|Publication number||US20060057573 A1|
|Application number||US 10/504,696|
|Publication date||Mar 16, 2006|
|Filing date||Feb 10, 2003|
|Priority date||Feb 15, 2002|
|Also published as||CA2476309A1, EP1481090A1, EP1481090A4, US8071288, US20080160535, WO2003070984A1|
|Publication number||10504696, 504696, PCT/2003/4142, PCT/US/2003/004142, PCT/US/2003/04142, PCT/US/3/004142, PCT/US/3/04142, PCT/US2003/004142, PCT/US2003/04142, PCT/US2003004142, PCT/US200304142, PCT/US3/004142, PCT/US3/04142, PCT/US3004142, PCT/US304142, US 2006/0057573 A1, US 2006/057573 A1, US 20060057573 A1, US 20060057573A1, US 2006057573 A1, US 2006057573A1, US-A1-20060057573, US-A1-2006057573, US2006/0057573A1, US2006/057573A1, US20060057573 A1, US20060057573A1, US2006057573 A1, US2006057573A1|
|Inventors||Larry Gold, Jonathan Smith, Dominic Zichi, Daniel Schneider, Chad Greef|
|Original Assignee||Somalogic, Inc|
|Export Citation||BiBTeX, EndNote, RefMan|
|Referenced by (19), Classifications (21), Legal Events (1)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The invention is directed towards nucleic acid ligands, methods of characterizing nucleic acid ligands, and methods and reagents for detecting target binding to nucleic acid ligands.
The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic Acid Ligands” each of which is specifically incorporated by reference herein. Each of these patents and applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX process-identified nucleic acid ligand is a specific ligand of a given target compound or molecule.
The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX process applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX process includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
One particularly important embodiment of the SELEX process is described in U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, and U.S. patent application Ser. No. 08/443,959 filed May 18, 1995, both entitled “Photoselection of Nucleic Acid Ligands,” and both now abandoned, and U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, WO 95/08003, U.S. Pat. No. 6,291,184, U.S. Pat. No. 6,458,539, and U.S. patent application Ser. No. 09/723,718, filed Nov. 28, 2000, each of which is entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” and each of which describe a SELEX process-based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. The resulting nucleic acid ligands are referred to interchangeably as “photocrosslinking nucleic acid ligands” and “photoaptamers.” These patents and patent applications are referred to in this application collectively as “the PhotoSELEX Process Applications.” In the photoSELEX process embodiment of the SELEX process, a modified nucleotide activated by absorption of light is incorporated in place of a native base in either RNA- or in ssDNA-randomized oligonucleotide libraries. One such photoreactive nucleotide whose photochemistry is particularly well-suited for this purpose is 5-bromo-2′-deoxyuridine (5-BrdU) (Meisenheimer and Koch (1997) Crit. Rev. Biochem. Mol. Biol. 32:101-140). The 5-BrdU chromophore absorbs ultraviolet (UV) light in the 310 nm range where native chromophores of nucleic acids and proteins do not absorb or absorb very weakly. The resulting excited singlet state intersystem crosses to the lowest triplet state which specifically crosslinks with aromatic and sulfur-bearing amino acid residues of a protein target in suitable proximity (Dietz and Koch (1987) Photochem. Photobiol. 46:971-8; Dietz and Koch (1989) Photochem. Photobiol. 49:121-9; Dietz et al. (1987) J. Am. Chem. Soc. 109:1793-1797; Ito et al. (1980) J. Am. Chem. Soc. 102:7535-7541; Swanson et al. (1981) J. Am. Chem. Soc. 103:1274-1276). Crosslinking may also occur via excitation of an aromatic residue of the protein in proximity to the bromouracil chromophore (Norris et al. (1997) Photochem. Photobiol. 65:201-207). Of particular importance, excited bromouracil in DNA is relatively unreactive in the absence of a proximal, oriented, reactive amino acid (Gott et al. (1991) Biochemistry 30:6290-6295; Willis et al. (1994) Nucleic Acids Res. 22:4947-4952; Norris et al. (1997) Photochem. Photobiol. 65:201-207) or nucleotide residue (Sugiyama et al. (1990) J. Am. Chem. Soc. 112:6720-6721; Cook and Greenberg (1996) J. Am. Chem. Soc. 118:10025-10030). The importance of orientation is evident in crystal structures of protein-nucleic acid complexes which show a lock and key arrangement of the bromouracil chromophore with the aromatic amino acid residue to which it crosslinks (Horvath et al. (1998) Cell 95:963-974; Meisenheimer and Koch (1997) Crit. Rev. Biochem. Mol. Biol. 32:101-140).
In a basic embodiment, the photoSELEX process comprises the following steps:
The photoSELEX process produces nucleic acid ligands that are single- or double-stranded RNA or DNA oligonucleotides. A photoreactive group may comprise a natural nucleic acid residue with a relatively simple modification that confers increased reactivity or photoreactivity to the nucleic acid residue. Such modifications include, but are not limited to, modifications at cytosine exocyclic amines, substitution with halogenated groups, e.g., 5′-bromo- or 5′-iodo-uracil, modification at the 2′-position, e.g., 2′-amino (2′-NH2) and 2′-fluoro (2′-F), backbone modifications, methylations, unusual base-pairing combinations and the like. For example, photocrosslinking nucleic acid ligands produced by the photoSELEX process can include a photoreactive group selected from the following: 5-bromouracil (BrU), 5-iodouracil (IU), 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuridine, 8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine. Preferably, the photoreactive group will absorb light in a spectrum of the wavelength that is not absorbed by the target or the non-modified portions of the oligonucleotide. In preferred embodiments of the photoSELEX process, the photoreactive nucleotides incorporated into the photocrosslinking nucleic acid ligands are 5-bromo-2′-deoxyuridine (5-BrdU) and 5-iodo-2′-deoxyuridine (5-IdU). These nucleotides can be incorporated into DNA in place of thymidine nucleotides.
Photocrosslinking nucleic acid ligands produced by the photoSELEX process have particular utility in diagnostic or prognostic medical assays. In one such embodiment, photocrosslinking nucleic acid ligands of targets implicated in disease are attached to a planar solid support in an array format, and the solid support is then contacted with a biological fluid to be analyzed for the presence or absence of the targets. The photocrosslinking nucleic acid ligands are photoactivated and the solid support is washed under very stringent, aggressive conditions (preferably under conditions that denature nucleic acids and/or proteins) in order to remove all non-specifically bound molecules. Bound target is not removed because it is covalently crosslinked to nucleic acid ligand via the photoreactive group. The ability to photocrosslink, followed by stringent washing, allows diagnostic and prognostic assays of unparalleled sensitivity and specificity to be performed. Arrays (also commonly referred to as “biochips” or “microarrays”) of nucleic acid ligands, including photocrosslinking nucleic acid ligands and aptamers, and methods for their manufacture and use, are described in U.S. Pat. No. 6,242,246, U.S. patent application Ser. No. 08/211,680, filed Dec. 14, 1998, now abandoned, WO 99/31275, U.S. patent application Ser. No. 09/581,465, filed Jun. 12, 2000, U.S. Pat. No. 6,503,715, and U.S. Pat. No. 6,458,543, each of which is entitled “Nucleic Acid Ligand Diagnostic Biochip.” These patents and patent applications are referred to collectively as “the biochip applications,” and are each specifically incorporated herein by reference in their entirety.
Automated methods and apparatus for the generation of photocrosslinking nucleic acid ligands are provided in U.S. patent application Ser. No. 09/993,294, filed Nov. 21, 2001, U.S. patent application Ser. No. 09/815,171, filed Mar. 22, 2001, U.S. patent application Ser. No. 09/616,284, filed Jul. 14, 2000, U.S. patent application Ser. No. 09/356,233, filed Jul. 16, 1999, U.S. patent application Ser. No. 09/232,946, filed Jan. 19, 1999, each of which is entitled “Method and Apparatus for the Automated Generation of Nucleic Acid Ligands.” Given the rapidity with which these highly parallel, automated methods can generate photocrosslinking nucleic acid ligands, it is desirable to have multiplexed methods for evaluation of the specificity and dose-response characteristics of those photocrosslinking nucleic acid ligands. The present invention includes such methods.
