Nucleic-acid programmable protein arrays

Details Nucleic-acid programmable protein arrays Nucleic-acid programmable protein arrays

2002-01-22

2004-10-05

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Enzymatic production of a protein or polypeptide

C435S283100, C435S287100, C435S287900, C435S288300, C435S288400

Reexamination Certificate

active

06800453

ABSTRACT:

BACKGROUND OF THE INVENTION
The swift pace of discovery of new gene products by genomics and proteomics efforts and the growing availability of vast repositories of genes necessitates a strategy for analyzing proteins in a high-throughput manner. The high-density array format lends itself well to ordered high-throughput experimentation and analysis and has therefore become an established and widely-used format for high-throughput analysis of nucleic acids. Nucleic acid microarrays have enabled researchers to compare the expression of thousands of genes simultaneously. By making such comparisons, the expression patterns of clusters of genes in a particular biological context can be rapidly identified, which in turn can indicate groups of proteins that may act in concert in a specific pathway or process.
Reports of the analysis of protein function on a large scale are only just emerging. For example, a large-scale analysis of gene function in
S. cerevisiae
has been performed using a transposon-tagging strategy for the genome-wide characterization of disruption phenotypes, gene expression, and protein localization (Ross-Macdonald et al.,
Nature
402:413-418, 1999). In addition, complete two-hybrid analysis has been done using a large matrix of proteins for the interaction mapping of
C. elegans
proteins involved in vulval development (Walhout et al (2000)
Science
287:116-122) and the
S. cerevisiae
genome (Uetz et al. (2000)
Nature
403, 623-631); and Schwikowski (2000)
Nature Biotech.
18:1257).
The concept of nonliving peptide and protein arrays has drawn considerable attention because this approach to high-throughput experimentation allows the direct analysis of discrete protein binding and enzymatic activities without the complications of adverse in vivo effects. For example, a low-density (96 well format) protein array has been developed in which proteins, spotted onto a nitrocellulose membrane and biomolecular interactions, were visualized by autoradiography Ge, H. ((2000)
Nucleic Acids Res.
28:e3, I-VII). In another example, a high-density protein array (100,000 samples within 222×222 mm) that was used for antibody screening was formed by spotting proteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999)
Anal. Biochem.
270:103-111). Proteins have been printed on a flat glass plate that contained wells formed by an enclosing hydrophobic Teflon mask, and the arrayed antigens were detected using enzyme-linked immunosorbent assay (ELISA) techniques (Mendoza et al. (1999)
Biotechniques
27:778-788.). A large-scale in vitro analysis of biochemical activity using affinity-purified yeast proteins has been performed in the context of an array of 6144 yeast strains, each bearing a plasmid expressing a different GST-ORF fusion (Martzen et al. (1999)
Science
286, 1153-1155). Proteins have been covalently linked to chemically derivatized flat glass slides in a high-density array (1600 spots per square centimeter), and protein-protein and protein-small molecule interactions were detected by fluorescence or radioactive decay (MacBeath and Schreiber (2000)
Science
289:1760-1763). De Wildt et al. generated a high-density array of 18,342 bacterial clones, each expressing a different single-chain antibody, for screening antibody-antigen interactions (De Wildt et al. (2000)
Nature Biotech.
18:989-994).
SUMMARY OF THE INVENTION
The inventors have discovered, among other things, that arrays of polypeptides can be generated by translation of nucleic acid sequences encoding the polypeptides at individual addresses on the array. This allows for the rapid and versatile development of a polypeptide microarray platform for analyzing and manipulating biological information.
In one aspect, the invention features an array including a substrate having a plurality of addresses. Each address of the plurality includes: (1) a nucleic acid (e.g., a DNA or an RNA) encoding a hybrid amino acid sequence which includes a test amino acid sequence and an affinity tag; and, optionally, (2) a binding agent that recognizes the affinity tag. Optionally, each address of the plurality also includes one or both of (i) an RNA polymerase; and (ii) a translation effector.
In a preferred embodiment, each test amino acid sequence in the plurality of addresses is unique. For example, a test amino acid sequence can differ from all other test amino acid sequence of the plurality by 1, or more amino acid differences, (e.g., about 2, 3, 4, 5, 8, 16, 32, 64 or more differences; and, by way of example, has about 800, 256, 128, 64, or 32, 16, 8, 4, or fewer differences). In another preferred embodiment, the test amino acid sequence encoded by the nucleic acid at each address of the plurality is identical to all other test amino acid sequences in the plurality of addresses. In a preferred embodiment, the affinity tag encoded by the nucleic acid at each address of the plurality is the same, or substantially identical to all other affinity tags in the plurality of addresses. In another preferred embodiment, the nucleic acid at each address of the plurality encodes more than one affinity tag. In yet another preferred embodiment, the affinity tag encoded by the nucleic acid at an address of the plurality differs from at least one other affinity tag in the plurality of addresses.
In a preferred embodiment, the affinity tag is fused directly to the test amino acid sequence, e.g., directly amino-terminal, or directly carboxy-terminal. In another preferred embodiment, the affinity tag is separated from the test amino acid by one or more linker amino acids, e.g., 1, 2, 3, 4, 5, 6, 8, 10, 12, 20, 30 or more amino acids, preferably about 1 to 20, or about 3 to 12 amino acids. The linker amino acids can include a cleavage site, flexible amino acids (e.g., glycine, alanine, or serine, preferably glycine), and/or polar amino acids. The linker and affinity tag can be amino-terminal or carboxy-terminal to the test amino acid sequence.
The nucleic acid can be a RNA, or a DNA (e.g., a single-stranded DNA, or a double stranded DNA). In a preferred embodiment, the nucleic acid includes a plasmid DNA or a fragment thereof; an amplification product (e.g., a product generated by RCA, PCR, NASBA); or a synthetic DNA.
The nucleic acid can further include one or more of: a transcription promoter; a transcription regulatory sequence; a untranslated leader sequence; a sequence encoding a cleavage site; a recombination site; a 3′ untranslated sequence; a transcriptional terminator; and an internal ribosome entry site. In one embodiment, the nucleic acid sequence includes a plurality of cistrons (also termed “open reading frames”), e.g., the sequence is dicistronic or polycistronic. In another embodiment, the nucleic acid also includes a sequence encoding a reporter protein, e.g., a protein whose abundance can be quantitated and can provide an indication of the quantity of test polypeptide fixed to the plate. The reporter protein can be attached to the test polypeptide, e.g., covalently attached, e.g., attached as a translational fusion. The reporter protein can be an enzyme, e.g., &bgr;-galactosidase, chloramphenicol acetyl transferase, &bgr;-glucuronidase, and so forth. The reporter protein can produce or modulate light, e.g., a fluorescent protein (e.g., green fluorescent protein, variants thereof, red fluorescent protein, variants thereof, and the like), and luciferase.
The transcription promoter can be a prokaryotic promoter, a eukaryotic promoter, or a viral promoter. In a preferred embodiment, the promoter is the T7 RNA polymerase promoter. The regulatory components, e.g., the transcription promoter, can vary among nucleic acids at different addresses of the plurality. For example, different promoters can be used to vary the amount of polypeptide produced at different addresses.
In one embodiment, the nucleic acid also includes at least one site for recombination, e.g., homologous recombination or site-specific recombination, e.g., a lambda att site or variant thereof; a lox site; or a FLP site. In a preferred emb

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