Surface-bound, double-stranded DNA protein arrays

Details Surface-bound, double-stranded DNA protein arrays Surface-bound, double-stranded DNA protein arrays

1998-08-11

2003-04-15

Marschel, Ardin H. (Department: 1631)

Chemical apparatus and process disinfecting, deodorizing, preser

Analyzer, structured indicator, or manipulative laboratory...

Means for analyzing liquid or solid sample

C422S050000, C435S004000, C435S006120, C435S007100, C435S007200, C436S501000, C536S025300

Reexamination Certificate

active

06548021

ABSTRACT:

FIELD OF INVENTION
The invention relates to nucleic acid protein arrays.
BACKGROUND OF THE INVENTION
Compact arrays or libraries of surface-bound, double-stranded oligonucleotides are of use in rapid, high-throughput screening of proteins to identify those that bind, or otherwise interact with, short, double-stranded DNA sequence motifs. Of particular interest are trans-regulatory factors that control gene transcription. Ideally, such an oligonucleotide array is bound to the surface of a solid support matrix that is of a size that enables laboratory manipulations, e.g. an incubation of a candidate protein with the nucleic acid sequences thereon, and that is itself inert to chemical interactions with experimental proteins, buffers and/or other components. In addition, it is desirable that the absolute number of unique nucleic acid sequences in the array be maximized, since methods of high-throughput screening are used in the attempt to minimize repetition of steps that are labor-intensive or otherwise costly.
A high-density, double-stranded DNA array complexed to a solid matrix is described by Lockhart (U.S. Pat. No.: 5,556,752); however, the DNA molecules therein disclosed are produced as unimolecular products of chemical synthesis. As synthesized, each member of the array contains regions of self-complementarity separated by a spacer (i.e. a single-strand loop), such that these regions hybridize to each other in order to produce a double-helical region. Further, it is required that those regions of complementary nucleic acid sequences that must hybridize in order to form the double-helical structure are physically attached to each other by a linker subunit.
SUMMARY OF THE INVENTION
The invention provides a synthetic array of surface-bound, bimolecular, double-stranded nucleic acid molecules, the array comprising a solid support and a plurality of bimolecular double-stranded nucleic acid molecule members, a member comprising a first nucleic acid strand linked to the solid support and a second nucleic acid strand which is substantially complementary to the first strand and complexed to the first strand by Watson-Crick base pairing, wherein for at least a portion of the members, each member comprises a recognition site within a nucleic acid sequence for a protein, wherein a recognition site within a nucleic acid sequence for a protein of a first member is different from a recognition site within a nucleic acid sequence for a protein of a second member and wherein a protein is bound to a member thereof.
The term “synthetic”, as used herein, is defined as that which is produced by in vitro chemical or enzymatic synthesis. The synthetic arrays of the present invention may be contrasted with natural nucleic acid molecules such as viral or plasmid vectors, for instance, which may be propagated in bacterial, yeast, or other living hosts.
As used herein, the term “nucleic acid” is defined to encompass DNA and RNA or both synthetic and natural origin. The nucleic acid may exist as single- or double-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNA copolymer, wherein the term “copolymer” refers to a single nucleic acid strand that comprises both ribonucleotides and deoxyribonucleotides.
As used herein, the term “bimolecular” refers to the fact that the 5′ end of the first strand and 3′ end of the second strand are not linked via a covalent bond, and thus do not form a continuous single strand. As used herein in this context, “covalent bond” is defined as meaning a bond that forms, directly or via a spacer comprising nucleic acid or another material, a continuous strand that comprises the 5′ end of the first strand and the 3′ end of the second strand, and thus includes a 3′/5′ phosphate bond as occurs naturally in a single-stranded nucleic acid. This definition does not encompass intermolecular crosslinking of the first and second strands.
When used herein in this context, the term “double-stranded” refers to a pair of nucleic acid molecules, as defined above, that exist in a hydrogen-bonded, helical array typically associated with DNA, and that under these umbrella terms are included those paired oligonucleotides that are essentially double-stranded, meaning those that contain short regions of mismatch, such as a mono-, di- or tri-nucleotide, resulting from design or error either in chemical synthesis of the oligonucleotide priming site on the first nucleic acid strand or in enzymatic synthesis of the second nucleic acid strand; it is contemplated that at least a portion of the members of the array have a second nucleic acid strand which is substantially complementary to- and base paired with the first strand along the entire length of the first strand.
As used herein, the terms “complementary” and “substantially complementary” refer to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Typically, sequences which are complementary will hybridize to each other under stringent conditions. Stringent hybridization conditions will typically include salt concentrations of less than about 1M, more usually less than about 500 mM, and preferably less than about 200 mM. Alternatively, stringent hybridization conditions typically include at least 10% formamide, preferably 20% and more preferably 40%. Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., more typically greater than about 30° C., and preferably in excess of about 37° C. Longer fragments may require higher hybridization temperatures for specific hybridization, while those that are rich in dA and dT may require lower temperatures. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Sequences that are substantially complementary may hybridize under stringent conditions; however, it is usually necessary to raise the concentration of salt, or lower the concentration of formamide or the hybridization temperature.
As used herein in reference to nucleic acid members of an array, the term “portion” refers to at least two members of an array. Preferably, a portion refers to a number of individual members of an array, such as at least 60%, 80%, 90% and 95-100% of such members.
As used herein, the terms “recognition site for a protein” and “recognition site within a nucleic acid sequence for a protein” refers to a nucleic acid sequence which is recognized and/or bound by a protein.
As used herein with regard to recognition sites within a nucleic acid sequence for a protein, the term “different” refers to two or more nucleic acid sequences which are recognized and/or bound by a protein or proteins, which recognition sites within a nucleic acid sequence for a protein differ in the identity of at least one nucleotide.
As used herein, the term “array” is defined to mean a heterogeneous pool of nucleic acid molecules that is affixed to a solid support in a spatially-ordered manner, such as a Cartesian distribution (in other words, arranged at defined points along the x- and y axes of a grid or specific ‘clock positions’ within- or degrees or radii from the center of a radial pattern) of nucleic acid molecules over the support, that permits identification of individual features during the course of experimental manipulation.
As used herein, the term “feature” refers to each nucleic acid sequence occupying a discrete physical location on the array; if a given sequence is represented at more than one such site, each site is classified as a feature. A feature comprises one or a

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