In one aspect, the invention provides a method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule is a protein, the method comprising:
In preferred embodiments, step d) is accomplished by exposing said biochip to conditions that denature nucleic acids and/or proteins.
Suitable detectable moieties include, but are not limited to, dyes (including fluorophores), enzymes (including, but not limited to, alkaline phosphatase and horseradish peroxidase), enzyme substrates, and radiolabels.
In preferred embodiments, at least one of said UPS reagents reacts with a group found on a protein, including but not limited to primary amines (preferably on lysine residudes), thiols, alcohols (including, but not limited to, alcohols groups on serine, threonine, tyrosine, and sugar moities on glycoproteins), and carboxylates.
In some embodiments, the UPS comprises an N-hydroxysuccinimide-activated dye, most preferably an N-hydroxysuccinimide-activated fluorophore, including but not limited to NHS-ALEXA fluorophores.
In other embodiments, the UPS comprises CBQCA (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde).
In still further embodiments, the UPS comprises a reagent that bears an amine reactive group selected from the list consisting of isocyanates, isothiocyanates, acyl azides, sulfonyl chlorides, aldehydes, 4-sulfo-2,3,5,6-tetrafluorophenol (STP) esters, NBD (7-nitrobenz-2-oxa-1,3-diazole) chloride, NBD fluoride, and dichlorotriazines.
In still further embodiments, the UPS comprises a biotin derivative capable of reacting with primary amines and a streptavidin derivatized with said detectable moiety.
In even further embodiments, the UPS comprises: a first biotin derivative capable of reacting with primary amines, streptavidin, and a second biotin derivative conjugated to said detectable moeity.
In yet further embodiments, the UPS comprises: 2-iminothiolane and a thiol-reactive derivative of a dye, preferably a maleimide derivative of a dye.
In still further embodiments, the UPS comprises: a hapten derivative capable of reacting with primary amines and an anti-hapten antibody conjugated to said detectable moiety. Alternatively, the detectable moiety may be conjugated to a secondary antibody that recognizes the anti-hapten antibody.
In further embodiments, the UPS comprises: a reagent that modifies amino acid side chains and an antibody that specifically recognizes said modified amino acid side chain. In this embodiment, the antibody may be conjugated to the detectable moiety. Alternatively, the detectable moiety may be conjugated to a secondary antibody that recognizes the anti-modified side chain antibody. Suitable reagents for the modification of amino acid side chains according to this embodiment include, but are not limited to nitrosylating agents (such as tetranitromethane) and acetylating agents (such as sulfo-N-hydroxysuccinimide acetate). Nitrosylated proteins may be recognized by anti-nitro tyrosine antibodies; acetylated proteins may be recognized by anti-acetylated lysine antibodies.
The UPS reagents and methods provided herein are especially useful in embodiments where multiplexed assays are performed using a biochip (also referred to as an “array” or “microarray”). In one such embodiment, the invention provides a method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule to be detected is a protein, the method comprising:
In a related embodiment, the invention provides a method for detecting the presence of a target molecule suspected of being contained in a test mixture, wherein said target molecule be to detected is a protein, the method comprising;
Using these methods, a single UPS can be used to detect all target proteins that have bound to nucleic acid ligands (both photocrosslinking and non-photocrosslinking) on the array.
In another aspect, the invention provides a biochip comprising an array of a plurality of nucleic acid ligands attached to a solid support, wherein a plurality of said nucleic acid ligands are specifically associated with a target molecule through non-Watson-Crick interactions, and wherein said target molecules are labeled with a detectable moiety.
In another aspect, the invention provides a method for simultaneously measuring the dose-response characteristics of a plurality of species of photocrosslinking nucleic acid ligands, each said species of photocrosslinking nucleic acid ligands having specific affinity for a cognate target protein, the method comprising:
Preferably, each said target protein is absent from at least one of the target protein mixtures. The target protein concentration profiles are preferably further configured such that for each pairwise combination of said cognate target proteins, at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least one order of magnitude higher than the second member of the pairwise combination and at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least one order of magnitude lower than the second member of the pairwise combination. More preferably, the target protein concentration profiles are configured such that for each pairwise combination of said cognate target proteins, at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least two orders of magnitude higher than the second member of the pairwise combination and at least one target protein mixture comprises the first member of the pairwise combination at a concentration that is at least two orders of magnitude lower than the second member of the pairwise combination.
The methods provided by the invention allow one simultaneously to: (1) evaluate the performance (dynamic range) of a plurality of photocrosslinking nucleic acid ligands; and (2) assess the specificity of each photocrosslinking nucleic acid ligand for its cognate target protein. Photocrosslinking nucleic acid ligands with the most desirable properties can then be selected for use in diagnostic and prognostic medical assays.
In another aspect, the invention provides a method for attaching a nucleic acid ligand to a solid support comprising:
In yet another aspect, the present invention provides a photocrosslinking nucleic acid ligand to Human Immunodeficiency Virus (HIV) gp120MN.
Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided:
As used herein, “nucleic acid ligand” is a non-naturally occurring nucleic acid having a desirable action on a target. Nucleic acid ligands are also sometimes referred to in this application as “aptamers.” A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule. In the preferred embodiment, the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule. Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.
As used herein, “candidate mixture” is a mixture of nucleic acids of differing sequence from which to select a desired ligand. The source of a candidate mixture can be from naturally-occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques. Modified nucleotides, such as nucleotides with photoreactive groups, can be incorporated into the candidate mixture. In addition, a candidate mixture can be produced by a prior SELEX process e.g., a first SELEX process experiment can be used to produce a ligand-enriched mixture of nucleic acids that is then used as the candidate mixture in a second SELEX process experiment. A candidate mixture can also comprise nucleic acids with one or more common structural motifs. For example, U.S. Provisional Patent Application Ser. No. 60/311,281, filed Aug. 9, 2001, entitled “Nucleic Acid Ligands With Intramolecular Duplexes” and incorporated herein by reference in its entirety, describes candidate mixtures comprising nucleic acids with intramolecular duplexes formed between their 5′ and 3′ ends.
In a preferred embodiment, each nucleic acid has fixed sequences surrounding a randomized region to facilitate the amplification process. As detailed in the automated SELEX process applications, the candidate mixture nucleic acids can further comprise fixed “tail” sequences at their 5′ and 3′ termini to prevent the formation of high molecular weight contaminants of the amplification process.
As used herein, “nucleic acid” means either DNA, RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3′ and 5′ modifications such as capping.
“SELEX” methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. The SELEX methodology is described in the SELEX Patent Applications. In some embodiments of the SELEX process, aptamers that bind non-covalently to their targets are generated. In other embodiments of the SELEX process, aptamers that bind covalently to their targets are generated.
As used herein, “Universal Protein Stain” or “UPS” refers to a reagent or reagents that labels proteins and not nucleic acids with a detectable moiety.
As used herein, “target protein concentration profile” refers to the description of the concentrations of individual target proteins present in a mixture of said target proteins. In preferred embodiments of the invention, for a particular collection of target proteins a plurality of target protein mixtures is produced, each mixture comprises a unique target protein concentration profile. If a specific target protein in the collection is absent from one of the mixtures, then the target protein concentration profile for that mixture will include the value 0 M for that target protein.
“SELEX target” or “target molecule” or “target” refers herein to any compound upon which a nucleic acid can act in a predetermined desirable manner. A SELEX target molecule can be a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell, tissue, etc., without limitation. Virtually any chemical or biological effector would be a suitable SELEX target. Molecules of any size can serve as SELEX targets. A target can also be modified in certain ways to enhance the likelihood of an interaction between the target and the nucleic acid. Embodiments of the SELEX process in which the target is a peptide are described in U.S. patent application Ser. No. 09/668,602, filed Sep. 22, 2000, entitled “Modified SELEX Processes Without Purified Protein,” incorporated herein by reference in its entirety.
