JP4716990B2 - How to use combinatorial artificial receptors - Google Patents

Description

Detailed Description of the Invention

  This application is a PCT international patent application in the name of Robert E. Carlson, a U.S. company, Receptors LLC, who is the applicant for designation in all countries other than the U.S. US provisional patent applications 60 / 499,752, 60 / 499,965, and 60 / 500,081 filed on September 3, 2003, and 60 / 526,511, 60 / filed on December 2, 2003. Claim priority to 526,699, 60 / 526,703, 60 / 526,708 and 60 / 527,190.

The present invention relates to methods of using artificial receptors such as combinatorial artificial receptor arrays. The receptor includes heterogeneous and immobilized combinations of building block molecules. In certain embodiments, a combination of 2, 3, 4, or 5 different building block molecules immobilized close to each other on a support provides a molecular structure that can be used in the present method. The method can then develop artificial receptors that can be used to detect receptor ligands. The method can find compounds that disrupt one or more binding interactions.

Background The preparation of artificial receptors that bind ligands such as proteins, peptides, carbohydrates, microorganisms, contaminants, pharmaceuticals, etc. with high sensitivity and properties is an active area of research. None of the conventional approaches have been particularly successful; only moderate sensitivity and specificity have been achieved, mainly due to the low binding affinity.

Antibodies, enzymes, and natural receptors generally have binding constants in the range of 10 ⁸ -10 ¹² resulting in both nanomolar sensitivity and targeted specificity. In contrast, conventional artificial receptors typically have binding constants of about 10 ³ to 10 ⁵ with predictable results of millimolar sensitivity and limited specificity.

  Several conventional approaches continue with attempts to achieve high sensitivity and specific artificial receptors. These approaches include, for example, affinity isolation, molecular imprinting, and rational and / or combinatorial design and synthesis of synthetic or semi-synthetic receptors.

  Such rational or combinatorial approaches have been limited by the relatively small number of receptors being evaluated and / or by reliance on a design strategy that focuses on only one building block, a homogenous design strategy. It was. The usual combinatorial approach forms a microarray containing 10,000 or 100,000 different spots on a standard microscope slide. However, such conventional methods for combinatorial synthesis provide a single molecule per spot. Using a single building block at each spot provides only a single potential receptor per spot. To create thousands of potential receptors would require the synthesis of thousands of building blocks.

  In addition, these conventional approaches are hampered by the current limited understanding of the leading actors that lead to efficient binding and the majority of potential structures for receptors that make such approaches problematic.

  There is a need for methods for detecting ligands and for detecting compounds that disrupt one or more binding interactions.

Overview The present invention relates to methods of using artificial receptors, such as combinatorial artificial receptor arrays. The receptor includes heterogeneous and immobilized combinations of building block molecules. In certain embodiments, a combination of 2, 3, 4, or 5 different building block molecules immobilized near each other on a support provides a molecular structure that can be used in the present methods. The method can then develop artificial receptors that can be used to detect receptor ligands. The method can find compounds that disrupt one or more binding interactions.

  The present invention includes a method for making an artificial receptor for binding a test ligand. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with a test ligand, detects binding of the test ligand to at least one candidate artificial receptor, and then detects binding of the test ligand. Selecting the selected at least one candidate artificial receptor as a working artificial receptor for the test ligand. Candidate artificial receptors can include a plurality of building blocks coupled to regions of the support.

  The present invention includes a method for detecting a test ligand. In one embodiment, the method of the invention contacts at least one working artificial receptor with a sample suspected of containing a test ligand and then monitors for binding to at least one working artificial receptor. Wherein binding to the at least one working artificial receptor is indicative of the presence of the test ligand in the sample. A working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the at least one working artificial receptor has been found to bind a test ligand. In one embodiment, the at least one working artificial receptor can include a plurality of artificial receptors.

  In one embodiment, the method of the invention contacts a plurality of candidate artificial receptors with a first test ligand; detects binding of the first test ligand to at least one candidate artificial receptor; It may then include classifying the location or composition of the at least one artificial receptor bound to the first test ligand. Each candidate artificial receptor can independently comprise a plurality of building blocks coupled to a region of the support.

  The present invention includes methods for detecting compounds that disrupt test ligand interactions. The method can include contacting at least one working artificial receptor with a test ligand and a candidate disruptor. Contact may be simultaneous, contact with the test ligand may come before contact with the candidate disruptor, or contact with the candidate disruptor ligand will come before contact with the test ligand. Also good. In one embodiment, the method can include contacting at least one working artificial receptor with a mixture comprising a test ligand and a candidate disruptor. The method can also include monitoring a candidate disruptor that reduces binding of the test ligand to the at least one working artificial receptor, wherein the decreased binding is due to the candidate disruptor being predominant. It shows that it is a disturbing substance. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the at least one working artificial receptor has been found to bind the test ligand.

