Photoactivatable nucleic acids

Details Photoactivatable nucleic acids Photoactivatable nucleic acids

1998-02-24

2003-01-14

Wilson, James O. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S025300, C536S025310, C536S025340, C536S024300, C536S024310, C536S024320

Reexamination Certificate

active

06506895

ABSTRACT:

TECHNICAL FIELD
The present invention relates to the immobilization of nucleic acids. In another aspect, the invention relates to solid supports, such as oligonucleotide (“oligo”) chips, incorporating such nucleic acids. In yet another aspect, the invention relates to photoreactive groups, to molecules derivatized with such groups, and to the attachment of such molecules to support surfaces by the activation of such groups.
BACKGROUND OF THE INVENTION
The development of oligonucleotide (“oligo”) probe arrays (more commonly known as “DNA chips” and “Gene Chip” (a registered trademark of Affymetrix, Inc.)) has made significant advances over the past few years, and is becoming the center of ever-increasing attention and heightened significance. See, for instance, Stipp, D.,
Fortune,
p.56, Mar. 31, 1997. See also Borman, S., C&EN, p.42, Dec. 9, 1996, and Travis, J.,
Science News
151:144-145 (1997).
Typically, oligonucleotide probe arrays display specific oligonucleotide sequences at precise locations in an information rich format. In use, the hybridization pattern of a fluorescently labeled nucleic acid target is used to gain primary structure information for the target. This format can be applied to a broad range of nucleic acid sequence analysis problems including pathogen identification, forensic applications, monitoring mRNA expression and de novo sequencing. See, for instance, Lipshutz, R. J., et al.,
BioTechniques
19(3):442-447 (1995). Such arrays sometimes need to carry several tens of thousands, or even hundreds of thousands of individual probes. The chips also need to provide a broad range of sensitivities in order to detect sequences that may be expressed at levels anywhere from 1 to 10,000 copies per cell.
A variety of approaches have been developed for the fabrication and/or use of oligonucleotide probe arrays. See, for instance, Weaver, et al. (WO 92/10092) which describes a synthetic strategy for the creation of large scale chemical diversity on a solid-phase support. The system employs solid-phase chemistry, photolabile protecting groups and photolithography to achieve light-directed, spatially addressable, parallel chemical synthesis. Using the proper sequence of masks and chemical stepwise reactions, a defined set of oligos can be constructed, each in a predefined position on the surface of the array.
Using this technology, Affymetrix, Inc. (Santa Clara, Calif.), has developed large microarrays of oligonucleotides affixed to silicon wafers. In use, a researcher will extract mRNA from a cell or other biological source, convert it to cDNA and label the sample with a fluorescent probe. Sequences complimentary to the chip-bound probe will hybridize to the wafer and allow the researcher to determine their relative amounts by measuring the fluorescence of each spot. To date, for instance, researchers have been able to quantitatively measure the expression of more than 1000 human genes in this manner.
One drawback of the Affymetrix approach is the limitation of the oligo length that can be affixed to the surfaces. With present techniques, it is common that every addition step involved in the synthesis of oligos will result in some errors or truncated sequences. With oligo chips, however, it is not possible to perform conventional post-synthesis purification techniques (e.g., HPLC) in order to remove truncated sequences since the oligo sequences remain bound to the support.
Synteni (Palo Alto, Calif.) produces arrays of cDNA by applying polylysine to glass slides. Arrays of cDNAs are printed onto the coated slides followed by exposure to UV light, in order to crosslink the DNA with the polylysine. Unreacted polylysine is then blocked by reaction with succinic anhydride. These arrays, called “Gene Expression Microarrays” (GEM™) are used by labeling cDNA prepared from a normal cell with a fluorescent dye, then labeling cDNA from an abnormal cell with a fluorescent dye of a different color. These two labeled cDNA probes are simultaneously applied to the microarray, where they competitively bind to the arrayed cDNA molecules. This two color coding technique is used to identify the differences in gene expression between two cell samples. (Heller, R. A., et al.,
Proc. Natl. Acad. Sci. USA,
94:2150-2155 (1997)).
Others have described a photo-crosslinking compound known as psoralen. Psoralen is a polycyclic compound having a planar configuration that intercalates within nucleic acid helices. When irradiated with UV light, the intercalated psoralen induces the formation of inter-strand linkages within the DNA molecule. Oligonucleotides derivatized at the 5′-terminus with psoralen have been used to crosslink double-stranded (Pieles, U. and U. Englisch,
Nucleic Acid Res.,
17(1):285-299, 1989), or triplex nucleic acids in solution (Takasugi, M., et al.,
Proc. Natl. Acad. Sci. USA,
88(13):5602-5606 (1991)). Psoralen derivatives have also been used to crosslink DNA-binding proteins to DNA (Sastry, S. S., et al.,
J. Biol. Chem.,
272(6):3715-3723 (1997)).
In a different application, psoralen derivatives have been used to covalently bond functional groups to the surface of solid supports such as polystyrene. Those functional groups, in turn, are then used to thermochemically attach compounds to the support surface (Goodchild, J.,
Bioconjugate Chem.,
1(3):165-187 (1990)). Currently, Nalge Nunc International uses this process to produce microplates that provide amine-functionalized surfaces for the thermochemical attachment of molecules such as nucleic acids. See, e.g., “DNA Assay Developments: Surface Chemistry and Formats for Molecular Screening and Diagnostics”, B. Sullivan, et al., Jun. 4, 1997, Nalge Nunc International Corporation product literature.
On a separate subject, the assignee of the present invention has previously described a variety of applications for the use of photochemistry, and in particular, photoreactive groups, e.g., for attaching polymers and other molecules to support surfaces. See, for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, 5,637,460, and 5,714,360 and International Patent Application Nos. PCT/US96/08797 (Virus Inactivating Coatings), PCT/US96/07695 (Capillary Endothelialization), and PCT/US97/05344 (Chain Transfer Agents).
To the best of Applicant's knowledge, however, the art does not teach, nor are there commercial products that involve, the activation of pendent photoreactive groups to attach nucleic acids to surfaces in a specific and controllable fashion. The attachment of a nucleic acid to a surface by irradiation would appear to be susceptible to radiation-induced damage, and would be inherently nonspecific. See, for instance, M. Pirrung, et al.,
J. Org. Chem.,
63:241-246 (1998), which states that “irradiation [during deprotection] with wavelengths<340 nm should be avoided . . . based on the potential photochemical damage to the DNA.”
In spite of the developments to date, there remains a need for methods and reagents that improve the immobilization of nucleic acids onto a variety of support materials, e.g., in order to form oligonucleotide probe arrays.
SUMMARY OF THE INVENTION
The present invention provides a composition comprising a photoactivatable nucleic acid derivative, in the form of a nucleic acid having one or more photoreactive groups bound thereto. The photoreactive group(s) are preferably covalently bound, directly or indirectly, at one or more points along the nucleic acid. Such groups can be activated in order to attach the nucleic acid to the surface of a solid support, such as the surface of a chip. A photoreactive group of this invention is separate and distinct from whatever group or bonds within the nucleic acid may be susceptible to radiation. In turn, the photogroup provides a derivatized nucleic acid that can be selectively and specifically activated in order to attach the nucleic acid to a support, and in a manner that substantially retains its desired chemical or biological function.
As used herein, unless otherwise indicated, a “photoreactive compo

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