“Tissue target” or “tissue” refers herein to a certain subset of the SELEX targets described above. According to this definition, tissues are macromolecules in a heterogeneous environment. As used herein, tissue refers to a single cell type, a collection of cell types, an aggregate of cells, or an aggregate of macromolecules. This differs from simpler SELEX targets which are typically isolated soluble molecules, such as proteins. In the preferred embodiment, tissues are insoluble macromolecules which are orders of magnitude larger than simpler SELEX targets. Tissues are complex targets made up of numerous macromolecules, each macromolecule having numerous potential epitopes. The different macromolecules which comprise the numerous epitopes can be proteins, lipids, carbohydrates, etc., or combinations thereof. Tissues are generally a physical array of macromolecules that can be either fluid or rigid, both in terms of structure and composition. Extracellular matrix is an example of a more rigid tissue, both structurally and compositionally, while a membrane bilayer is more fluid in structure and composition. Tissues are generally not soluble and remain in solid phase, and thus partitioning can be accomplished relatively easily. Tissue includes, but is not limited to, an aggregate of cells usually of a particular kind together with their intercellular substance that form one of the structural materials commonly used to denote the general cellular fabric of a given organ, e.g., kidney tissue, brain tissue. The four general classes of tissues are epithelial tissue, connective tissue, nerve tissue and muscle tissue.
Examples of tissues which fall within this definition include, but are not limited to, heterogeneous aggregates of macromolecule such as fibrin clots which are acellular; homogeneous or heterogeneous aggregates of cells; higher ordered structures containing cells which have a specific function, such as organs, tumors, lymph nodes, arteries, etc.; and individual cells. Tissues or cells can be in their natural environment, isolated, or in tissue culture. The tissue can be intact or modified. The modification can include numerous changes such as transformation, transfection, activation, and substructure isolation, e.g., cell membranes, cell nuclei, cell organelles, etc. Sources of the tissue, cell or subcellular structures can be obtained from prokaryotes as well as eukaryotes. This includes human, animal, plant, bacterial, fungal and viral structures.
As used herein, “solid support” is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, plastics, paramagnetic beads, charged paper, nylon, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold and silver. Any other material known in the art that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces, grooved surfaces, and cylindrical surfaces e.g., columns. Multiple nucleic acid ligands, each specific for a different target, may be attached to specific locations (“addresses”) on the surface of a solid support in an addressable format to form an array, also referred to as a “microarray” or as a “biochip.” By way of non-limiting example only, an array may be formed with a planar solid support, the surface of which is attached to nucleic acid ligands. By way of non-limiting example only, an array may be also be formed by attaching nucleic acid ligands to beads, and then placing the beads in an array format on another solid support, such as a microtiter plate.
“Partitioning” means any process whereby ligands bound to target molecules can be separated from nucleic acids not bound to target molecules. More broadly stated, partitioning allows for the separation of all the nucleic acids in a candidate mixture into at least two pools based on their relative affinity to the target molecule. Partitioning can be accomplished by various methods known in the art. Nucleic acid-protein pairs can be bound to nitrocellulose filters while unbound nucleic acids are not. Columns which specifically retain nucleic acid-target complexes can be used for partitioning. For example, oligonucleotides able to associate with a target molecule bound on a column allow use of column chromatography for separating and isolating the highest affinity nucleic acid ligands. Beads upon which target molecules are conjugated can also be used to partition nucleic acid ligands in a mixture. If the beads are paramagnetic, then the partitioning can be achieved through application of a magnetic field. Surface plasmon resonance technology can be used to partition nucleic acids in a mixture by immobilizing a target on a sensor chip and flowing the mixture over the chip, wherein those nucleic acids having affinity for the target can be bound to the target, and the remaining nucleic acids can be washed away. Liquid-liquid partitioning can be used as well as filtration gel retardation, and density gradient centrifugation.
As used herein, “PhotoSELEX” is an acronym for Photochemical Systematic Evolution of Ligands by EXponential enrichment, and refers to embodiments of the SELEX process in which photocrosslinking nucleic acid ligands (also referred to as “photoaptamers” or “photocrosslinking aptamers”) are generated. In the photoSELEX process, a photoreactive nucleotide activated by absorption of light is incorporated in place of a native base in either RNA- or in ssDNA-randomized oligonucleotide libraries, the nucleic acid target molecule mixture is irradiated causing some nucleic acids incorporated in nucleic acid-target molecule complexes to crosslink to the target molecule via the photoreactive functional groups, and the selection step is a selection for photocrosslinking activity. The photoSELEX process is described in great detail in the PhotoSELEX Process Applications.
The SELEX Patent Applications and the PhotoSELEX Process Applications describe and elaborate on the aforementioned processes in great detail. Included are targets that can be used; methods for the preparation of the initial candidate mixture; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixtures. The SELEX Patent Applications and the PhotoSELEX Process Applications also describe ligand solutions obtained to a number of target species, including protein targets wherein the protein is or is not a nucleic acid binding protein.
Note that throughout this application, various publications, publications, and patent applications are mentioned; each is incorporated by reference to the same extent as if each was specifically and individually incorporated by reference.
Multiplexed Evaluation of Photocrosslinking Nucleic Acid Ligands
In one embodiment of the invention, photocrosslinking nucleic acid ligands that are identified as possessing potentially useful affinity and photocrosslinking activity are rapidly assayed using a multiplexed assay on a nucleic acid ligand array, also referred to herein interchangeably as a “microarray” or “biochip.” Nucleic acid ligands can be immobilized onto a wide variety of solid support surfaces that can be used in assay protocols including, but not limited to, the following formats: microtitre wells, microscope slides, silicon wafer chips, flow through chips, and microbeads. Methods for immobilizing nucleic acids on solids supports are well known in the art.
For each protein target, one or more identified photocrosslinking nucleic acid ligands can be immobilized to discrete addresses on the array. A plurality of identical arrays is preferably constructed, and then each individual array can be contacted with the analyte solution (for example, a mixture of protein targets (also herein referred to as a “cocktail”) or a biological fluid, including, but not limited to, serum, tissue culture supernatant, urine, and tissue homogenate). The array can be used to either (1) test the multiplexed performance of the nucleic acid ligand; or (2) assay a sample for analyte protein concentration. If the arrays are used to test multiplexed performance of the nucleic acid ligand, then each protein target for which a corresponding photocrosslinking nucleic acid ligand is present on the array is included in the target protein mixtures that are incubated with the array.
Following the incubation of the analyte with the microarray, the photocrosslinking nucleic acid ligands are photocrosslinked to their cognate protein targets, and the arrays are washed under stringent conditions in order to remove non-specifically bound proteins. Protein binding by the arrays can the be quantified by using a Universal Protein Stain (UPS) as described below in the section entitled “Universal Protein Stains.” Examples 3 and 4 provide exemplary methods for processing the multiplexed arrays.
The mutiplex assays described herein allow a two-fold evaluation of photocrosslinking nucleic acid ligands by allowing one to: (1) evaluate the performance (dynamic range) of a photocrosslinking nucleic acid ligand in the array format and (2) assess its specificity for the cognate target protein. If the arrays are used to assess the performance of nucleic acid ligands, mixtures of target proteins can be produced, each mixture having a different target protein concentration profile, and the mixtures may be used as samples to contact the arrays. By varying the absolute concentrations of each protein, it is possible to obtain a dose-response curve for each photocrosslinking nucleic acid ligand. It is possible to evaluate simultaneously the specificity of each photocrosslinking nucleic acid ligand. The multiplexed assays can also be performed in the presence and absence of serum in order to further screen for non-specific interactions. In this way, it is possible to evaluate simultaneously many photocrosslinking nucleic acid ligands on a single series of arrays. Photocrosslinking nucleic acid ligands with the most desirable properties can then be selected for use in diagnostic and prognostic medical assays.
The multiplexed assays of the invention allow for dramatic streamlining of the photocrosslinking nucleic acid ligand evaluation process, saving both time and resources. For example, running separate assays for each photocrosslinking nucleic acid ligand would require, say, 10 measurements each in buffer and serum. To evaluate 5 photocrosslinking nucleic acid ligands to each of 100 different proteins would require 2×10×5×100=10,000 experiments. A multiplex assay can simultaneously measure 500 photocrosslinking nucleic acid ligand responses to target protein mixtures (“cocktails”). Measurement of 10 target protein mixtures, each having a specific target protein concentration profile, both in buffer and serum, requires only 20 experiments and will provide the same data as 10,000 experiments plus yield additional information about specificity.