  The present invention includes a method for making an affinity support for a test ligand. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with the test ligand; detects binding of the test ligand to at least one candidate artificial receptor; At least one candidate artificial receptor for which is detected as a working artificial receptor for the test ligand; the working artificial receptor is then bound to a second support to form an affinity support Can include. The candidate artificial receptor can include a plurality of building blocks coupled to a region of the first support.

  The present invention includes a method for isolating a test ligand. In one embodiment, the method of the present invention may comprise contacting a sample containing the test ligand with a working artificial receptor. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the working artificial receptor has been found to bind the test ligand.

  The present invention includes a method for making a reaction support for a reactant. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with the reactant; detects binding of the test ligand to at least one candidate artificial receptor; At least one candidate artificial receptor detected is selected as a working artificial receptor for the test ligand; at least one working artificial receptor is contacted with the reactant and then the reaction of the reactant is monitored Selecting a working artificial receptor for which the reaction has been observed as a reactive artificial receptor; and then binding the reactive artificial receptor to a second support to form a reactive support. obtain. The candidate artificial receptor can include a plurality of building blocks coupled to a region of the first support. In one embodiment, the method can include contacting at least one working artificial receptor with a second reactant.

  The present invention includes a method for reacting reactants. In one embodiment, the method of the present invention may include contacting a sample containing the reactant with a working artificial receptor. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the working artificial receptor has been found to bind the test ligand. In one embodiment, the method can include contacting the working artificial receptor with a second reactant.

  The present invention includes a method of making a catalyst support for a reactant. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with the reactant; and detects the catalytic action of the reactant in the presence of the at least one candidate artificial receptor. Selecting at least one candidate artificial receptor for which catalytic action has been detected as a working artificial receptor for the test ligand; then binding the working receptor to a second support to form a catalytic support; Forming. The candidate artificial receptor can include a plurality of building blocks coupled to a region of the first support. In one embodiment, the method can include contacting at least one working artificial receptor with a second reactant.

  The present invention includes a method of catalyzing a reaction. In one embodiment, the method of the present invention can include contacting a sample containing a reactant with a working artificial receptor. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the working artificial receptor has been found to bind the test ligand and catalyze the reaction.

  The present invention includes a method of creating a surface that does not bind a test ligand. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with the test ligand; detects a loss of binding of the test ligand to at least one candidate artificial receptor; ligand binding The test deficiency may comprise selecting at least one candidate artificial receptor detected as a non-binding surface for the test ligand. The candidate artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the test ligand includes a plurality of test ligands.

  The present invention includes a method for detecting a compound that disrupts the interaction of a test ligand with a second ligand. In one embodiment, the method of the invention may comprise binding the test ligand to at least one working artificial receptor. The method can include contacting at least one working artificial receptor with a second ligand and a candidate disruptor. Contact may be simultaneous and contact with the second ligand may come before contact with the candidate disruptor, or contact with the candidate disruptor may occur before contact with the second ligand. You may come. In one embodiment, the method can include monitoring a candidate disruptor that reduces binding of the second ligand to the test ligand, wherein the decreased binding is due to the candidate disruptor being predominant. It shows that it is a disturbing substance. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the at least one working artificial receptor has been found to bind the test ligand.

Detailed Description Definitions As used herein, the term “peptide” refers to a compound comprising two or more amino acid residues linked by amide bond (s).

  As used herein, the terms “polypeptide” and “protein” refer to a peptide comprising about 20 or more amino acid residues linked by peptide bonds.

  As used herein, the term “proteome” refers to the expression profile of a protein of an organism, tissue, organ, or cell. A proteome can be specific for a particular state (eg, growth, health, etc.) of the organism, tissue, organ, or cell.

  A reversibly immobilized building block on the support allows the building block to be separated from the support without destroying or unacceptably degrading the building block or the support. The building block is bonded to the support through a mechanism that That is, immobilization can be reversed without destroying the building block or the support or unacceptably degrading. In one embodiment, immobilization can be reversed by only a slight or ineffective level of degradation of the building block or the support. Reversible immobilization can use readily reversible covalent bonds or non-covalent interactions. Suitable non-covalent interactions include ionic interactions, hydrogen bonds, van der Waals interactions, and the like. A readily reversible covalent bond refers to a covalent bond that can be formed and broken under conditions that do not break or unacceptably degrade the building block or the support.

  For example, the combination of building blocks immobilized on a support can be a candidate artificial receptor, a leading artificial receptor, or a working artificial receptor. That is, a heterogeneous building block spot on a slide or a plurality of building blocks coated on a tube or well can be a candidate artificial receptor, a leading artificial receptor, or a working artificial receptor. A candidate artificial receptor can be a lead artificial receptor, which can be a working artificial receptor.

  As used herein, the term “candidate artificial receptor” refers to an immobilized combination of building blocks that can be tested to determine whether a particular test ligand binds to the combination. In one embodiment, the combination includes one or more reversibly immobilized building blocks. In one embodiment, the candidate artificial receptor can be a heterogeneous building block spot on a slide or a plurality of building blocks coated on a tube or well.

