA leaving groups

RNA leaving groups

RNA leaving groups

Experimental

RNA (0.1 OD) 12.5 µl DNA (0.1 OD) 2.3 µl 3 '→ 5'-phosphodiesterase / buffer solution (2 mU) 10.0 µl Σ 24.8 µl

Incubation temperature: 40 ° C

The advantages of the method are listed again below:

• - The process works electrophoresis-free and is therefore very fast.
• - No additional marker is required for detection and no radioactivity. In order to all marking steps are omitted.
• - The method not only uses the determination of the mass for data interpretation, but also the peak intensities. This also includes spectrometers with low resolution that too are easy to operate, deployable and the process becomes inexpensive.
• - It is not necessary to determine the exact mass. Therefore, very long polymers can also be used sequenced.
• - The method can be used to predict the secondary structure of biopolymers will.
• - The method can be used for sequencing modified biopolymers.
• - The method can be used to determine organisms (fingerprint / footprint).
• - The method can be automated and parallelized.

summary

A new method for sequencing biopolymers using mass spectrometry is being used described. The sequencing of biopolymers is either tedious or requires one accurate mass determination of fragments. This is particularly difficult for long polymers. The rate of hydrolysis of phosphodiester, peptide or glycoside bonds with Exo- / endonucleases, -peptidases, -glycosidases or other hydrolytically acting Substances is used in our procedure for sequence analysis of nucleic acids or other Biopolymers used. Separation and detection of the generated fragments is carried out with Mass spectrometry by determining the mass and different peak intensities. Of the The primary advantage of the method is that it is no longer necessary to determine the exact mass and also low resolution mass spectrometers can be used for analysis and separation of the fragments are extremely fast since they are electrophoresis-free and the Sequence of modified nucleic acids can be determined. Other advantages are that unlabeled nucleic acids can be used and no radioactivity is required. The method can be parallelized by simultaneous sequence analysis of several nucleic acids which increases the sequencing speed. The method can still go to Recognition and identification of organisms are used (fingerprint, footprint), whereby the fingerprint and footprint are more precise than with previous methods: According to the Hydrolysis of a phosphodiester bond detected both cleavage fragments while conventional methods can only detect a labeled fragment. After all, the Method for elucidating the secondary structure of nucleic acids using mass spectrometry contribute. These principles can also be applied to sequencing or Apply secondary structure determination of other biopolymers, such as B. peptides and Oligosaccharides.

literature

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Claims (8)

1. A method for sequencing biopolymers using mass spectrometry, mainly ribonucleic acids (RNA) or nucleic acids (DNA) or peptides or Oligosaccharides by digesting the RNA or DNA or peptide or Oligosaccharide strand with specific exo- / endonucleases, peptidases, -carboxyesterases, -amidases or -glycosidases or other sequence or base-specific cleavage compounds, with the separation and detection of the generated fragments following kinetics by mass spectrometry, mainly MALDI, occurs and different peak intensities, caused by enzymatic or chemical hydrolysis of the corresponding individual bonds in the Mass spectra can be used to interpret the sequence data. 2. The application of the method according to claim 1 for the sequencing of modified and unmodified DNA, RNA, peptides and oligosaccharides. Modifications can be made at Nucleosides and nucleotides - compared to adenosine, guanosine, uridine, cytidine and Thymidine or its phosphates and oligomeric changes to the base, to the sugar or on the phosphate group or the phosphate backbone, in the case of peptides Variations in monomers known as "20 natural amino acids" from peptides and in the case of oligosaccharides, the well-known "natural" saccharides. 3. The use of enzymes or other sequence- or base-specific cleavage Compounds that hydrolyze bonds at different rates have and for sequencing nucleic acids (RNA / DNA), peptides or Oligosaccharides are used in a method according to claims 1 and 2. 4. A method for the simultaneous sequencing of several ribonucleic acids (RNA), Nucleic acids (DNA), peptides and oligosaccharides according to Claim 1, characterized characterized in that the digestion of the RNA, DNA, peptide or to be examined Oligosaccharide strand with specific exo- / endonucleases, -peptidases, -amidases, -carboxyesterases or exo- and endoglycosidases or other sequence or base-specific cleaving compounds takes place simultaneously, with the separation and Detection of the generated fragments following kinetics by mass spectrometry, mainly MALDI, and different peak intensities, caused by enzymatic or chemical hydrolysis of the corresponding individual bonds in the Mass spectra can be used to interpret the sequence data. 5. A method for sequencing RNA, DNA, peptides and oligosaccharides Digestion of the strand to be examined with specific endonucleases, endonucleases, -carboxyesterases, -amidases, endoglycosidases and others sequence- or base-specific Endo-cleavage compounds, with the separation and detection of the generated Fragments by mass spectrometry, principally MALDI, is done. 6. A method for the analysis and determination of organisms according to claim 1, characterized in characterized in that their RNA or DNA is subject to "fingerprinting" or "footprinting" are subjected and the separation and detection of the fragments generated by Mass spectrometry, primarily MALDI, takes place. 7. A method for determining the secondary structure of nucleic acids (DNA / RNA), peptides and oligosaccharides by digesting the biopolymers with exo- / endonucleases, peptidases, carboxyesterases, amidases and glycosidases or other sequence or base-specific cleavage compounds and separation and detection of the generated Fragments with mass spectrometry, primarily MALDI, according to claim 1, characterized characterized in that secondary structure areas (e.g. loops, double-stranded areas) only be attacked slowly or not at all or much faster by these enzymes. 8. Combination of the methods according to claims 1-7 for the analysis of (ribo-) nucleic acids, Peptides and oligosaccharides.