Metallic solid supports modified with nucleic acids

Details Metallic solid supports modified with nucleic acids Metallic solid supports modified with nucleic acids

1997-02-28

2001-07-24

Houtteman, Scott W. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

Reexamination Certificate

active

06265155

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to metallic solid supports comprising blocking moieties and modified nucleic acids, and to methods of using them.
BACKGROUND OF THE INVENTION
The detection of specific nucleic acid sequences is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
In contrast, specificity remains a problem in many currently available gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. Variations in the concentrations of probes, of targets and of salts in the hybridization medium, in the reaction temperature, and in the length of the probe may alter or influence the specificity of the probe/target interaction.
It may be possible under some limited circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization. New experimental techniques for mismatch detection with standard probes include DNA ligation assays where single point mismatches prevent ligation and probe digestion assays in which mismatches create sites for probe cleavage.
Finally, the automation of gene probe assays is an area of high interest. Such assays generally rely on the hybridization of a labelled probe to a target sequence followed by the separation of the unhybridized free probe. This separation is generally achieved by gel electrophoresis or solid phase capture and washing of the target DNA, and is generally quite difficult to automate easily.
Immobilization of biomolecules on solid phases is widely used. Of particular interest is the immobilization of nucleic acids on solid surfaces for use in nucleic acid detection systems. There are a number of known techniques for the immobilization of nucleic acid on solid supports, including Hegner et al., FEBS Letters 336(3):452 (1993); Millan et al., Anal. Chem. 65:2317 (1993); Southern et al., Nucleic Acids Res. 22(8):1368 (1994); Maskos et al., Nucleic Acids Res. 20(7):1679 (1992); Palecek, Electroanalysis 8:7 (1996); Hashimoto et al., Anal. Chem. 66:3830 (1994); Su et al., Anal. Chem. 66(6):769 (1994); Chrisey et al., Nucleic Acids Res. 24(15):3031 (1996); Williams et al., Nucleic Acids Res. 22(8):1365 (1994); Xu et al., J. Am. Chem. Soc. 117:2627 (1995); Millan et al., Electroanalysis 4:929 (1994); Lee et al., Science 266:771 (1994); Millan et al., Anal. Chem. 66:2943 (1994); and Xu et al., J. Am. Chem. Soc. 116:8386 (1994).
It is an object of the present invention to provide novel compositions for the detection of nucleic acids, and methods of using the compositions.
SUMMARY OF THE INVENTION
In accordance with the objects outlined above, the present invention provides solid supports having a metallic surface. The metallic surface of the solid supports comprise blocking moieties, and at least one modified nucleic acid. The blocking moieties have at least a first and a second end, and is attached at the first end to the metallic surface. The modified nucleic acid comprises a linker moiety having a first and a second end. The first end of the linker moiety is attached to the solid support and the second end is attached to a nucleic acid.
In an additional aspect, the present invention provides solid supports having a metallic surface comprising a covalently immobilized monolayer, wherein a subset of the molecules forming the monolayer are covalently linked to nucleic acid.
In a further aspect the invention provides blocking moieties having the formula comprising:
SCM is a sulfur-containing moiety which is attached to the metallic surface. R
1
and R
2
are independently selected from the group consisting of hydrogen and substituent groups, n is an integer from 3 to 50, and X is a terminal group.
In an additional aspect the invention provides modified nucleic acids having the formula comprising:
In a further aspect the invention provides solid supports having metallic surfaces comprising a mixed monolayer of blocking moieties and modified nucleic acids.
In an additional aspect, the invention provides methods of hybridizing probe nucleic acid to target nucleic acid. The methods comprise adding target nucleic acid to a solid support having a metallic surface comprising blocking moieties and modified nucleic acids as is described herein, under conditions where the probe nucleic acid and the target nucleic acid will hybridize to form a hybridization complex.
In a further aspect, the methods comprise additionally detecting the hybridization complex.
DETAILED DESCTION OF THE INVENTION
The present invention is directed to the discovery that nucleic acids attached to metallic surfaces exhibit excellent characteristics in hybridization assays, in a stable, reproducible, rapid manner. That is, when a metallic surface such as gold is modified with nucleic acids, attached via linkers, and blocking moieties, which serve to shield the nucleic acids from the metallic surface, excellent hybridization characteristics are seen. Thus, the present invention provides new and useful compositions for the immobilization of nucleic acids on solid supports.
Accordingly, the present invention provides metallic solid supports comprising blocking moieties and modified nucleic acids.
By “solid support comprising a metallic surface” or grammatical equivalents herein is meant a surface that has a metallic layer. Suitable metallic layers include any metals to which thiol groups may be attached, with gold and copper being preferred, and gold being particularly preferred. Thus, any material which can be made to contain a metallic layer or film can be used as a solid support. Accordingly, the entire surface may be metal, or only a thin layer or film of metal on the top of a different material may be used. Thus, for example, glass, plastic, polymers, graphite, or metals other than gold and copper can be used as a support, with at least a portion of one side of the support having a metallic surface.
The deposition of gold onto any number of materials is known, using techniques including vapor deposition, electroplating, sputter coating, and chemical deposition. In addition, the surface can be polished as is known in the art.
The metallic solid supports described herein are generally depicted as a flat surface, which is only one of the possible conformations of the metallic solid support and is for schematic purposes only. In addition, the metallic surface on the solid support may be a single continguous surface, or may be divided up into smaller locations, in any number of ways. That is, the metallic solid supports can be used to form arrays, where different regions (“addresses”) on the surface contain different nucleic acids. As will be appreciated by those in the art, this may be done in a variety of ways. In one embodiment, the different addresses are physically separated by areas that do not contain a metallic layer. Alternatively, the different addresses may be separated by the addition of other materials, such as polymers, glass, silicon and other materials to the metallic layer. Alternatively,

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