  As used herein, the term “leading artificial receptor” refers to a predetermined concentration of test ligand, eg, 10, 1, 0.1, or 0.01 μg / ml, or 1, 0.1, or Refers to an immobilized combination of building blocks that bind the test ligand at 0.01 ng / ml. In one embodiment, the combination includes one or more reversibly immobilized building blocks. In one embodiment, the lead artificial receptor can be a heterogeneous building block spot on a slide or a plurality of building blocks coated on a tube or well.

  As used herein, the term “acting artificial receptor” refers to a combination of building blocks that bind test ligands with selectivity and / or sensitivity effective to classify or identify test ligands. . That is, binding to the combination of building blocks means that the test ligand belongs to the category of test ligands or is a specific test ligand. For example, a working artificial receptor can bind a ligand at a concentration of, for example, 100, 10, 1, 0.1, 0.01, or 0.001 ng / ml. In one embodiment, the combination includes one or more reversibly immobilized building blocks. In one embodiment, the working artificial receptor can be a heterogeneous building block spot or tube on a slide, a plurality of building blocks coated on a well, slide, or other support or scaffold.

  As used herein, the term “acting artificial receptor complex” refers to a plurality of artificial receptors, each of which is a combination of building blocks, that classifies or identifies the test ligand. The test ligand is bound by an effective selectivity and / or sensitivity pattern. That is, binding to several receptors of the complex means that the test ligand belongs to the test ligand category or is a specific test ligand. Each individual receptor in the complex can bind a ligand at different concentrations or with different affinities. For example, the individual receptors in the complex bind the ligand at a concentration of 100, 10, 1, 0.1, 0.01 or 0.001 ng / ml, respectively. In one embodiment, the combination includes one or more reversibly immobilized building blocks. In one embodiment, the working artificial receptor complex comprises a plurality of heterogeneous building block spots or regions on a slide; a plurality of wells each coated with a different combination of building blocks; or a different combination of building blocks. It can be a plurality of coated tubes.

  As used herein, the term “significant number of candidate artificial receptors” provides an opportunity to discover a working artificial receptor, a working artificial receptor complex, or a leading artificial receptor. Refers to sufficient candidate artificial receptors to do. About 100 to about 200 candidate artificial receptors are a significant number to discover artificial receptor complexes that act appropriately to distinguish two proteins (eg, cholera toxin and phycoerythrin) obtain. In other embodiments, a significant number of candidate artificial receptors may include about 1,000 candidate artificial receptors, about 10,000 candidate artificial receptors, about 100,000 candidate artificial receptors, or more.

  Although not limited to the present invention, the significant number of candidate artificial receptors required to provide an opportunity to discover a working artificial receptor is required to find a working artificial receptor complex. It is believed that it may be greater than the significant number. While not limited to the present invention, the significant number of candidate artificial receptors required to provide an opportunity to discover leading artificial receptors is the number of candidate artificial receptors required to find a working artificial receptor. It may be greater than a significant number. While not limited to the present invention, the significant number of candidate artificial receptors required to provide an opportunity to find a working artificial receptor for a test ligand with few characteristics has many characteristics. It is believed that this may be the case for the test ligand.

  As used herein, the term “building block” refers to an artificial receptor molecule comprising one or more linkers, one or more frameworks, and a moiety that can be imagined or include one or more recognition elements. Refers to ingredients. In one embodiment, the building block includes a linker, a framework, and one or more recognition elements. In one embodiment, the linker comprises a site suitable for reversibly immobilizing the building block, eg, on a support, surface or lawn.

  As used herein, the term “linker” connects the building block to a support via, for example, covalent bonds, ionic interactions, electrostatic interactions, or hydrophobic interactions. Refers to a part of a building block or a functional group on it that can be used (or reversibly) attached.

  As used herein, the term “framework” refers to the part of the building block that contains the linker or to which the linker is attached and to which one or more recognition elements are attached.

  As used herein, the term “recognition element” refers to the part of the building block that is attached to the framework but not covalently attached to the support. Without being limited to the present invention, the recognition element can provide or form one or more groups, surfaces, or spaces for interacting with the ligand.

  As used herein, the term “plurality of building blocks” refers to two or more building blocks of different structure in a mixture, in a kit, or on a support or scaffold. Each building block has a particular structure, and the use of a plurality of building blocks, or a plurality of building blocks, refers to one or more of these particular structures. A building block or blocks does not refer to a plurality of molecules each having the same structure.

  As used herein, the term “building block combination” refers to a plurality of building blocks that are in a spot, region, or candidate, lead or acting artificial receptor together. The building block combination may be a subset of the building block set. For example, the building block combination may be one of 2, 3, 4, 5, or 6 building block possible combinations from N (eg, N = 10-200) building block sets.

  As used herein, the terms “homogeneous immobilization building block” and “homogeneous immobilization building blocks” refer to a support having only a single building block immobilized thereon or therein, or Refers to a spot.

  As used herein, the term “activated building block” refers to a building block that has been activated, eg, to prepare to form a covalent bond to a functional group on a support. Building blocks containing carboxyl groups can be converted to building blocks containing activated ester groups that are activated building blocks. An activated building block containing an activated ester group can react with, for example, an amine to form a covalent bond.

