Field of the Invention
The present invention relates to the fields of microarray devices and hybridization technologies. Specifically, the present invention provides hybridization devices having a surface and a saturated film of adsorbed oligonucleotides effective to hybridize to a complementary nucleic acid as a non-helical asymmetrical duplex.
Description of the Related Art
The double helix is known to form as a result of the hybridization of complementary nucleic acid strands in aqueous solution. In the helix, the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and there, are available for interaction with cationic groups. Cation-coated solid supports are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cation surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Oligonucleotides of less than 100 nucleotides are better suited for hybridization on such supports than full length genes.
Microarray technology has revolutionized applied genomics (Cheung V G et al., 1999, Nature Genetics 21:15-19; Duggan D J et al., 1999, Nature Genetics 21:10-14). It is based upon hybridization of a surface-bound nucleic acid to a nucleic acid in solution to form a Watson-Crick double helix by a mixed phase reaction between complementary nucleic acid strands. The secondary structure of the resulting double helix is determined, in part, by base pairing and base stacking, in conjunction with the constraints imposed on phosphodiester backbone conformation and sugar pucker. Those interactions serve to define local base pairing and also the overall pitch of the helix. Although, in solution, the average pitch of the helix is near to 10 base pairs, structural studies have revealed a high degree of variability and flexibility of pitch angle, including the modeling-based prediction that a flat, non-helical ribbon-like structure might form under conditions of extreme mechanical distension (Leger J F et al., 1999, Phys. Rev. Lett. 83:1066-1069; Bensimon D et al., 1995, Phys. Rev. Lett. 74:4754-4757; Smith S B et al., 1996, Science 271:795-799; Lebrun A et al., 1996, Nucl. Acids Res. 24:2260-2267; or upon the disruptive binding of an intercalator (Gao O et al., 1991, Proc. Natl. Acad. Sci. USA 88:2422-2426).
Nucleic acids may be covalently or noncovalently immobilized on a surface. Several means of covalent attachment of nucleic acids are known in the art. For example, oligonucleotides may be covalent coupling to a surface by chemical or photochemical crosslinking as commonly practiced following Northern and Southern blotting of nucleic acids onto nylon or nitrocellulose membranes. Covalent attachment also may be achieved using pre-synthesized oligonucleotides that are fabricated with a chemical linker at one or both ends of the oligonucleotide. U.S. Pat. No. 6,048,695 to Bradley and Cai discloses a method wherein a linker is added to an oligonucleotide for the purpose of making a covalent bond with reactive moieties on the surface. See e.g. U.S. Pat. No. 6,048,695 and references therein. Thus, oligonucleotide probe attachment occurs with the surface through the linker, rather than by direct adsorptive interaction of the probe with the surface.
Still another means of covalently attaching nucleic acids to a surface is by photolithography. U.S. Pat. No. 5,959,098 to Goldberg et al. discloses a method of derivatizing a surface to provide photoprotected functional groups. Nucleic acids are synthesized directly on the surface by selectively illuminating surface regions to remove the protecting groups. The deprotected regions are then available for covalent attachment of nucleotide monomers having photoprotected functional groups. Repetition of these steps results in oligonucleotides covalently linked to the surface. Further examples of array fabrication include U.S. Pat. No. 6,221,653 to Caren and Luebke (inkjet printing of the nucleic acid building blocks) and U.S. Pat. No. 6,024,925 (microfluidics robot to prepare sample arrays for analysis in a mass spectrometer).
Methods for noncovalently immobilizing nucleic acids typically require a bridging agent. In some cases that bridging agent is a salt or detergent. For example, U.S. Pat. No. 5,610,287 to Nikiforov and Knapp discloses a noncovalent immobilization method comprising contacting a glass or hydrophilic polystyrene solid support with a combination of a nucleic acid and a cationic bridging agent (sodium chloride or a detergent). See e.g. '287, abstract. The method of attachment is based upon interaction of the detergent flocculent with the surface, rather than direct adsorptive interaction between the oligonucleotide and the surface.
Alternatively, the bridging agent may be a high-affinity interaction pair such as avidin and biotin or digoxigenin and an anti-digoxigenin antibody. For example, a biotinylated nucleic acid may be immobilized on a streptavidin-coated surface. See e.g. Belosludtsev Y et al., 2001, Biochem Biophys Res Commun. 282:1263-1267; Holmstrom et al., 1993, Anal. Biochem. 209(2):278-283. In this method, a biotin-modified linker is added to an oligonucleotide for the purpose of making a bond with avidin or avidin-like groups coated into the surface. Attachment occurs with the surface through the biotin-modified linker, rather than by direct adsorptive interaction of the probe with the surface.
