Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-11
2001-09-11
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S023100, C536S024300, C536S024330, C536S025300, C536S025320
Reexamination Certificate
active
06287780
ABSTRACT:
This invention concerns mass markers for labelling nucleic acids and other molecules. In particular the invention concerns compounds comprising specific monomeric, oligomeric and polymeric mass markers. Using this invention, mixtures of analytes can be labelled with mass markers for simultaneous detection by mass spectrometry.
At present commercially favoured detection systems for analysing nucleic acids are based on fluorescent labelling of DNA. Fluorescent labeling schemes permit the labelling of a relatively small number of molecules simultaneously, typically 4 labels can be used simultaneously and possibly up to eight. However, the costs of the detection apparatus and the difficulties of analysing the resultant signals limit the number of labels that can be used simultaneously in a fluorescence detection scheme. PCT/GB98/00127 describes arrays of nucleic acid probes covalently attached to cleavable labels that are detectable by mass spectrometry which identify the sequence of the covalently linked nucleic acid probe. The labelled probes of this application have the structure Nu-L-M where Nu is a nucleic acid covalently linked to L, a cleavable linker, covalently linked to M, a mass label. The detachable mass labels of PCT/GB98/00127, comprising the L-M components of the nucleic acid probes have a number of advantages over other methods of analysing nucleic acids. An advantage of using mass labels is the possibility of generating large numbers of labels that have discrete peaks in a mass spectrum, allowing similar numbers of distinct molecular species to be labelled simultaneously. Fluorescent dyes are expensive to synthesise whereas mass labels can comprise relatively simple polymers permitting combinatorial synthesis of large numbers of labels at low cost.
A critical feature of the mass labelled nucleic acid probes disclosed in PCT/GB98/00127 is the design of the cleavable mass labels (L-M). A number of features are required of a molecule that is to be a good mass label. A label should:
Be easily detachable from DNA.
Be fragmentation resistant in mass spectrometer.
Form a single ion peak in the mass spectrum.
Permit very sensitive detection.
Be easily distinguishable from background contamination, such as DNA. It should be clear that a mass peak is from a mass label.
Be compatible with conventional automated oligonucleotide synthesisers.
Be easy to synthesise in a combinatorial manner to minimise number of chemical steps and the number of reagents necessary to generate large number of labels.
Be compatible with existing mass spectrometry instrumentation without requiring physical modification.
Linkers to allow a mass label to be easily cleaved from its associated nucleic acid are disclosed in PCT/GB98/00127 and in GB patent application numbers 9815163.2 and 9815164.0. Compounds which improve the sensitivity of detection of a mass label by mass spectrometry are disclosed in PCT/GB98/00127 and in GB patent application number 9815166.5.
Other uses of mass modified nucleic acids are known in the art using probes with mass modifications that are not removable for direct analysis of nucleic acids by mass spectrometry. The application of mass modified peptide nucleic acids for multiplexed detection of single nucleotide polymorphisms by mass spectrometry is discussed in the following references: Ross, P. L. et al. Anal. Chem. 69:4197-4202, 1997 and Griffin, T. J. et al. Nature Biotechnology 15:1368-1372, 1997. PCT/US94/00193 discloses the use of mass modified nucleic acids in a method of DNA sequencing by mass spectrometry to permit multiplexed detection of a number of different templates.
It is thus an object of this invention to overcome problems associated with the prior art and to provide mass labelled nucleic acids that can be analysed with existing mass spectrometers. In particular, an object of this invention is to enable analysis by electrospray ionisation, thermospray ionisation, Matrix Assisted Laser Desorption Ionisation (MALDI) and tandem mass spectrometry, whether through direct analysis of mass modified nucleic acids or through analysis of labels that are cleavably detachable from their corresponding nucleic acids.
It is also an object of this invention to provide mass label entities with appropriate functionalities for attachment to nucleic acids or other analyte molecules.
It is a further object of this invention that the provided mass labels be compatible with conventional automated oligonucleotide synthesis chemistries. It is another object of this invention to provide mass labels that are compatible with the cleavable linkers disclosed in PCT/GB98/00127 and in the GB patent application numbers 9815163.2 and 9815164.0 and also mass labels that are compatible with the sensitising groups disclosed in PCT/GB98/00127 and in GB patent application number 9815166.5.
Accordingly, the present invention provides a compound having the following formula:
N-L-M
wherein N comprises one or more nucleic acid bases, L is either a direct bond between N and M or L comprises a linker moiety, and M comprises a mass marker comprising an aryl ether.
The invention further provides an array of mass markers, each mass marker in the array being as defined above, each set of mass markers comprising a plurality of different mass markers, each mass marker in any one set differing in mass from all other mass markers in that set by a mass of at least substantially 4 Daltons and each mass marker in any one set having the same number of aryl ether units as each of the other mass markers in that set and a different number of aryl ether units from the each of the markers in any other set.
The invention further provides a method for characterising a nucleic acid or other molecule, which method comprises identifying a mass marker by mass spectrometry, which mass marker is relatable to a specific nucleic acid base or base sequence, or a specific atom or group in a molecule, to identify the mass marker and thereby identify the base or base sequence, or the specific atom or group wherein the mass marker is as defined above.
The invention also provides use of a mass marker identifiable by mass spectrometry for the characterisation of a nucleic acid or other molecule, which mass marker is as defined above.
In certain aspects of this invention, the labels are cleavably detachable from their associated nucleic acid and are detachable by mass spectrometry. In other aspects of this invention, the labels are not cleavably detachable from their associated nucleic acid and are used to modify the mass of the analyte to which they are attached. Specifically this invention relates to compounds that can be used as mass modifying groups to generate arrays of labels or arrays of mass modified nucleic acids with distinct masses. In addition these compounds have favourable properties for use as mass labels including fragmentation resistance, thermal stability, chemical inertness and ease of ionisation.
REFERENCES:
patent: 0 611 075 A (1994-08-01), None
patent: 2 041 919 A (1980-09-01), None
patent: WO 97/27327 (1997-07-01), None
patent: WO 98/31830 (1998-07-01), None
Baker et al, “Irreversible Enzyme Inhibitors. 195: Inhibitors of Thymidine Kinase from Walker 256 Carcinoma Derived from Thymidine 5′-Acetate”,Journal of Medicinal Chemistry, vol. 15, No. 9, 1972, pp. 940-944.
Nestler et al, “A General Method for Molecular Tagging of Encoded Combinatorial Chemistry Libraries”,Journal of Organic Chemistry, vol. 59, No. 17, 1994, pp. 4723-4724.
Ohlmeyer et al, “Complex synthetic chemical libraries indexed with molecular tags”Proceedings of the National Academy of Sciences, USA, vol. 90, Dec. 1993, pp. 10922-10926.
Geysen et al, “Isotope or mass encoding of combinatorial libraries”,Chemistry and Biology, vol. 3, No. 8, Aug. 1996, pp. 679-688.
Search Report, PCT/GB98/03842, Apr. 4, 1999.
Johnstone Robert Alexander Walker
Schmidt Gunter
Thompson Andrew Hugin
Brax Group Limited
Burns Doane Swecker & Mathis L.L.P.
Riley Jezia
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