  As used herein, the term “naive” as used with respect to one or more building blocks refers to building blocks that have not been determined or found to bind to the test ligand of interest. For example, the recognition element (s) on the naive building block has not been determined or known to bind to the test ligand of interest. A building block that is or contains a known ligand (eg, GM1) for a particular protein (test ligand) of interest (eg, cholera toxin) is not naive with respect to the protein (test ligand).

  As used herein, the term “immobilized” as used with respect to a building block coupled to a support is used to refer to a support so that they do not move onto or release from the support. Refers to a building block that is stably oriented upward. The building blocks can be immobilized by covalent bonds, by ionic interactions, by electrostatic interactions such as ion pairs, or by hydrophobic interactions such as van der Waals interactions.

  As used herein, a “region” of a support, tube, well, or surface means that the “region” of the support, tube, well, or surface means that the support, tube, well, or surface. Refers to the adjacent part of the surface. A building block coupled to a region can refer to a building block proximate to the other of the region.

  As used herein, a “bulky” group on a molecule is larger than a moiety containing 7 or 8 carbon atoms.

  As used herein, a “small” group on a molecule is hydrogen, methyl, or another group that is smaller than a moiety containing 4 carbon atoms.

  As used herein, the term “lawn” refers to a layer, spot, or region of functional groups on a support, eg, of sufficient density to place a coupled building block proximate to the other. Point to. Functional groups can include groups capable of forming covalent, ionic, electrostatic, or hydrophobic interactions with building blocks.

As used herein, the term “alkyl” includes saturated alkyl groups including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. Refers to an aliphatic group. In certain embodiments, a straight chain or branched chain has no more than 30 carbon atoms in its backbone (eg, C ₁ -C ₁₂ for straight chain, C ₁ -C ₆ for branched chain) . Similarly, a cycloalkyl can have 3-10 carbon atoms in the ring structure, eg, 5, 6, or 7 carbons in the ring structure.

  As used herein, the term “alkyl” refers to both “unsubstituted alkyl” and “substituted alkyl”, the latter of which is hydrogen on one or more carbons of the hydrocarbon backbone. Refers to an alkyl moiety having a substituent that replaces. Such substituents are, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ester, formyl, or ketone), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphonate, phosphinate. , Amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamyl, sulfonamide, sulfonyl, heterocyclyl, arylalkyl, or an aromatic or heteroaromatic moiety. The sites substituted on the hydrocarbon chain can themselves be substituted if appropriate. For example, substituted alkyl substituents may include substituted and unsubstituted forms of the above groups.

  As used herein, the term “arylalkyl” refers to an alkyl group substituted with an aryl group (eg, an aromatic group or a heterocyclic aromatic group).

  As used herein, the terms “alkenyl” and “alkynyl” are unsaturated fats similar to alkyl groups in length and optional substitution, respectively, except that each contains at least one double or triple bond. Refers to a group.

  The term “aryl” as used herein refers to 0 to 4 heteroatoms such as, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. Includes 5-, 6- and 7-membered monocyclic aromatic groups which may include. An aryl group having a heteroatom in the ring structure may be referred to as an “aryl heterocycle” or “heteroaromatic compound”. The aromatic ring can be substituted with substituents as described above for alkyl groups at one or more ring positions. The term “aryl” also means that at least one of the rings is an aromatic ring, eg, two or more carbons, other ring (s) are cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl. A polycyclic system having two or more rings common to two adjacent rings, which are “fused rings”.

  As used herein, the term “heterocycle” or “heterocyclic group” refers to 3- to 12-, for example, 3- to 7-membered rings in which the ring structure contains 1 to 4 heteroatoms. Refers to a member ring structure. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, Indole, indazole, purine, quinolidine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenalsazine, phenothiazine, furazane, phenoxazine, pyrrolidine , Oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactone, Lactams such as Sechijinon and pyrrolidinones, sultams, sultones, and the like. Heterocycles can be substituted at one or more positions with substituents as described for alkyl groups.

  As used herein, the term “heteroatom” means an atom of any element other than carbon or hydrogen, such as nitrogen, oxygen, sulfur and phosphorus.

Artificial Receptor Overview FIG. 1 schematically illustrates an embodiment using four different building blocks in a spot on a microarray to create a ligand binding site. This figure shows a group of four building blocks at the corners of the square forming the unit cell. A group of four building blocks can be expected as vertices on any square. FIG. 1 shows that a spot or region of a building block can be expected as a plurality of unit cells, in this figure square unit cells. In addition, a group of unit cells of four building blocks having other rectangular shapes can be formed on a support.

  Each of the immobilized building block molecules can provide one or more “arms” extending from the “framework”, each comprising a group that interacts with a ligand or with another immobilized building block portion. obtain. FIG. 2 provides a molecular arrangement of building blocks in which a combination of four building blocks, each containing a framework with two arms (referred to as “recognition elements”), forms a site for binding a ligand. Such sites formed by building blocks such as those exemplified below can bind small molecules such as drugs, metabolites, contaminants, etc. and / or larger ligands such as macromolecules or microorganisms Can be combined.

