Compositions and methods using platinum compounds for...

Details Compositions and methods using platinum compounds for... Compositions and methods using platinum compounds for...

2002-03-01

2004-11-30

Riley, Jezia (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024300, C536S026600

Reexamination Certificate

active

06825330

ABSTRACT:

BACKGROUND OF THE INVENTION
Labeled biomolecules are essential to a wide array of methods used for biological research, medical diagnosis and therapy. Labeled biomolecules permit a researcher or clinician to detect the location, size, amount or other properties of biomolecules of interest. Commonly labeled biomolecules include, among others, nucleotides, oligonucleotides, nucleic acids, amino acids, peptides and polypeptides, proteins, carbohydrates and lipids.
Nucleic acid hybridization is one of the most frequently used methods requiring labeled nucleic acid probes, and is used in both research and diagnostic medicine. Methods utilizing nucleic acid hybridization include, for example, fluorescent in-situ hybridization (FISH), DNA in-situ hybridization (DISH; Singer & Ward, 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 7331-7335), RNA in-situ hybridization (RISH; Singer et al., 1986, BioTechniques 4: 230-250), multi-color fluorescent in-situ hybridization (MFISH), gene mapping (Pitta et al., 1990, Strategies 3: 33), Southern and Northern blots (Southern, 1975, J. Mol. Biol. 98: 503-517; Alwine et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 5350-5354), microarray-based assays (Callow et al., 2000, Genome Res. 10: 2027-2029) and diagnostic array assays, among others. Radioisotopes are perhaps the most commonly used detectable labels for nucleic acid hybridization. However, there is a need in the art for non-isotopic alternatives to radiolabeling because isotopic labels are expensive, dangerous to handle and have increasingly expensive disposal costs.
An ideal non-radioactive labeled probe for nucleic acid hybridization should have the following properties: 1) label that is easily attached to the probe; 2) stability under various separation/purification conditions such as gel electrophoresis, HPLC, TLC or column purification; 3) stability under nucleic acid hybridization conditions, such as exposure to solutions containing detergents and formamide, and temperatures up to 100° C.; 4) label that does not interfere with hybridization to a complementary target; 5) label that is detectable at very low amounts, ideally 1 attomole of nucleic acid or less; 6) applicability to solution or solid-phase hybridization assays such as those performed on membranes, microtiter plates and microarrays (gene chips); 7) adaptability to homogeneous assay formats wherein the hybridized probe is detectable and distinguishable from unhybridized probe in solution; 8) long shelf-life for storage; and 9) compatibility with automated analysis and high throughput instruments.
Enzymatic Labeling Methods
Labels are generally attached to nucleic acids using either enzymatic or chemical means. Enzymatic methods are useful for both end-labeling of existing strands of nucleic acid and for the template-dependent internal labeling of nucleic acid strands polymerized in vitro or in vivo. There are several different approaches taken to the enzymatic generation of end-labeled nucleic acid probes. Polynucleotide kinase is often used to label the 5′ end of a polynucleotide strand with a radioactive phosphate (e.g.,
32
P or
33
P) transferred from the gamma position of labeled ATP. Kinase labeling results in, at best, a single radioactive phosphate label moiety per polynucleotide strand. Alternatively, end-labeled probes comprising more than one label moiety per strand can be generated by a 3′-tailing reaction catalyzed by terminal transferase (Roychoudhury et al., 1980, Nucleic Acids Res. 6: 1323-1333). However, the optimal reaction conditions vary for the incorporation of the various nucleotides by terminal transferase, and conditions vary for each different probe to be labeled. Another alternative for end-labeling probes is to use PCR to make 5′ end-labeled DNA probes through incorporation of a 5′-labeled primer. This approach can rapidly generate a significant quantity of extended, end-labeled probes. However, the PCR method requires first generating the end-labeled primer at high specific activity.
There are also a number of approaches for generating internally labeled probes using enzymes. One of the commonly used enzymatic techniques is “Random-Primed” labeling, (Feinberg & Vogelstein, 1983, Anal. Biochem. 132: 6-13; Feinberg & Vogelstein, 1984, Anal. Biochem. 137: 266-267). The resulting labeled probe is a mixture of different sized sequences of the template. The ratio between primer and template, and the amount of labeled and unlabeled nucleotide are varied to obtain the highest specific activity probes. An alternative internal labeling method is “Nick Translation” (Rigby et al., 1977, J. Mol. Biol. 133: 237-251). In this procedure, a mixture of the endonuclease Dnase I and 5′→3′ DNA polymerase Pol I is used, and the ratio of these two enzymes and the percentage of labeled nucleotide determines the amount and length of the labeled DNA. Another alternative for generating an internally-labeled probe is the Polymerase Chain Reaction (PCR; Mullis et al., 1986, Cold Spring Harb. Symp. Quant. Biol. 51 Pt. 1: 263-273), performed in the presence of a mixture of labeled and unlabeled nucleotides. For mRNA analysis, reverse transcription (RT) in the presence of labeled deoxyribonucleotides is the traditional method to create labeled cDNA (Varmus & Swanstrom, 1979, in
Molecular Biology of Tumor Viruses
, vol.2:
RNA Tumor Viruses
(Weiss et al., Eds) pp. 369-512, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
In any enzymatic labeling method, the reaction conditions and the ratio of labeled to unlabeled nucleotides should be optimized for every labeling reaction, thereby complicating the labeling procedure. Each of the enzymatic labeling methods described above, and other enzymatic methods known to those skilled in the art, tend to work well for the incorporation or attachment of radiolabeled species. In general, isotopic labels do not interfere with the function of the enzymes to label nucleic acids. In contrast, labeling moieties other than isotopes tend to be large and can interfere with the labeling reactions, such that relatively poor incorporation rates and low total incorporation of the label is observed. The relatively poor recognition and incorporation of nucleotides labeled with non-isotopic moieties, such as fluorescent molecules, can sometimes be compensated by increasing the absolute concentration and modifying the ratio of the labeled nucleotide in the reaction. However, even when this approach works, the cost of the labeling reaction is increased. In addition, variation in enzyme quality, resulting for example from storage or frequent freeze/thaw cycles, can result in variable labeling reaction efficiency.
A common alternative solution to the problem of poor incorporation of nucleotides labeled with larger non-isotopic markers is to enzymatically incorporate a nucleotide modified with a small affinity moiety, such as biotin or digoxigenin, into a probe sequence. The probe may then either be directly reacted with a labeled affinity binding partner, such as avidin or anti-digoxigenin antibodies, or it may be hybridized to target nucleic acid and then reacted with a labeled affinity binding partner. The biotin/avidin system is characterized by subpicogram sensitivity (Feinberg & Vogelstein, 1983, supra; Feinberg & Vogelstein, 1984, supra). A disadvantage of this method is its high non-specific background due to the inherent positive charge exhibited by avidin at neutral pH and the endogenous ubiquity of vitamin H (biotin) in biological samples. This method also suffers from problems discussed above related to variations in enzyme activity leading to variable labeling reaction efficiency.
Chemical Labeling Methods
Chemical labeling methods are an alternative to the enzymatic labeling methods. Among methods for direct derivatization of nucleic acids with detectable markers are photolabeling reactions using aryl azide compounds (Forster et al., 1985, Nucleic Acids Res. 13: 745-761) psoralen, angelicin, acridine dyes (Chimino et al., 1985, Ann. Rev. Biochem. 54: 1151-1193

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