In one non-limiting example of this embodiment of the invention, the dynamic ranges of a single photocrosslinking nucleic acid ligand is assessed by collecting data for 9 different protein concentrations in target protein mixtures starting at 10−6 M and decreasing a log in concentration to 10−14 M. A tenth target protein mixture will contain no cognate protein i.e., the target protein concentration profile for that mixture will be 0 M. It will be apparent to those skilled in the art that the exact number and ranges of these can be adjusted for more suitable limits. For a highly multiplexed assay, it may be desirable to evaluate the dynamic range of 100 or more different proteins simultaneously, so at least ten target protein mixtures are required as in the single ligand case. For example, for photocrosslinking nucleic acid ligands with negligible non-specific binding, one can make ten target protein mixtures where the first ten target proteins are absent in the first target protein mixture, the next ten target proteins are present at the lowest concentration in the range and so on, with the last ten target proteins at the highest concentration. The next target protein mixture could then have the first ten target proteins at the lowest concentration, the next ten at the second lowest, and so on, with the last 10 target proteins absent in this target protein mixture. Repeating this pattern eight more times would result in 10 complex target protein mixtures, each having a unique target protein concentration profile. Each target protein would sample the entire concentration range desired. Although this strategy could potentially measure the dynamic range of 100 different protein targets, a different protein distribution strategy will simultaneously yield specificity data as well.
Non-specific binding/crosslinking and interference is a possibility in multiplexed assays, although the unique characteristics of photocrosslinking nucleic acid ligands reduce this to an extremely low level compared to other reagents used in diagnostic chip-based assays. In order to minimize further the likelihood of such effects in multiplexed assays, it is desirable to detect these effects and eliminate those photocrosslinking nucleic acid ligands that demonstrate crosslinking to noncognate proteins (or at least minimize such effects by proper photocrosslinking nucleic acid ligand choice). In one embodiment of the invention, this may be achieved by appropriate configuration of the target protein concentration profiles of the individual target protein mixtures. Since any protein in a target protein mixture may affect the signal of any other protein, in preferred embodiments each pairwise combination of target proteins is tested in varying concentrations across all of the protein target mixtures—some high with respect to the first protein and some high with respect to the second since photocrosslinking nucleic acid ligand cross-reactivity is certainly not symmetric. For example, for ten proteins 1-10, the pairwise combinations for protein 1 would be: [1,2], [1,3), [1,4] . . . [1,10]. In especially preferred embodiments, each protein pair differs by plus and minus at least one log of concentration (i.e. at least one order of magnitude higher and lower) in some number of the target protein mixtures, more preferably by plus and minus at least two logs of concentration, and even more preferably by plus and minus at least three logs of concentration. For example, with reference to the aforementioned pairwise combination members 1 and 2, in at least one target protein test mixture, protein 1 would be present at a concentration that is two orders of magnitude higher than protein 2; and in at least one target protein mixture, protein 1 would be present at a concentration that is at least two orders of magnitude lower than protein 2. This will ensure that one can detect cross-reactivity in the evaluation assays. In addition, this constraint on target protein mixture composition will also require that no two proteins have the same concentration in any two target protein mixtures.
Consider ten proteins 1-10 present at ten different concentrations in ten target protein mixtures A-J. For the first protein there are 10!=3628800 ways to distribute the ten concentrations among ten target protein mixtures. There are 10!-1 ways for the second since it cannot have the same distribution among the protein mixtures as the first, and so on. A key aspect of configuring the target protein concentration profiles should be related to maximizing the difference in distributions for each of the 100 proteins, as described above. Table 1 below enumerates exemplary target protein concentration profiles in the ten resulting target-protein mixtures A-J. For example protein 1 is absent from the mixture A, has a concentration of 10−11 in the mixture B, 10−9 in the mixture C, etc. The target protein concentration profiles displayed in the table ensures that each protein pair has concentrations separated by three logs, both plus and minus, at least twice in the ten target protein mixtures. For instance, in mixture A, protein 6 is at least three logs less than proteins 10, 8, 9, 2, 7, and 5. In mixture E protein 6 is at least three logs higher than proteins 5, 10, 8, 7, and 4.
TABLE 1 Exemplary target protein concentration profiles Target Protein Concentration Profiles (M) Mixture 0 10−14 10−13 10−12 10−11 10−10 10−9 10−8 10−7 10−6 A 1 6 4 3 10 8 9 2 7 5 B 2 7 5 4 1 9 10 3 8 6 C 3 8 6 5 2 10 1 4 9 7 D 4 9 7 6 3 1 2 5 10 8 E 5 10 8 7 4 2 3 6 1 9 F 6 1 9 8 5 3 4 7 2 10 G 7 2 10 9 6 4 5 8 3 1 H 8 3 1 10 7 5 6 9 4 2 I 9 4 2 1 8 6 7 10 5 3 J 10 5 3 2 9 7 8 1 6 4
Out of 3,628,800 possible target protein concentration profiles, it is possible to find 100 that provide the best chance of revealing cross-reactivity of proteins for non-cognate photocrosslinking nucleic acid ligands. These distributions are generated using Monte Carlo techniques, by randomly sampling distributions and accepting only those that satisfy the criteria of having multiple comparisons between each pair of proteins. Maximizing the number of times one measures a cognate protein in the presence of excess “background” proteins will provide a reasonable test of specificity. In the absence of significant cross-reactivity a reasonable standard curve will be generated. Spikes in these curves at low cognate protein levels, or losses in sensitivity will be indicative of cross-reactivity.
In the absence of any non-specific binding or interference, multiplexed evaluation assays performed according to the aforementioned methods will produce standard curves for each photocrosslinking nucleic acid ligand, wherein each curve will reveal the dynamic range of the photocrosslinking nucleic acid ligand. In order to determine the effect of non-specific binding, the present invention now provides a model to evaluate the fraction of the complex mixture that binds to each photocrosslinking nucleic acid ligand.
The model is based on a standard treatment of equilibrium binding according to the following equation:
P i +L j ⇄P i :L j K ij =[P i :L j ]/[P i ][L j] (1)
where [Pi] is the concentration of unbound protein analyte i (i varies from 1 to 100) in the sample, [Lj] is the concentration of unbound photocrosslinking nucleic acid ligand j (varies from 1 to 500, i.e., there are five photocrosslinking nucleic acid ligands for each protein measured in one assay) on the chip, [Pi: Lj] is the concentration of immobilized protein/photocrosslinking nucleic acid ligand complex, and Kij is the equilibrium association constant for the reaction. If there was no nonspecific binding the matrix of association constants K is diagonal for a protein chip in which there is only one photocrosslinking nucleic acid ligand per protein; off diagonal entries indicate cross-reactivity and most likely have association constants several logs lower than the specific (diagonal) interaction term.
The mass balance equations, in terms of total protein and photocrosslinking nucleic acid ligand concentrations, are
[P i]total =[P i]+Σj [P i :L j ]=[P i](1+Σj K ij [L j]) (2)
[L j]total =[L j]+Σi(P i :L j ]=[L j](1+Σi K ij [P i]) (3)
where the second equality is obtained by substituting [Pi]Kij[Lj] for [Pi: Lj] (see Eq. (1)). Substitution of Eq. (3) into Eq. (2) yields a system of equations for the free protein concentrations
[P i ]=[P i]total(1+Σj K ij [L j]total/(1+Σk K kj [P k]))−1 (4)
The system of equations can be solved iteratively to self-consistency in the free protein concentrations [Pi]. The concentrations of the bound photocrosslinking nucleic acid ligands are then calculated with the following
[P i :L j ]=[P i ]K ij [L j]total/(1+Σk K kj [P k]))−1 (5)
The washing and crosslinking will reduce the amount of protein detected from that originally bound. The fraction of loss due to washing will depend on the off rate of the individual complexes; high affinity interactions with slow off rates will lose little specific bound protein whereas non-specific binders should be substantially reduced. The crosslinking efficiencies for aptamers recovered from an enriched pool will certainly vary from 0-100%; it is possible to eliminate the non-crosslinking photocrosslinking nucleic acid ligands since they will not remain on the array following the harsh washing. Finally, each protein will produce a signal proportional to the number of stain molecules that specifically bind to the captured proteins, for example, as a function of the number of lysines contained in the protein and their reactivity.