  The artificial receptors of the present invention can include a building block reversibly immobilized on a support or surface. Reversible immobilization of the building blocks allows movement of the building blocks to different positions on the support or surface, or exchange of building blocks on and out of the surface. For example, when reversibly bound to or immobilized on the support, the combination of building blocks can bind to the ligand. Reversal of building block coupling or immobilization provides an opportunity for building block reordering to improve ligand binding. Furthermore, the present invention allows the addition of additional or different building blocks that can further improve ligand binding.

  FIG. 3 schematically shows an embodiment using an initial artificial receptor surface (A) having four different building blocks on the surface, represented by the shaded shape. This initial artificial receptor surface (A) (1) experiences binding of ligand to the artificial receptor, (2) shuffles the building blocks on the receptor surface, leading artificial receptors (B) Get. Shuffle refers to reversible coupling or immobilization of building blocks and allows rearrangement on the receptor surface. After forming the lead artificial receptor, additional building blocks can be exchanged on and / or out of (3) the receptor surface (C). Exchange refers to a building block that leaves the surface and enters the solution that contacts the surface, and / or a building block that leaves the solution that contacts the surface and becomes part of the artificial receptor. Additional building blocks can be selected for structural diversity (eg, randomly) or can be selected based on the structure of the building blocks in the lead artificial receptors to provide additional ways to improve binding . The original and further building blocks can then be (4) shuffled and replaced to provide a higher affinity artificial receptor on the surface (D).

Methods of Using the Artificial Receptor A working artificial receptor can be generated that is specific for a given test ligand or specific for a particular portion of a given test ligand. Different and fixed combinations of building block molecules form the working artificial receptor. For example, a combination of 2, 3, 4, or 5 different building block molecules immobilized in close proximity to the other on the support provides a molecular structure that acts as a candidate and acting artificial receptor. The building block can be naive to the test ligand. Once multiple candidate artificial receptors occur, they can be tested to determine which are specific or useful for a given ligand.

  Specific or working artificial receptors or receptor complexes can then be used in a variety of different methods and systems. For example, the receptor can be used in methods and / or devices for binding or detecting a test ligand. According to further examples, the receptor can be used in methods and / or devices for chemical synthesis. Methods and systems for chemical synthesis can include methods and systems for site-specific and stereospecific chemical synthesis. The receptors can also be used to develop compounds that disrupt or model binding interactions. Methods and systems for developing therapeutic agents can include methods and systems for pharmaceutical and vaccine development.

  In one embodiment, the methods and systems of the invention can be used to detect multiple ligands of interest. For example, an unknown biological sample can be characterized by the presence of a specific ligand combination. Such methods can be useful in assays to detect specific pathogens or disease states. As a further example, such embodiments can be used to determine a genetic profile of interest. For example, cancer tissue can be detected, or a genetic predisposition to cancer can be detected.

  Artificial receptors of the invention include: genomic and / or proteomic (protein isolation and characterization) analysis; pharmaceutical development (such as identification of sequence-specific small molecule reads, protein characterization for protein interactions); Detector for test ligands (such as clinical or electric field analysis of cocaine or other addictive drugs); Dependent drug diagnosis or treatment; Hazardous waste analysis or improvement; Chemical exposure warning or intervention; Disease diagnosis or treatment; It may be part of a product used in cancer diagnosis or treatment (such as clinical analysis of prostate specific antigen); biological agent warning or intervention; food chain contamination analysis or improvement and clinical analysis of food hybrids.

Methods of binding or detecting test ligands In one embodiment, the present invention may include methods and / or devices for binding or detecting test ligands. For example, the artificial receptors of the present invention can be used in various assays that currently use antibodies. The artificial receptors of the present invention can be specific for a given ligand, such as an antigen or immunogen. Therefore, the artificial receptor of the present invention can be used in a manner similar to enzyme immunoassay, enzyme-linked immunoassay, immunodiffusion, immunoelectrophoresis, latex agglutination and the like. Test ligands detectable by such methods include addictive drugs, biological agents (such as harmful agents), markers for biological agents, markers for medical conditions, and the like. Methods and systems for detection can include methods and systems for all types of clinical chemistry, environmental analysis, and diagnostic assays.

  For example, the artificial receptor can be contacted with a sample comprising or suspected of containing at least one test ligand. The building blocks that make up the artificial receptors can be naive to the test ligand. The binding of one or more test ligands to the artificial receptor can then be detected. The binding results can then be interpreted to provide information about the sample. In one embodiment, the invention includes a method for detecting a ligand in a sample comprising contacting an artificial receptor specific for the test ligand with a sample suspected of containing the test ligand. The method can also include detecting or quantifying the binding of the test ligand to the artificial receptor. For example, using an artificial receptor that binds (eg, tightly) the molecule, cell, or microorganism under appropriate conditions in a form where the binding itself is sufficient to indicate the presence of the molecule or organism. Can do. Such formats can include artificial receptors that are probed with positive and control samples.