Methods of adsorptive, non-covalent immobilization of long, single or double stranded DNA molecules onto membrane surfaces are the basis for a device referred to as a “Southern” or “Northern” blot. Standard practice of the blotting technology art has shown that, where probe length is less than about 100 bases, the known adsorptive methods of attachment are too weak to support probe attachment that is sufficiently stable to form a hybridization device. Thus, the standard for attachment of short nucleic acid probes in blotting applications has involved covalently crosslinking the nucleic acid to the solid support (as by photochemical cross linking) or other means of non-adsorptive linkage (such as chemical crosslinking). Known methods to increase the strength of adsorptive, non-covalent immobilization of short nucleic acids to membrane supports have been shown to render the DNA unsuitable for a hybridization device and hence the conventional blotting methods involving short nucleic acid probes all employ a covalent means of immobilization. Other types of porous material, including porous beads and related small particle porous substrates, are known to behave as do membranes, that is, long DNA probes may be attached by adsorptive interaction, but short probes must be attached by non-adsorptive means.
Adsorptive, non-covalent immobilization of long, single or double stranded DNA molecules onto non-membranous surfaces, most particularly the planar substrates (often referred to as slides), may be achieved by known methods and used for the fabrication of DNA microarrays. The adsorptive probe interaction is the basis for fabrication and use of DNA microarrays in which long (greater than 100 bases) nucleic acid probes are spotted into planar surfaces to form the microarray. Standard practice of the microarray fabrication art has shown that long nucleic acid probes may be attached to surfaces by means of adsorptive association with polycation-coated surfaces (usually poly-lysine).
However when short nucleic acids (less than 100 bases) are to be attached to microarray surface, the known adsorptive methods of microarray attachment are found to be too weak to provide for stable probe attachment to form a microarray-based hybridization device. Thus the standard of the art for attachment of oligonucleotide probes (less than 100 bases) in microarray applications has involved covalent attachment of the nucleic acid to the microarray support, generally by covalent linkage of the oligoncucleotide terminus (3′ or 5′) to the solid support or other means of non-adsorptive interaction. Known methods to increase the strength of adsorptive, non-covalent immobilization of short nucleic acids to microarray supports have been shown to render the DNA unsuitable for these applications. This may be due to a loss of the ability of the oligonucleotide to form a duplex or unsuitably high levels of non-specific target binding to the microarray.
Oligonucleotides (short single stranded pieces of nucleic acid (DNA, RNA) of less than 100 bases in length) are well suited as probes to be attached to a solid support as the basis for a hybridization device. However, no method is presently known to directly adsorb the oligonucleotide onto a solid support by adsorption, in a way that yields a probe that can be used for hybridization. In fact, the literature indicates that direct absorption of oligonucleotides is not expected to work. For example, Lindsay asserts that methods of attaching DNA over about 100 bases in length to mica using aminopropyltriethoxysilane for structural analysis by atomic force microscopy result in DNA that is bound too strongly for studies of processes in situ. See Lindsay S M, The Scanning Probe Microscope in Biology, draft available at http://green.la.asu.edu/review/chap_7(3-5).htm.
A recent study of adsorbed oligonucleotides at a hydrocarbon coated silica surface concluded that oligonucleotide (<100 bases) adsorption would necessarily prohibit base pair specific hybridization, and thus make the product useless as a hybridization device. See Wirth M J, http://www.udel.edu/chem/wirth/oligos.html (“Any specific adsorption to sites on the surface interferes with the hybridization process. In practice, surfaces tend to have groups that hydrogen bond to the bases on oligonucleotides. Such hydrogen bonding to the substrate gives background noise that reduces the sensitivity of detecting oligonucleotides.”)
Furthermore, Bradley and Cai argue that adsorptive methods to create DNA hybridization devices adversely affect the quality of performance of the device due to the electrostatic charge on the glass surface. See U.S. Pat. No. 6,048,695, col. 1, line 46 to col. 2, line 8.
Thus, several lines of argument in the literature assert a hybridization device based on direct adsorption of oligonucleotide probes to a solid support is not feasible. To the best of the inventors' knowledge, there is no evidence for or discussion of such an oligonucleotide adsorption based hybridization device described in the literature or available commercially. All known oligonucleotide based hybridization devices (including microarrays, membranes, bead supports and all other configurations involving probe attachment to a support) are based on one of the above-described attachment methods.
Thus, there is a recognized need in the art to provide improved, low cost, hybridization devices utilizing direct adsorption of oligonucleotides. More specifically, the prior art is deficient in biomolecular hybridization devices comprising oligonucleotide probes effective to hybridize to a cognate target as a nonhelical, asymmetrical duplex. The present invention fulfills this long-standing need and desire in the art.