For the following discussion, washing and crosslinking losses, as well as the stain signal enhancement will be treated as a factor unique to each complex. The final signal measured for each photocrosslinking nucleic acid ligand on the array is given as
RF j=Σl f ij [P i :L j ]+b (6)
where RFj is the relative fluorescence measured for photocrosslinking nucleic acid ligand j, fij accounts for the washing, crosslink efficiency, and staining of each protein/photocrosslinking nucleic acid ligand complex Pi: Lj, and b is the instrument background that sets the absolute lower limit of detection.
This completes the model. Using the ten target protein mixtures defined above as an example, one can explore the proposed assay qualitatively. To illustrate, consider one photocrosslinking nucleic acid ligand at a concentration of 10−11 M with a target protein specific interaction Kij=1011 M−1 (Kd=10 pM) and fij/b=5.0×1013 (this is somewhat arbitrary and only affects the lower limit of detection—saturation of the photocrosslinking nucleic acid ligand sets the upper limit here).
To explore the behavior of the response in the presence of a single non-specific interaction, nine curves were generated, one for each of the other proteins in the target protein mixtures; only one protein was allowed to cross-react in each simulation. For the same photocrosslinking nucleic acid ligand as above, the nonspecific interaction was set three logs less than the specific one, Kij=108 M−1 (Kd=10 nM), and fij/b=5.0×1012, ten percent of the specific. The same target protein mixtures were used in each calculation.
Since the data are plotted on a log/log scale the deviations from linearity due to the non-specific interactions are usually quite apparent. Two curves display deviations that may be difficult to discern in the presence of noise, but that is entirely a consequence of the target protein mixtures used here; further enhancements can be achieved by a more careful choice of protein mixtures.
The model provided herein shows that the dynamic range and potential non-specific interactions of photocrosslinking nucleic acid ligands in a multiplexed evaluation assay can be obtained from a set of 10 measurements. It is important to note that there is no restriction on using ten target protein mixtures—twenty could be used to ensure more comparisons at low levels of cognate protein to every other protein in the mixture to maximize the detection of non-specific interactions. Doubling the number of experiments would not compromise the efficiency of the approach. Crossreactivity of photocrosslinking nucleic acid ligands is assessed for every target protein in the target protein mixtures.
Many surface attachment chemistries may be used for nucleic acid ligand immobilization including, but not limited to: thiol-modified nucleic acid ligands bound to thiol-reactive surfaces including gold; acrydite-modified nucleic acid ligands bound to thiol containing surfaces; biotinylated nucleic acid ligands bound to streptavidin surfaces; amine-modified nucleic acid ligands bound to carboxylate, isothiocyanate, N-hydroxy-succinimide, or epoxide-activated surfaces. A wide variety of surface coatings have been demonstrated for nucleic acid ligand arrays including: epoxide on glass, epoxide on silicon, Accelr8 N-hydroxy-succinimide-activated organic polymer, Surmodics N-hydroxy-succinimide-activated acrylamide polymer, Rosatech amine-reactive organic polymer, gold coated with organic self-assembled monolayers (SAMs), and Matrix thiol-containing acrylamide polymer.
Printing (also referred to as “spotting”) buffer components that are routinely evaluated in initial screens include buffer (NaPO4, NaBO4, NaCO3 are commonly used), detergent (sarkosyl, Tween20, and SDS), hydrophilic additives (PEG, Me-PEG, glycerol), and organic solvents (dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N-methylpyrrolidone (NMP)).
Following printing of the nucleic acid ligands onto the modified surface, in preferred embodiments post printing treatments are carried out to eliminate residual functional groups and to modify the environment around the nucleic acid ligand features. Typical post-printing treatments carried out to cap residual functionality on the surface include alkaline washes to hydrolyze labile groups and acetylation of primary amines or free sulfhydryls.
Additional post printing treatments can include the reaction of surface functional groups with alkyl or polyethylene glycol chains to modify the environment around the nucleic acid ligands themselves (see Example 5 below). For example, in some embodiments, poly(ethylene glycol) (PEG) spacers are inserted between the nucleic acid ligands and the surface of the solid support. It has been found by the inventors that PEG spacers promote specific protein binding activity by the nucleic acid ligands in microarrays. Similarly, the use of PEG molecules cospotted with nucleic acid ligands to create an array promotes the specific binding activity of the nucleic acid ligands in microarrays. Without being limited by a single theory or hypothesis, it is believed that the PEG molecule minimizes denaturation and improves specific activity of the nucleic acid ligand by providing a long, flexible, neutrally charged and hydrophilic spacer between each nucleic acid ligand and surface of the array.
Specifically, the conjugation of bifunctional linkers that include, but are not limited to, PEG spacers (and may also include fluoro compounds) to 5′-derivatized nucleic acid ligands facilitates the attachment to the surface, and also moves the nucleic acid ligand to a more hydrophilic environment removed from the substrate itself. Surface-specific inactivation effects are observed for some nucleic acid ligands in the absence of PEG spacers and protein binding activity has been regained when the PEG spacer is used for the nucleic acid ligand immobilization. PEG polymers, co-spotted on the surface, have been used to reduce non specific interactions on various surfaces. Methods and reagents for coupling PEG molecules to nucleic acids and to solid supports are well known in the art. For example, NHS-PEG-vinyl sulfone may be reacted with a 5′ amine derivatized nucleic acid ligand, leading to coupling of the PEG vinyl-sulfone moiety to the nucleic acid ligand. The resulting vinyl sulfone-PEG nucleic acid ligands may then be coupled to a solid support that is labeled with thiol groups.
It will be understood that the aforementioned methods for array synthesis can be used with both photocrosslinking nucleic acid ligands and with non-photocrosslinking nucleic acid ligands.
Example 5 provides an exemplary protocol for PEGylation of nucleic acid ligands.
Photoaptamer Assay Protocols
The present invention provides methods for designing multiplexed assays that employ arrays of photocrosslinking nucleic acid ligands. Exemplary methods for processing those multiplexed arrays are now provided.
In preferred embodiments of the invention, multiplexed detection of protein target binding to an array of photocrosslinking nucleic acid ligands is carried out by first equilibrating the arrays with SELEX buffer, a blocking nucleic acid, such as TRNA, and a blocking protein, such as methylated casein carrier. Protein analyte is incubated with the surface, preferably for enough time to approach equilibrium (either under static conditions or with flow across the array surface). To ensure minimal noncognate protein background, the affinity-bound proteins are preferably washed with buffer, most preferably SELEX buffer (40 mM HEPES, pH 7.5, 111 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.05% TWEEN-20), to remove noncognate, low affinity proteins from the array. In some embodiments, this pre-crosslinking wash is carried out at increased ionic strength to increase the likelihood of disrupting such non-specific interactions.
The array is then exposed to UV light (preferably at 308 nm from a monochromatic light source, or using wavelengths above 312 nm, selected by applying a cutoff filter to a mercury lamp) to photocrosslink nucleic acid ligands that have bound to their cognate protein target. Preferably, crosslinking is carried out with a thin layer of aqueous buffer over the array surface.
Most preferably, the array is then washed harshly under conditions that denature nucleic acids and/or proteins. Suitable harsh conditions are achieved by combinations of, for example, salt (for example 20 mM NaH2PO4, pH 7.4, 150 mM NaCl, 0.1% SDS, 1 mM EDTA), detergent, hydroxide (for example 20 mM NaOH), acid, chaotropic agents (for example 8M urea), heat (for example 40° C.), and flow. The harshness of the washing is limited only by the ability of the organic surface coating to maintain structural integrity. The immobilization chemistries provided above and the photocrosslinks themselves are both stable to such washes.
Following the harsh washing of the slide to remove nonspecifically bound protein, the array is stained with a UPS in order to label all proteins with a detectable moiety. To minimize background signals, in preferred embodiments the array is again washed harshly following the UPS treatments. The array is then “read” by quantifying the signal from the detectable moiety. In preferred embodiments, a standard microarray fluorescent reader with the appropriate excitation source and filters is used.