  FIG. 4 schematically illustrates an embodiment of a method for evaluating candidate artificial receptors for binding test ligands, such as molecules or cells. The method can include creating an array of candidate artificial receptors. A working artificial receptor can be identified by contacting the array with a test ligand and identifying which receptor binds the test ligand. The building blocks that make up the artificial receptors can be naive to the test ligand. Such methods can use labeled test ligands. The method can include fabricating an array or device comprising the working artificial receptor or receptor complex. In one embodiment, the method can include using the array or device to detect or characterize the test ligand in a sample, such as a biological, laboratory, or environmental sample.

  FIG. 5 schematically illustrates an embodiment of the invention using an array of candidate artificial receptors. This embodiment of the method can use an array containing a significant number of the artificial receptors of the invention to generate an assay or system for characterizing or detecting a test ligand. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a test ligand, eg, a molecule or a cell. The building blocks that make up the artificial receptors can be naive to the test ligand. The molecule or cell may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in a method for characterizing a biological sample or in a method for characterizing or detecting a molecule or cell (eg, working artificial receptor or working artificial receptor complex). Can be selected.

  As shown in FIG. 5, test ligands can be identified by methods using single or multiple primary or acting artificial receptors. Multiple primary or working artificial receptors suitable for identifying test ligands can be used in an array format test. A single lead or working artificial receptor can be configured on the support as a strip with positive and / or negative control receptors that can also be configured as a strip.

  In one embodiment, the method can include generating or using a selected working artificial receptor or receptor complex on a substrate. The substrate can include a working artificial receptor for a single test ligand or a working artificial receptor for multiple test ligands. For example, the method can include contacting the artificial receptor with a sample. A substrate comprising a working artificial receptor for a single test ligand can be used in a method or system for detecting the test ligand. Binding to the working artificial receptor indicates that the sample contains the test ligand. Substrates comprising working artificial receptors for a plurality of test ligands can be used in a method or system for detecting one, some, or all of the test ligands. Binding to the working artificial receptor for a particular test ligand or ligand indicates that the sample contains such a test ligand or ligand.

  The working artificial receptor or receptor complex can be configured to provide a pattern that indicates the presence of one or more of the test ligands. The method can include detecting the binding pattern of the sample and comparing it to a binding pattern from a known sample. FIG. 6 schematically shows a specific binding pattern on an array of working artificial receptors. In one embodiment, all artificial receptors for a test ligand can be arranged in a line across the substrate. Referring to FIG. 6, the receptor specific for IL-2 is in line 12 on the array 10 of working artificial receptor complexes. A working artificial receptor that binds a test ligand (eg, IL-2) is shown as shaded 24. The working artificial receptor that did not bind the test ligand is shown as open circle 26.

  The method of using the shown array can include detecting binding on the working artificial receptor line 12 via fluorescence or another means described herein. In the illustrated embodiment, detecting binding of working artificial receptor on line 12 indicates that the sample contains IL-2. Furthermore, a lack of signal from other working artificial receptors in array 10 indicates that the sample does not contain IFN-gamma, IL-10, TGF-beta, IL-12, or TGF-alpha. Thus, methods using such arrays can determine whether a sample is a specific type of biological sample or contains a specific type of molecule or cell.

  When designed for use with an electric field assay kit, the device 30 may have spots arranged such that a positive result produces an easily recognizable pattern 36 such as a plus sign. Thus, a readily recognizable pattern can indicate that a particular test ligand is present in the sample. Alternatively, artificial receptors or spots for a particular target 42 can be randomly arranged on the third array 40. Thus, when a detection device or array is used, the results of the test may not be immediately apparent to the observer, but bind receptors or spots at different locations can be Or read immediately by a programmable machine to correlate with cellular identity.

  In one embodiment, the present invention includes a method for detecting or characterizing a biological sample, molecule, or cell. This embodiment of the method selects an artificial receptor that binds a biological sample, molecule, or cell from an array of artificial receptors, contacts the artificial receptor with a test composition, and then the test composition Detecting binding of the artificial receptor to the. In such embodiments, binding indicates the presence of the biological sample, molecule, or cell in the test composition. In one embodiment, the present invention includes a method for detecting or characterizing a biological sample, molecule, or cell. This embodiment of the method can include contacting an array of artificial receptors with a test composition and detecting binding to the artificial receptors. Binding indicates the presence of the biological sample, molecule, or cell in the test composition.

  The methods of the invention can develop or use multiple working receptors that are specific for a particular test ligand, eg, a biological sample, molecule, or cell. That is, the working artificial receptor can be specific for a particular test ligand, but different receptors can have different distinct antigens (eg, proteins or carbohydrates), ligands, functional groups, or test ligands. Can interact with other structural features. Such a method may provide a robust assay for the presence of a test ligand. For example, such a robust assay can reduce the chance of false positive or false negative results in comparison to assays that rely on a single unique receptor to detect a given test ligand. Furthermore, this embodiment of the method develops or uses a working receptor that exhibits high binding affinity due to interaction with multiple antigens or ligands on the same test ligand (eg, multivalent binding). Can do.

