Metal ion-binding mass markers for nucleic acids

Details Metal ion-binding mass markers for nucleic acids Metal ion-binding mass markers for nucleic acids

2001-03-16

2003-06-24

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S094000, C436S173000

Reexamination Certificate

active

06582916

ABSTRACT:

This invention concerns compounds which comprise mass markers for detection by mass spectrometry. The invention relates to methods for characterising analytes, such as nucleic acids and other molecules, using markers that are cleavably detachable from their associated analyte and that are detectable by mass spectrometry. Specifically this invention relates to chemical entities that improve the sensitivity of detection by mass spectrometry of detachable mass labels.
PCT/GB98100 127 describes arrays of cleavable labels that are detectable by mass spectrometry which identify the sequence of a covalently linked nucleic acid probe. These mass labels have a number of advantages over other methods of analysing nucleic acids. At present commercially favoured systems are based on fluorescent labelling of DNA. Fluorescent labelling schemes permit the labelling of a relatively small number of molecules simultaneously, typically four 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. An advantage of using mass labels is the possibility of generating large numbers of labels which 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 labelling techniques disclosed in PCT/GB99/00127 is the design of mass markers. A number of features are required of a molecule that is to be a good mass marker. A marker 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, such that it can be clearly determined that a mass peak is from a mass label.
Be compatible with conventional automated oligonucleotide synthesisers.
Be compatible with existing mass spectrometry instrumentation without requiring physical modification.
A feature up to present of the analysis of nucleic acids by mass spectrometry is the need to condition the nucleic acid prior to analysis. This involves removing all metal ions, particularly magnesium and sodium as these readily form adducts with nucleic acids. These sample conditioning steps require additional preparation steps and instrumentation to allow automation of analysis. Indirect analysis of markers by mass spectrometry avoids many of the problems of direct analysis of, for example, DNA, whilst retaining the benefits of the mass spectrometer, such as high throughput, automation and high sensitivity.
However even with indirect labelling there is a need to ensure that there is little background contamination or that the labels are easily detectable over any background signals. It is thus desirable to provide mass markers that are detectable with high sensitivity over a background of contaminating material to reduce the requirements for sample conditioning and to simplify the use of mass labelling techniques. It is also desirable that mass markers are detected preferentially over background material.
A feature of the analysis of complex mixtures of analytes is competition during ionisation leading to suppression of certain ion peaks. In the use of multiple mass markers it is desirable to reduce these sorts of effects by providing an array of mass labels in which there is no competition during ionisation so that a high proportion of labels are ionised during mass spectrometry improving sensitivity and the signal to noise ratio.
It is an object of this invention to solve the above problems and to provide improved mass markers which are compatible with existing mass spectrometers particularly electrospray ionisation and tandem mass spectrometry, that have the desired features disclosed above.
It is also an object of this invention to provide compounds which have desirable features as mass labels which further simplify the manipulations of the molecules to which the are linked prior to being able to perform mass spectrometry.
Accordingly, the present invention provides a compound having the following formula:
N—L—M
wherein N comprises a nucleic acid, L is either a direct bond between N and M or L comprises a cleavable linker, and M comprises a mass marker having a metal ion-binding moiety.
The invention further provides a method for characterising an analyte, which method comprises:
(a) providing a compound in which the analyte is attached by a cleavable linker to a mass marker relatable to the analyte,
(b) cleaving the mass marker from the analyte; and
(c) identifying the mass marker, thereby characterising the analyte.
wherein the mass marker comprises a metal ion-binding moiety.
The invention also provides use of a mass marker identifiable by mass spectrometry for the characterisation of an analyte, wherein the mass marker comprises a metal ion-binding moiety.
The mass labelled molecules used in the present invention have the following properties:
(1) The ability to bind to a metal ion to generate a charged species.
(2) Stability to permit participation in combinatorial synthesis of multiple distinct mass markers and stability in a conventional automated nucleic acid synthesiser.
(3) Fragmentation resistance under conditions within a mass spectrometer.
(4) Improved detection by mass spectrometry.
Thus the compounds and methods of this invention achieve preferential ionisation over background material through the.binding of a metal ion, effectively pre-ionising the label prior to mass spectrometry.
This ion-binding feature also ensures that there is no competition for Ionisation between labels as it is relatively trivial to ensure that there are sufficient metal ions in the buffers that are used in the analytical protocols preceding mass spectrometry.
By pre-ionising the mass labels used in this invention very gentle ionisation conditions can be used in the mass spectrometer reducing the ionisation of contaminating material. In this way the signal to noise ratio of mass spectrometry analysis steps is greatly improved.
A further feature of the metal ion binding mass markers is high sensitivity of detection. It is known that in typical ionisation procedures such as Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI) only about one in a thousand molecules ionise although accurate data for precise quantities are not available. MALDI is known to be worse in this respect than ESI. Despite this mass spectrometers achieve extraordinary levels of sensitivity. This is because the detection apparatus used is sensitive to the arrival of a single ion. The labels of this invention will be pre-ionised, potentially achieving much higher ionisation, of the order of 1 in 10 molecules. This further enhances the signal to noise ratio in the mass spectrometry detection and may also increase the sensitivity of detection by up to two orders of magnitude.
The various aspects of the invention will now be described in more detail.


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