Universal Protein Stains
A Universal Protein Stain (UPS) comprises a reagent or reagents that label all proteins with a detectable moiety but does not label nucleic acids or other components of an array, such as the derivatized surface used to immobilize the nucleic acid ligands. The detectable moiety may be detected via fluorescent, chemiluminescent signal or any other quantifiable signal depending on its identity. It is preferred, but not required, that at least one of the UPS reagents reacts covalently with the protein. Any reactive chemical group found on proteins, but not found on nucleic acids or the slide substrate, can serve as the site of covalent attachment. In the detection of protein targets, these groups include, but are not limited to, primary amines (lysine), thiols (cysteine, which may be produced by the reduction of disulfide linkages), alcohols (serine, threonine, tyrosine and sugar moieties on glycoproteins (including the products of oxidation of cis diols on such sugars)), and carboxylates (glutamic and aspartic acid).
The detectable moiety may be, without limitation, a dye (most preferably a fluorophore), a radiolabel, a quantum dot, an enzyme, an enzyme substrate, or any other substance that may be used in any manner to generate a quantifiable signal. In the case where the detectable moiety is an enzyme (for example, alkaline phosphatase), the quantifiable signal may be generated in the presence of the enzyme substrate and any additional factors necessary for enzyme activity. In the case where the detectable moiety is an enzyme substrate the quantifiable signal may be generated in the presence of the enzyme and any additional factors necessary for enzyme activity. Suitable reagent configurations for attaching the detectable moiety to the protein include, but are not limited to, covalent attachment of the detectable moiety to the protein, non-covalent association of the detectable moiety with another UPS component that is covalently attached to the proteins, and covalent attachment of the detectable moiety to a UPS component that is non-covalently associated with the protein.
In its most basic embodiment, the UPS is a single chemical reagent that reacts covalently with functional groups unique to proteins and in doing so covalently attaches the detectable moiety to the protein. Preferred UPSs according to this embodiment comprise dyes with groups capable of reacting covalently with functional groups that are unique to proteins. Such groups may be added to the dyes by derivatization, or may be present on the unmodified dye. N-hydroxysuccinimide-activated dyes (also known as NHS-activated dyes) react with amine groups, and are especially preferred UPSs. Another especially preferred UPS is CBQCA (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde) which also reacts with amines in the presence of cyanide or thiols to form highly fluorescent isoindoles. Other amine reactive groups suitable for use in UPS reagents include, but are not limited to, isocyanates, isothiocyanates, acyl azides, sulfonyl chlorides, aldehydes, 4-sulfo-2,3,5,6-tetrafluorophenol (STP) esters, and arylating agents such as NBD (7-nitrobenz-2-oxa-1,3-diazole) chloride, NBD fluoride, and dichlorotriazines.
In some embodiments, the UPS comprises a plurality of reagents. For example, the UPS can comprise a first reagent that reacts covalently with the proteins, and one or more further reagents that attach the detectable moiety, either directly or indirectly, covalently or non-covalently, to the protein via a chemical group or other functionality introduced by the first reagent. For example, in one embodiment a suitable UPS comprises (a) a biotin derivative that reacts with proteins; and (b) a streptavidin-detectable moiety conjugate, for example a fluorescent streptavidin derivative or a streptavidin-enzyme conjugate. The biotin derivative reacts with amine groups, thereby covalently attaching biotin to proteins; the streptavidin-detectable moiety conjugate binds to the immobilized biotin groups, thereby localizing the detectable moiety to sites of protein binding on the array.
In another embodiment, a suitable UPS comprises: i) biotin or a biotin derivative, conjugated to a reactive group that is capable of covalently attaching the biotin or biotin derivative to the bound protein targets; ii) avidin and/or streptavidin; and iii) a biotin-detectable moiety conjugate, for example a fluorescent biotin derivative. Preferably, the biotin derivative in i) is an amine-reactive biotin derivative, most preferably NHS-Biotin, wherein the biotin is optionally separated from the NHS by spacer atoms (Calbiochem, Inc.). Reaction of the NHS group with primary amines on bound protein targets leads to the covalent attachment of biotin to protein targets that are bound to nucleic acid ligands on the array. The array can then be treated with the streptavidin or avidin. Since streptavidin and avidin can each bind four biotins, the addition of these proteins provides three biotin binding sites for each biotin originally coupled to the bound protein target by the NHS-biotin. The biotin-detectable moiety derivative of iii) can then be added, whereupon it binds tightly to the unoccupied biotin binding sites on the streptavidin or avidin.
In another embodiment, the UPS comprises (a) a hapten, such as dinitrophenol (DNP), that is derivatized with a group that reacts with proteins; and (b) an anti-hapten antibody conjugated to a detectable moiety, for example a fluorescent anti-hapten antibody or an enzyme-anti-hapten antibody conjugate. In still further embodiments, the UPS may comprise: (a) a hapten, such as dinitrophenol (DNP), that is derivatized with a group that reacts with proteins; and (b) an anti-hapten antibody; (c) a secondary antibody that binds to the anti-hapten antibody (for example, an antibody preparation that reacts with all immunoglobulins from the animal species used to prepare the anti-hapten antibody), which secondary antibody is conjugated to a detectable moeity.
Where the UPS comprises multiple reagents, those skilled in the art will understand that in some cases the reagents should be added sequentially, whereas in other cases they may be added together.
In another embodiment, the UPS comprises an agent for the reduction of disulfides (e.g., cysteine) to thiol groups, and further comprises a thiol-reactive compound. Suitable thiol-reactive groups that may be used in UPSs include, but are not limited to, iodoacetamides, maleimides, benzylic halides and bromomethylketones. For example, one suitable UPS according to this embodiment comprises (a) a reducing agent; and (b) a detectable moiety derivatized with a thiol-reactive group. Another example of a UPS according to this embodiment comprises (a) a reducing agent; (b) a biotin derivative that reacts with thiol groups; and (c) a streptavidin-detectable moiety conjugate.
In a further embodiment, the UPS comprises an agent for oxidizing cis-diols at protein glycosylation sites, and further comprises a compound bearing a hydrazide reactive group. For example, one suitable UPS according to this embodiment comprises: (a) an oxidizing agent for the oxidation of cis-diols in sugars; (b) a hydrazide derivative of a detectable moiety.
In another embodiment, carboxylates on protein can be reacted with a UPS comprising (a) EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide)/sulfo-NHS(N-hydroxysulfosuccinimide) which reacts with carboxylates to form NHS-ester groups; and (b) a detectable moiety conjugated to a group that reacts with the NHS-ester groups, for example amine group(s) or hydrazide group(s). In one such embodiment, a UPS may comprise: (a) EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide)/sulfo-NHS(N-hydroxysulfosuccinimide); (b) a biotin derivative with an amine group; (c) a streptavidin-enzyme conjugate or fluorescent streptavidin derivative.
Those skilled in the art will recognize that it may be necessary to “block” unreacted functional groups in certain embodiments of the invention. For example, in the example described above involving EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide)/sulfo-NHS(N-hydroxysulfosuccinimide), unreacted NHS-ester groups on the protein may remain after the addition of the bitotin derivative with the amine group; those unreacted NHS-ester groups might then react with the enzyme, leading to its inactivation. Blocking of unreacted NHS-ester groups in this embodiment can be achieved using a small molecule with amine group, such as ethanolamine.
In some embodiments, the UPS comprises a reagent(s) that reacts with a group unique to proteins, including thiol groups produced by reduction of disulfide linkages and oxidized cis diols, and in doing so introduces a functional group into the protein. For example Traut's reagent (2-iminothiolane) and N-succinimidyl 3-[2-pyridyldithio]-propionamido (SPDP) heterobifunctional linker both react with amine groups, thereby introducing thiol groups. The newly-introduced thiol group can then be reacted with other reagents as described above in order to label the protein with a detectable moiety. For example, one suitable UPS according to this embodiment comprises: (a) Traut's reagent; and (b) a dye derivatized with a thiol-reactive group. Many other reagents for derivatizing proteins with functional groups, including homobifunctional linker and heterobifunctional linkers, are well known to those skilled in the art.