  In one embodiment, the method uses an array comprising a significant number of the artificial receptors of the invention to generate an assay or system for characterizing or detecting a polynucleotide, eg, DNA or RNA. Can do. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the polynucleotide, eg, DNA or RNA. The building blocks that make up the artificial receptors can be naive to DNA or RNA. For example, the polynucleotide, such as DNA or RNA, can exhibit characteristic binding to one or several of the candidate artificial receptors from the array. One or several receptors can be used, for example, in a method for characterizing a biological sample, or in a method for characterizing or detecting the polynucleotide such as DNA or RNA (eg, acting artificial receptor or acting Artificial receptor complex).

  In one embodiment, the method can generate an assay or system that characterizes or detects a polypeptide or peptide using an array comprising a significant number of the artificial receptors of the invention. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the polypeptide or peptide. The building blocks that make up the artificial receptors can be naive to the polypeptide or peptide. The polypeptide or peptide may exhibit characteristic binding to one or several of the candidate artificial receptors from the array. One or several artificial receptors characterize a biological sample, or artificial receptors that can be used in methods for characterizing or detecting a polypeptide or peptide (eg, working artificial receptors or working artificial receptor complexes). Body).

  In one embodiment, the method can generate an assay or system for characterizing or detecting oligosaccharides or polysaccharides using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding oligosaccharides or polysaccharides. The building blocks that make up the artificial receptors can be naive to oligosaccharides or polysaccharides. Oligosaccharides or polysaccharides can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors may be used to characterize a biological sample or to be used in a method for characterizing or detecting oligosaccharides or polysaccharides (eg, working artificial receptors or working artificial receptor complexes). Body).

  In one embodiment, the method can generate an assay or system for characterizing or detecting cells, such as, for example, hepatocytes, using an array comprising a significant number of the present artificial receptors. . The method can include, for example, evaluating an array that includes a significant number of candidate artificial receptors for binding to cells such as hepatocytes. The building blocks that make up the artificial receptors can be naive to the cells. For example, cells such as hepatocytes may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors characterize a biological sample, or artificial receptors that can be used in a method for characterizing or detecting cells such as hepatocytes (eg, working artificial receptors or working artificial receptors). Body complex).

Methods for binding or detecting addictive drugs In one embodiment, the present invention may include methods and / or devices for binding or detecting addictive drugs. Methods and systems for detection can include methods and systems for all types of clinical chemistry, electric field analysis, and diagnostic assays. For example, the artificial receptor can be contacted with a sample containing or suspected of containing at least one addictive drug. The binding of one or more addictive drugs to the artificial receptor can then be detected. The binding results can then be interpreted to provide information about the sample. In one embodiment, the present invention provides a method for detecting an addictive drug in a sample comprising contacting an artificial receptor specific for the addictive drug with a sample suspected of containing the addictive drug. including. The method can also include detecting or quantifying binding of the addictive drug to the artificial receptor.

  FIG. 4 schematically illustrates an example of a method for evaluating candidate artificial receptors for binding to a test ligand. This embodiment of the method of the invention can be used to detect test ligands such as addictive drugs. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with an addictive drug and identifying which receptor binds the addictive drug. The method can include generating an array or device comprising the working artificial receptor or receptor complex. In one embodiment, the method can include using an array or device to detect or characterize the addictive drug in a sample, such as a biological, laboratory, or evidence sample.

  FIG. 5 schematically illustrates an embodiment of the method of the invention using an array of candidate artificial receptors. Embodiments of the invention can generate an assay or system for characterizing or detecting addictive drugs using an array comprising a significant number of the artificial receptors of the invention. The method can include evaluating an array including a significant number of candidate artificial receptors for binding an addictive drug. The building blocks that make up the artificial receptors can be naive to addictive drugs. An addictive drug can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors may be used to characterize living or field samples, or artificial receptors that can be used in methods for characterizing or detecting addictive drugs (eg, working or working artificial receptors) Complex).

  In one embodiment, the method can include generating or using the selected working artificial receptor or receptor complex on a substrate. The substrate may include an artificial receptor that acts on a single addictive drug or an artificial receptor that acts on multiple addictive drugs. For example, the method can include contacting the artificial receptor with a sample. A substrate comprising a working artificial receptor for a single addictive drug can be used in a method or system for detecting the addictive drug. Binding to the working artificial receptor includes the addictive drug. Substrates comprising working artificial receptors for multiple addictive drugs can be used in a method or system for detecting one, some, or all of the addictive drugs. Binding to the working artificial receptor for a particular addictive drug or addictive drug indicates that the sample contains such addictive drug or addictive drug.

  The working artificial receptor or receptor complex can be configured to provide a pattern that indicates the presence of one or more of the addictive drugs. The method can include detecting the binding pattern of the sample and comparing it to binding patterns from known samples. FIG. 6 schematically shows the binding pattern on an array of working artificial receptors. Such patterns and schemes can be used to identify various test ligands including addictive drugs.

