Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1997-12-10
2003-10-21
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S006120, C536S022100, C536S023100, C536S024300, C536S024310, C536S024330, C536S025300, C536S025320, C436S172000
Reexamination Certificate
active
06635452
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of chemical analysis. More particularly, it concerns a new class of nonvolatile, releasable tag reagents for use in the detection and analysis of target molecules i.e., by mass spectrometry.
2. Description of Related Art
Chemical labels, otherwise known as tags or signal groups, are widely used in chemical analysis. Among the types of molecules used are radioactive atoms, fluorescent reagents, luminescent reagents, metal-containing compounds, electron-absorbing substances and light absorbing compounds. Chemical signal groups can be combined with reactivity groups so that they might be covalently attached to the target, the substance being detected. In many cases, however, chemical moieties present on the target may interfere with the detection of the signal group or not allow for measurement of the signal group in an optimal detection environment.
Indirect detection of the target is oftentimes, therefore, preferred. For example, the signal group may be the product of the degradation of the target or a derivative of the target (Bueht et. al., 1974; Senft, 1985; U.S. Pat. Nos. 4,650,750; 4,709,016; 4,629,689). Volatile releasable tag compounds that can be analyzed using various forms of electron-attachment mass spectrometry, often with gas chromatography-mass spectrometry (GC-MS), have been described (Wang et al., 1996; U.S. Pat. Nos. 5,360,819; 5,516,931). Despite the broad range of volatile mass labels reported, a transition from liquid to gas phase is required for analysis which places significant synthetic and size parameters on the label. Isotopic mass labels have also been described, such as using tin or sulfur isotopes, with various mass spectrometric sampling approaches (Arlinghaus et al. 1997; U.S. Pat. No. 5,174,962). The isotopic labeling often limits the extent of multiplexing and provides a more complex analysis requirement.
Mass spectral analysis of signal groups involves none of the concerns related to radioactive signal groups, such as their short half-lives and their safety and disposal issues. Another key advantage to detection of signal groups via mass spectrometry is that it allows a great ability to multiplex, to detect for more than one signal group in a complex mixture, and therefore more than one target at a time. Brummel et al. (1994; 1996) have demonstrated the use of mass spectrometry in the direct analysis of combinatorial libraries of small peptides. However, use of this technology is limited to analysis of the entire reacting compound by mass spectrometry.
Detection of multiple fluorescent labels has been used to analyze nucleic acid sequences. Nucleic acid hybridization probes are modified to contain fluorescent chromophores that when excited by light emit a unique color spectrum signature. In fluorescence based sequencing systems, four different chromophores can be multiplexed within a sample and individually detected with the aid of software deconvolution. The practical upper limit for fluorescence multiplexing is likely to be around 10 different labels due to the broad overlapping spectrum produced by existing fluorescent chromophores. Clearly the development of nonvolatile releasable mass labels, detectable over the usable range of a mass spectrometer, would represent a significant advantage by permitting the multiplexing of tens, hundreds and perhaps even thousands of different mass labels that can be used to uniquely identify each desired target.
At present, while tools are available through which target molecules may be detected, there remains a need for further development of these systems in order to analyze a large number of targets simultaneously. This will allow for the systematic analysis of target molecules with predetermined properties and functions.
SUMMARY OF THE INVENTION
It is, therefore, a goal of the present invention to provide compositions and methods relating to the use of release tag compounds for detection and analysis of target molecules.
The present invention relates to the use of nonvolatile, releasable tag compounds, containing releasable mass labels, in chemical analysis, and to the use of these reagents in conjunction with probes which react with or bind noncovalently to a molecule whose presence is to be detected. The releasable tag reagents thus may indirectly detect target molecules, including biomolecular targets. The mass label may be released from the probe following reaction with or binding of the probe to the target and detected by mass spectrometry. The mass value of the label identifies and characterizes the probe and, therefore, the target molecule. In the case of a mass-labeled oligonucleotide probe used to target a polynucleotide, the detection of mass-labels rather than the nucleic acid probes or the nucleic acid targets themselves means that biochemical analysis procedures can be greatly simplified. The need for slow, laborious, costly, and/or complex solid-phase and/or solution-phase cleanup and desalting procedures can be minimized or even eliminated.
Therefore, in accordance with the present invention, there is provided a release tag compound comprising Rx, Re and M, wherein Rx is a reactive group, Re is a release group, and M is a mass label detectable by mass spectrometry. As used herein the term “a” encompasses embodiments wherein it refers to a single element as well as embodiments including one or more of such elements. For example, the phrase “a reactive group” may refer to a single reactive group, but also encompasses embodiments including more than one reactive group.
Although the mass label may typically be a synthetic polymer or a biopolymer or some combination thereof, in some embodiments, the mass label may generally be any compound that may be detected by mass spectrometry. In particular embodiments, the mass label may be a biopolymer comprising monomer units, wherein each monomer unit is separately and independently selected from the group consisting essentially of an amino acid, a nucleic acid, and a saccharide with amino acids and nucleic acids being preferred monomer units. Because each monomer unit may be separately and independently selected, biopolymer mass labels may be polynucleic acids, peptides, peptide nucleic acids, oligonucleotides, and so on.
As defined herein “nucleic acids” refer to standard or naturally-occurring as well as modified
on-natural nucleic acids, often known as nucleic acid mimics. Thus, the term “nucleotides” refers to both naturally-occurring and modified
onnaturally-occurring nucleotides, including nucleoside tri, di, and monophosphates as well as monophosphate monomers present within polynucleic acid or oligonucleotide. A nucleotide may also be a ribo; 2′-deoxy; 2′, 3′-deoxy as well as a vast array of other nucleotide mimics that are well-known in the art. Mimics include chain-terminating nucleotides, such as 3′-O-methyl, halogenated base or sugar substitutions; alternative sugar structures including nonsugar, alkyl ring structures; alternative bases including inosine; deaza-modified; chi, and psi, linker-modified; mass label-modified; phosphodiester modifications or replacements including phosphorothioate, methylphosphonate, boranophosphate, amide, ester, ether; and a basic or complete internucleotide replacements, including cleavage linkages such a photocleavable nitrophenyl moieties. These modifications are well known by those of skill in the art and based on fundamental principles as described Saenger (1983), incorporated herein by reference.
Similarly, the term “amino acid” refers to naturally-occurring amino acid as well as any modified amino acid that may be synthesized or obtained by methods that are well known in the art.
In another embodiment, the mass label may be a synthetic polymer, such as polyethylene glycol, polyvinyl phenol, polyproplene glycol, polymethyl methacrylate, and derivatives thereof. Synthetic polymers may typically contain monomer units selected from the group consisting essentially of ethylen
Becker Christopher H.
Monforte Joseph A.
Pollart Daniel J.
Shaler Thomas A.
Heller Ehrman White & McAuliffe LLP
Riley Jezia
Seidman Stephanie L.
Sequenom Inc.
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