To enhance the signal achieved by staining the protein target, in some embodiments amplification may be exploited using a UPS that includes an enzyme as the detectable moiety. Amplification, a consequence of turnover of multiple substrates by each enzyme, yields better signal to noise ratios for protein quantification in systems with minimal general backgrounds. In these embodiments, the quantifiable signal is generated upon addition of the enzyme substrate and any additional factors necessary for enzyme activity. One suitable UPS for labeling proteins with an enzyme comprises (a) a biotin derivatized with a group that reacts with a group unique to proteins; and (b) a streptavidin-enzyme conjugate. Another suitable UPS comprises (a) a hapten derivative that reacts with a group unique to proteins (for example, a dinitrophenol derivative); and (b) an anti-hapten antibody which is conjugated to the enzyme.
One suitable enzyme that may be used in a UPS system according to the embodiment provided above is alkaline phosphatase, particularly when used with the soluble, non-fluorescent substrate 2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF-97 phosphate). Alkaline phosphatase cleaves this substrate to produce the insoluble and fluorescent ELF alcohol which precipitates locally, thereby providing a quantifiable signal at the site of immobilization of the enzyme.
Another suitable enzyme that may be used in a UPS system according to the embodiment described above is horseradish peroxidase (HRP), particularly when used in the tyramide signal amplification (TSA) system. HRP accepts various fluorescently-labeled tyramide derivatives as substrates. The resulting tyramide radical product is highly reactive and forms covalent bonds with localized tyrosine and other aromatic amino acids, leading to a localized fluorescent signal at the sites of protein binding on the array.
In embodiments of the invention that employ a UPS reagent with an N-hydroxy succinimide group, the desired reaction of the NHS group (for example, reaction of a N-hydroxysuccinimide-activated dye with amine groups on protein) competes with hydrolysis of the NHS group. In order to minimize NHS hydrolysis, the NHS reagent is preferably stored in dry DMSO prior to dilution into aqueous buffer, and the diluted reagent is preferably used immediately after dilution. In addition, in preferred embodiments, a dry organic solvent such as dimethylformamide (DMF) may be included in the reaction, thereby allowing more efficient utilization of the NHS group, and consequently more efficient protein labeling. Non-nucleophilic organic bases such as DIEA (diisopropylethylamine) may also be included in the UPS reaction to catalyze the reaction of the lysine amine side chain with N-hydroxysuccinimidyl esters.
Organic solvents have strong protein denaturing properties, because of their ability to solubilize hydrophobic sidechains, and thus maximize the availability of protein sidechains to active UPS reagents that react with groups on those protein sidechains. The identity of the solvent also has large effects on the degree of staining of the solid support on which the nucleic acid ligand array is immobilized, depending on the characteristics of the surface layer of the solid support and properties of the solvent. Thus, in preferred embodiments of the invention, the UPS reaction(s) are carried out in the presence of an organic solvent(s) selected in least in part for its ability to suppress reactivity of the surface layer of the solid support with components of the UPS. For example, the surface reactivity of OptiChem surfaces on Accelr8 slides for NHS-activated dyes under aqueous conditions is suppressed in the polar aprotic organic solvents DMF or DMSO (possibly due to the dehydration and collapsing of the surface gel layer).
It will be appreciated by those skilled in the art that using organic solvents for the UPS reaction(s) according to the methods provided herein can lead to dramatic improvements in the labeling of proteins relative to the same reactions carried out in aqueous solution. Specifically, if a suitable organic solvent(s) is used for the UPS reaction(s), improvements in the stability of the UPS reagent(s), the availability of the protein sidechain groups, and in the suppression of reactivity of the solid support surface layer may be simultaneously realized. For example, the use of organic solvent in conjunction with a UPS comprising a NHS-activated-dye both increases the staining of the protein (by at least 10-fold due to the increased availability of amines in protein sidechains and due to the decreased hydrolysis of the NHS group) and decreases the staining of the slide surface (by approximately 5-fold), thus increasing the sensitivity of the staining protocol.
The sensitivity of a UPS can be limited by background signals due to reaction of the UPS with the surface of the solid support on which the array is immobilized, or with a small fraction of the DNA bases in nucleic acid ligand features. In some embodiments, the UPS includes a reagent that covalently reacts with amino acids, leading to the formation of a modified amino acid side chain that does not occur naturally. The UPS further comprises a reagent, preferably an antibody, that binds to the modified amino acid, but not to the unmodified amino acid. The antibody may be directly conjugated to a detectable moiety, for example, a fluorophore or an enzyme. Alternatively, additional UPS reagents may be added to indirectly localize the detectable moiety to the bound antibody. For example, a fluorescently labeled secondary antibody that recognizes the first antibody may be used.
In one embodiment employing modified amino acids, the UPS comprises tetranitromethane (which nitrosylates tyrosine and other aromatic amino acids) and further comprises an anti-nitro tyrosine antibody. In another embodiment, the UPS comprises sulfo-NHS acetate (which acetylates lysine residues) and further comprises an anti-acetylated lysine antibody.
Examples of UPS reagents are provided in Example 3 and Example 4.
The UPS reagents described herein can be used to detect target protein binding to nucleic acid ligands, including photocrosslinking nucleic acid ligands and non-photocrosslinking nucleic acid ligands. Preferably, the UPS reagents are used to detect target protein binding to nucleic acid ligands that are immobilized on a solid support. In especially preferred embodiments, the UPS reagents are used to detect target protein binding to multiplexed arrays of nucleic acid ligands. In such arrays, target identity is determined by the position (“address”) on the array to which protein binds. In some embodiments using multiplexed arrays, a single UPS can be used to detect the binding of hundreds or thousands of different target proteins.
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
Protocols for Arraying Photocrosslinking Nucleic Acid Ligands on a Solid Support
The following description provides an exemplary and non-limiting method for arraying amine-terminated nucleic acid ligands, including photocrosslinking nucleic acid ligands, on a Surmodics N-hydroxy-succinimide-activated slide surface.
Photoaptamers are prepared for printing by making a 10 μM solution of each nucleic acid ligand in 150 mM NaPO4 pH 8.5, 0.001% Sarkosyl. 15 μL of each aptamer are placed into wells of a 384 well microtiter plate. Controls include 30N12 random DNA, print buffer containing no DNA, and 30N12 random DNA+0.2 μM NH-Model-Cy3 for use as corner markers. The microtiter plate is centrifuged briefly to draw all materials to the bottoms of their wells, and the plate is ready for printing.
The printing process may be carried out using a commercial printer, such as a Packard Gene Array contact spotter according to the following procedure (contact spotter maintained at 22° C. and 62% RH):
More preferably, the printing procedure may be carried out according to the following procedure:
The specific printing protocol varies for each surface used for nucleic acid ligand immobilization.
Microbeads are another substrate for nucleic acid ligand binding. Carboxy-derivatized beads are washed with NaOH, then activated with 250 mM EDC in 100 mM MES (2-(N-Morpholino)-ethanesulfonic acid) buffer at pH 6.0. The activated beads are then treated with 250 mM Sulfo-N-hydroxy-succinimide in 100 mM MES buffer at pH 6.0 for 3 hours at room temperature. The aptamer is coupled to the beads by addition of a solution of 10 μM 5′-amine nucleic acid ligand in 500 mM CTAB (hexadecyltmethylammonium bromide), 2% PEG (8 kDa) in 100 mM bicarbonate buffer at pH 9.0. A variety of bead types has been used as substrates including beads derivatized with carboxy, amine, streptavidin, and epoxide functionality.
Target Incubation, Photocrosslinking, and Detection with UPS
The following exemplary protocol allows the multiplexed detection of protein targets that have bound and become photocrosslinked to their cognate photocrosslinking nucleic acid ligand. The UPS in this example is the NHS-derivative of the fluorophore ALEXA 555 (Molecular Probes, Inc.); however, those skilled in the art will understand that this example is broadly applicable to other UPS reagents, and is not limited to NHS-derivatives.
Comparison of UPSs
Two different photocrosslinking nucleic acid ligands (6.7 and 6.40; see
Using a Surmodics slide with photocrosslinking nucleic acid ligands to bFGF, angiogenin, and endostatin, additional UPS reagents were assayed. The target protein concentrations were 10 nM bFGF, 50 nM endostatin, and 50 nM angiogenin.
Nitrosylation of tyrosine, followed by staining with a anti-nitrosylated tyrosine primary, then followed by staining with a fluorescent secondary antibody was also investigated as a UPS.
