WO1998045700A2 - Method for the mass spectrometric sequencing of biopolymers - 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. These principles can also be applied to sequencing or secondary structure determination of other biopolymers, like, for example, peptides and oligosaccharides.

Description

 Process for the mass spectrometric sequencing of biopolymers

description

The 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

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

We 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

REPLACEMENT 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

These 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 (RULE 26) Examples

Example 1: Sequencing an 8mer with 5 '→ 3' phosphodiesterase and (see Fig. 1)

Sequencing an 8-mer with RNase CL3 (see FIG. 2)

Table 1: Masses and sequences (5 '→ 3' direction) of the sequencing fragments of a δmer, which were generated by enzymatic digestion

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

b) with RNase CL3 (from chicken liver)

Experimentelles:

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 (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

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 ^* = ^{, 3} C labeled

Uπdin on all five carbon atoms of the sugar building block),

1 20mer (5s-rιbosomal RNA) 5'-pUGCCUGGCGGCCGUAGCGCGGUGGUCCCAC

CUGACCCCAUGCCGAACUCAGAAGUGAAACGCCGUAGCGCCGAUGGUAGUG UGGGGUCUCCCCAUGCGAGAGUAGGGAACUGCCAGGCAU-OH-3 '

In 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

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 ul

5 '→ 3'-phosphodiesterase (24mU) 6.0 ul

Σ 1 5.0 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

1 7.0 ul

Incubation temperature: 50 ° C Incubation time: 10 minutes

Example 2: Sequencing of a 1 6mer with 5 '- »3' phosphodiesterase (see Fig. 3)

Sequencing a 1 δmer with 3 '→ 5' phosphodiesterase (see Fig. 4)

Table 2: Masses and sequences (5 '→ 3' direction) of the sequencing fragments of a 1 6 mer (recorded with DE-MALDI)

with 5 '- * 3' phosphodiesterase (from calf spleen)

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

Experimental:

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

21.2 l

Incubation temperature: 22 ° C

RNA 16mer (0.1 OD) 15.2 µl

3 '→ 5'-phosphodiesterase / buffer solution (0.6 mU) 3.0 ul

18.2μl

Incubation temperature: 40 ° C

REPLACEMENT BOOT (RULE 26) Example 3: Sequencing a 22mer with 5 '→ 3' phosphodiesterase (see Fig. 5)

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

Table 3: Masses and sequences (5 '→ 3' direction) of the sequencing fragments of a 22mer

REPLACEMENT BUTT (RULE 26) a) with 5 '→ 3'phosphodiesterase (from calf spleen)

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

10 rt u

15

a n r> n

20th

25th

Experimental:

RNA 22mer (0.1 OD) 9.0 ul

5 '- * 3'-phosphodiesterase (24mU) 6.0 ul

15.0μl

Incubation temperature: 22 ° C

RNA 22mer (0.1 OD) 9.0 ul

3 '→ 5'-phosphodiesterase / buffer solution (0.6 mU) 3.0 ul

Σ 12.0μl

Incubation temperature: 40 ° C

Example 4: Simultaneous sequencing of two oligoribonucleotides (8mer and

22mer; 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 ul RNA 8mer (0.1 OD) 9.0 ul

5'- → 3'-phosphodiesterase (20mU) 5.0 ul

23.0 ul

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 ul

RNase CL3 / buffer solution (400 mU; denaturing) 8.0 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

24.2 ul

Incubation temperature: 50 ° C Incubation time: 1 minute

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

Table 4: Fragments and masses of the digestion of an RNA 9mer (5'-H0-GCAUGUGAC-OH-3 ') with 3' → 5'-phosphodiesterase (from 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)

Tabelle 6: DNA-Abgangsgruppen:

Table 6: DNA leaving groups:

Table 7: RNA leaving groups.

Experimental:

RNA (0.1 OD) 1 2.5 ul

DNA (0.1 OD) 2.3 ul

3 '→ 5'-phosphodιesterase / buffer solution (2mU) 1 0.0 μl

24.8 ul

Incubation temperature: 40 ° C

The advantages of the method are listed again below

- The process works without electrophoresis and is therefore very fast

- No additional marker is required for detection and no radioactivity. This eliminates all marking steps

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.

REPLACEMENT BUTT (RULE 26) literature

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(4) Maxam, A.M. and Gilbert, W., Proc. Natl. Acad. Be. USA, (1977) 74, 560-564

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(7) Zhang, Y., Liu, W. Feng, Y. and Wang, T. P., Anal. Biochem., (19δ7) 163, 513

(8) Sanger, F. Nicklen, S., Coulson, A.R., Proc. Natl. Acad. Be. USA, (1977) 74, 5463-5467 (9) Hahn, C, S., Strauss, E., G. and Strauss, J., H. in Methods of Enzymology,

180, 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

(12) Wolter, M.A., Engels, J.W., Eur. Mass Spectrom., (1995) 1, 583-590,

(13) Ni, J., Pomerantz, S.C., Rozenski, J., Zhang, Y. and McCIoskey, J.A., Anal. Chem., (1996) 68, 1989-1999

(14) Limbach, P.A., McCIoskey, J.A., Crain, P.F., Nucleic Acids Res. Symp. Ser., (1994) 31, 127-128

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(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

(18) Little, D.P., Chorush, R.A., Speir, J.P., Senko, M.W., Kelleher, N.L.,

REPLACEMENT BUTT RULE 26) McLafferty, FW, J. Am. Chem. Soc. (1994) 116, 4893-4897

(19) M. Karas, F. Hillenkamp, Anal. Chem., (1983) 60, 2299

(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-

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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REPLACEMENT BUTT (RULE 26)

Claims

 claims I claim A 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 Compounds, 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 The 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 Oligomeric 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 The 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 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 Strands with specific exo / endonucleases, peptidases, amidases, carboxyesterases or exo- and endoglycosidases or other sequence- or base-specific cleaving compounds takes place simultaneously, the REPLACEMENT 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 A 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 A 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 A method for the secondary structure determination of nucleic acids (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 Secondary structure areas (eg loops, double-stranded areas) are attacked only slowly or not at all or much faster by these enzymes Combination of the method according to claims 1-7 for the analysis of (ribo) nucleic acids, peptides and oligosacchases