Biomolecule and/or cellular arrays on metal surfaces

#The invention is directed to the fabrication of biomolecular or cellular arrays on metal surfaces for use in the study of interactions between large molecules, between cells and large molecules, and between cells, such as nucleic acid-protein interactions or cellular interactions with antigens.

#The binding of proteins to DNA plays a pivotal role in the regulation and control of gene expression, replication and recombination. In addition, enzymes that recognize and modify specific oligonucleotide sequences are critical components of biological nucleic acid manipulation and repair systems. An enhanced understanding of how these proteins recognize certain oligonucleotide sequences would aid in the design of biomedical systems which could, for example, be used to regulate the expression of therapeutic proteins. For this reason, the study of protein-nucleic acid interactions (i.e., protein-DNA and protein-RNA interactions) is a rapidly growing area of molecular biology, aided in part by recent advances in NMR and X-ray structural determination methods. At the same time, the explosive increase in the amount of available genomic and extra-genomic (i.e., ribosomal) sequence information obtained from large-scale nucleic acid sequencing efforts creates a need to survey this vast amount of new sequence data for protein binding sites. The present invention addresses this need by using surface plasmon resonance (SPR) imaging techniques as a rapid and efficient method for screening the sequence or structure-specific binding of proteins to large arrays of nucleic acid molecules immobilized at chemically-modified metal surfaces.

#Arrays of DNA molecules attached to planar surfaces are currently employed in hybridization adsorption experiments to sequence DNA, Pease et al. (1994) Proc. Natl. Acd. Sci. USA 91:5022–5026; to screen for genetic mutations, Winzeler et al. (1998) Science 281:1194–1197: and in DNA computing applications, Frutos et al. (1997) Nucleic Acids Res. 25:4748–4757; and Frutos et al (1998) J. Am. Chem. Soc. 120:10277–10282. These arrays are exposed to solutions containing fluorescently labeled complementary DNA sequences, rinsed, and then “read-out” using fluorescence imaging methods.

#FIG. 1 is a schematic representation of a fabrication scheme for constructing multi-element DNA arrays according to the present invention. A clean gold surface is reacted with an amine-terminated alkanethiol and subsequently reacted with a protecting group to create a hydrophobic surface. This surface is then exposed to UV radiation through a quartz mask and rinsed with solvent to remove the alkanethiol+protecting group from specific areas of the surface, leaving bare gold pads. These bare gold areas on the sample surface are filled in with the alkanethiol, resulting in an array of alkanethiol pads surrounded by a hydrophobic protecting-group background. Solutions of nucelic acid are then delivered by pipette onto the specific array locations and are covalently bound to the surface via a bifunctional linker. In the final two steps, the protecting groups on the array background are removed and replaced by functional groups which prohibit the non-specific binding of analyte proteins to the background.

#FIG. 2 depicts a surface reaction scheme showing the steps involved in the reversible modification of the array background. The starting amine-terminated alkanethiol surface is reacted with the Fmoc-NHS protecting group to form a carbamate linkage thus creating a hydrophobic Fmoc-terminated surface. After nucleic acid immobilization, the surface is deprotected, resulting in the return of the original alkanethiol surface. In the final array fabrication step, the deprotected alkanethiol surface is reacted with PEG-NHS to form an amide bond which covalently attaches PEG to the array surface (to prevent non-specific binding of analyte proteins to the background).

#FIG. 3 depicts PM-FTIRRAS spectra in the mid-IR region for the surfaces involved in the array background modification. (A) The starting MUAM surface. (B) After reaction with Fmoc-NHS, bands indicative of the carbamate linkage and the Fmoc ring stretch appear in the spectrum. (C) The surface is deprotected and reverts back to the MUAM surface as evidenced by the similarities between spectra A and C. (D) After reaction with PEG-NHS, bands indicative of the amide linkage as well as those associated with the ethylene glycol groups are present.

#Abbreviations and Commercial Suppliers:

#The following abbreviations and terms are used throughout the specification and claims. All other terms have their standard, accepted meaning in the relevant art.

#“biomolecule”=any molecule found in biological material, expressly including, but not limited to nucleic acids, proteins, peptides, antibodies, enzymes, cell-wall components such as phospholipids, etc., and modified and synthetic forms thereof, such as labeled biomolecules and recombinant biomolecules.