Preparation of Vinyl-Sulfone-PEG Nucleic Acid Ligand Conjugates
The preparation of vinyl sulfone-PEG-nucleic acid ligand conjugates for several 5′amine-derivatized nucleic acid ligands (92.4, 328.43, 334.46, 71.25, 12.48, 6.40, 457.4, 311.37) is achieved by treating the nucleic acid ligand with 6 equivalents of the bifunctional NHS-PEG(3400 kDa)-vinyl sulfone (Shearwater) in 2 μL dry DMSO in a 10 uL aliquot of 100 mM borate buffer at pH 9.2 for 30 minutes. Conjugated nucleic acid ligands are purified on a pre-equilibrated ion exchange column (Q5 from Sartorius) to remove excess PEG reagent, then eluted with 1M NaCl, then desalted on a Sephadex G50 desalting column. The vinyl sulfone-PEG-nucleic acid ligands were printed onto thiol activated surfaces (Apogent).
Array of 67 Photocrosslinking Nucleic Acid Ligands
Sixty-seven photocrosslinking nucleic acid ligands to 25 different protein targets were arrayed according to the method of Example 1. Each photocrosslinking nucleic acid ligand was spotted in triplicate. The array also comprises the following addresses:
Eight duplicate arrays were produced with this layout. The sequence of each photoaptamer on the array is provided in
Protein mixtures were produced as detailed in Table 2 (which provides a key for the individual concentrations designated Level 1-8) and Table 3 (which shows the target protein concentration profiles (Level 1-8) in each of the individual target protein mixture (Tubes 1-8)).
TABLE 2 Protein Level Range (moles/L) Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Level 7 0 1E-11 3.16E-11 1E-10 3.16E-10 1E-09 1E-08 TABLE 3
Target Protein Concentration Profiles of the
Individual Target Protein Mixtures
Detection of HIV gp120MN in Serum
Using the techniques in Examples 1 and 3, photocrosslinking nucleic acid ligands to gp120MN, bFGF, and thrombin were arrayed on slides. One slide was incubated with 5% serum only; the other was incubated with 5% serum+100 nM gp120MN.
Array of Photocrosslinking Nucleic Acid Ligands Directed Toward 14 Target Protein Analytes
Arrays were printed onto slides according to Example 1. Each slide comprised eight duplicate arrays. Each photocrosslinking nucleic acid ligand was spotted four times in succession, giving a total of 96 photocrosslinking nucleic acid ligand features on each array. The layout of the array is given in
Protein mixtures containing 14 different proteins (endostatin, luciferase, thrombin, IL-4, tPA, catalase, C3, IL-8, von Willebrand Factor, bFGF, HIV gp120MN, IGFBP-3, angiogenin, and VEGF) were produced. The protein mixtures were designed to test aptamer sensitivity and specificity. Eight protein mixtures were designed such that each protein is present at least once in excess of each of the other proteins in the mixture. The concentration range of the individual proteins was 10 pM-10 nM for the majority of the proteins. Three of the proteins (bFGF, HIV gp120MN, and von Willebrand Factor) were present from 10 pM-2 nM because high concentrations caused some nonspecific reactions. The total added protein concentration in each mixture was approximately 25 nM. An example of the protein mixture multiplex design is given in Table 3 (which provides a key for the individual concentrations designated Level A-H) and Table 4 (which shows the target protein concentration profiles of the individual protein mixtures (Tubes 1-8)).
TABLE 3 Target Protein Concentration Key Protein Level Range (moles/L) Level A Level B Level C Level D Level E Level F Level G Level H bFGF, gp 120, von Willebrand 0.00E + 00 1.00E − 11 2.40E − 11 5.80E − 11 1.40E − 10 3.40E − 10 8.30E − 10 2.00E − 09 All other proteins 0.00E + 00 1.00E − 11 3.20E − 11 1.00E − 10 3.20E − 10 1.00E − 09 3.20E − 09 1.00E − 08 TABLE 4 Target Protein Concentration Profiles of the Individual Target Protein Mixtures Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 gp120MN E B C H D A G F bFGF C H D A G F E B von Willerand F E B C H D A G angiogenin B C H D A G F E IL4 H D A G F E B C Luciferase D A G F E B C H tPA A G F E B C H D C3 G F E B C H D A IL8 E F D A G B C H IGFBP3 F D A G B C H E endostatin D A G B C H E F VEGF A G B C H E F D thrombin G B C H E F D A catalase B C H E F D A G
Protein mixtures were added to four sample matrices:
Each individual protein mixture (in sample matrix) was incubated with an array and processed according to the method of Example 3 using N-hydroxy-succinimide-Alexa-555 as the UPS. Thus, in total there were 32 different assay samples (each the eight protein mixtures in each of the four sample matrices). As each slide had eight duplicate arrays, the entire assay was carried out using only four slides, with each slide providing the dose response curve for 14 proteins targets in a particular sample matrix.
This example demonstrates that nucleic acid ligand microarrays can be used to measure specific protein concentrations in complex protein mixtures, including serum and urine specimens. Dose response curves were generated simultaneously to 14 proteins from 8 arrays printed on a single slide. The sensitivity and reproducibility of the photoaptamer arrays has been demonstrated by subnanomolar measurement of proteins in multiple sample matrices. The results demonstrate that photoaptamer arrays possess the qualities required for use in an analytical laboratory setting, namely: high throughput, ability to assay multiple analytes with consistent conditions, ability to use minimal sample volume (<100 μL), ability to provide reproducible results, and minimal matrix interference.
Detection of Cognate Proteins in 5% Serum
Using the techniques in Examples 1 and 3, photocrosslinking nucleic acid ligands to bFGF, VEGF, endostatin, and catalase were arrayed on slides. Microarrays were exposed to 5% serum (filtered through a 0.2 micron filter to remove particulates, but without any other pretreatment) with some samples spiked with additional bFGF, VEGF, endostatin, and catalase in concentrations varying from 1 pM to 1 nM. The dose response for VEGF, endostatin, and catalase in the matrix of 5% serum is clear with minimal interference from the serum. The use of 5% serum in samples requires extensive harsh wash treatment following the UV photocrosslinking step defined as: 10 mM DTT, 0.1% SDS, 70 mM TRIS buffer at pH 11.0, and 500 mM NaCl flowing across the microarray for 30 minutes at 40° C. Nonfouling surfaces provided by Accelr8 and others yield the most definitive dose response curves in the presence of untreated serum. Dose response curves for VEGF, endostatin, and catalase in 5% serum on Accelr8 slide surfaces are shown in
Detection of Cognate Proteins on Beads
When beads are loaded with nucleic acid ligands as described in Example 2, then exposed to cognate protein, dose response curves can be generated that are similar to those observed on flat surfaces.
Detection of Endogenous Serum Proteins
In addition to detecting proteins spiked into serum, photocrosslinking nucleic acid arrays are able to detect endogenous levels of target proteins in test fluids such as serum or tissue culture supernatants. Samples of 1% serum, 5% delipidated serum, 50% serum base matrix or 50% filtered serum were added to arrays (produced according to Example 1), allowed to equilibrate, crosslinked, then washed under native conditions to avoid denaturing the captured proteins. Antibodies specific to endostatin and thrombin were allowed to bind to the captured proteins, and these antibodies were detected by use of an Alexa-555-labeled-anti-rabbit secondary antibody. The reaction of photoaptamers with endogenous protein in tissue culture supernatant or untreated serum was observed; no other photoaptamers on the array reacted with the endogenous proteins. See
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|U.S. Classification||435/6.11, 435/21|
|International Classification||G01N33/53, G01N33/68, C12N15/09, G01N33/58, C12Q1/25, C12Q1/28, C12M1/00, C12Q1/42, G01N33/543, A61K38/00, G01N21/78, C12Q1/68, C12M1/34, G01N33/566, C12N15/115|
|Cooperative Classification||C12N15/115, G01N33/58|
|European Classification||G01N33/58, C12N15/115|
|Nov 3, 2004||AS||Assignment|
Owner name: SOMALOGIC, INCORPORATED, COLORADO
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GOLD, LARRY;SMITH, JONATHAN DREW;ZICHI, DOMINIC;AND OTHERS;REEL/FRAME:015330/0783;SIGNING DATES FROM 20041007 TO 20041021