  The methods of the present invention can develop or use multiple acting receptors that are specific for a particular addictive drug or characteristics of the addictive drug. That is, the acting receptor can be specific for a particular addictive drug, but different receptors interact with different distinct ligands, functional groups, or structural features of the addictive drug. obtain. Such a method can provide a robust assay for the presence of addictive drugs. For example, such a robust assay can reduce the chance of false positive or false negative results in comparison to assays that rely on a single unique receptor to detect a given addictive drug. In addition, this embodiment of the present invention develops or uses acting receptors that exhibit higher binding affinity due to interaction with multiple ligands or features of the dependent drug (eg, multivalent binding). can do.

  Suitable addictive drugs include cannabinoids (e.g., hashish and marijuana), inhibitors (e.g., barbituric acid hypnotics, benzodiazepines, gammahydroxybutyric acid, metacaron), dissociative anesthetics (e.g., ketamine, PCP, and PCP) Analogues), hallucinogens (e.g. LSD, mescaline, thyrosibine), opiates or opioids (e.g. codeine, fentanyl, fentanyl analogues, heroin, morphine, opium, oxycodone chloride, hydrocodone tartrate), stimulants ( Examples include amphetamine, cocaine, methylenedioxy-methamphetamine, methamphetamine, methylphenidate, nicotine), inhalants (eg, solvents) and the like.

  Suitable addictive drugs include motor performance enhancers such as stimulants and beta blockers, anabolic agents, oxygen carrier promoters, masking agents, and inhalants. Suitable stimulants include caffeine and amphetamine. Suitable beta blockers include salbutamol (used in asthma inhalers) and the like. Suitable anabolic agents include steroids (eg anabolic steroids), steroid analogs, and growth hormone. Suitable oxygen carrier promoters include erythropoietin and the like.

Methods for binding or detecting isomers In one embodiment, the present invention may include methods and / or devices for binding or detecting an isomer or multiple isomers of a compound. Methods and systems for detection can include methods and systems for clinical chemistry, environmental analysis, and all types of diagnostic assays. For example, the artificial receptor can be contacted with a sample that contains or is suspected of containing at least one isomer of a compound. The binding of one or more isomers of the compound to the artificial receptor can then be detected. The binding result can then be interpreted as providing information about the isomer. In one embodiment, the present invention detects an isomer of a compound in a sample comprising contacting an artificial receptor specific for the isomer with a sample suspected of containing the isomer. Including methods. The method can also include detecting or quantifying the binding of the isomer to the artificial receptor.

  The methods of the invention can be used for stereoisomers (eg, geometric isomers or optical isomers), optical isomers (eg, enantiomers and diastereomers), geometric isomers (eg, cis- and trans-isomers). It can be applied to isomers such as The methods of the invention can be used to develop acting or lead artificial receptors or working artificial complexes that can bind to one or more isomers of a compound (eg, an enantioselective receptor environment). ). For example, the artificial receptor or complex can bind to one stereoisomer of a compound, but binds only weakly or not to the other stereoisomer of the compound. For example, the artificial receptor or complex can bind to one geometric isomer of a compound, but binds weakly or not to the other geometric isomer. For example, the artificial receptor or complex can bind to one optical isomer of a compound, but binds weakly or not at all to another optical isomer. For example, the artificial receptor or complex can bind to one enantiomer of a compound, but binds weakly or not to the other enantiomer. For example, the artificial receptor or complex can bind one diastereomer of a compound, but binds weakly or not at all to the other diastereomer.

  FIG. 4 schematically illustrates an example of a method for evaluating candidate artificial receptors for binding to a test ligand. This embodiment of the method of the invention can be used to detect test ligands, such as isomers of compounds. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with an isomer and identifying whether the receptor binds the isomer. The method can include generating an array or device comprising the working artificial receptor or receptor complex. In one embodiment, the method can include using an array or device to detect or characterize isomers in a sample, such as a biological, laboratory, or clinical sample.

  FIG. 5 schematically illustrates an embodiment of the method of the invention using an array of candidate artificial receptors. This embodiment of the method can use an array containing a significant number of the artificial receptors of the invention to generate an assay or system for characterizing or detecting isomers. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding isomers. The building blocks that make up the artificial receptors can be naive to the isomers. The isomer may exhibit characteristic binding to one or several of the candidate artificial receptors from the array. One or several artificial receptors characterize living, clinical, or research samples, or artificial receptors that can be used in methods for characterizing or detecting isomers (eg, working artificial receptors or actions As an artificial receptor complex).

  In one embodiment, the method can include generating or using a selected working artificial receptor or receptor complex on a substrate. The substrate may comprise an artificial receptor that acts on a single isomer or an artificial receptor that acts on multiple isomers. For example, the method can include contacting the artificial receptor with a sample. A substrate comprising a working artificial receptor for a single isomer can be used in a method or system for detecting the isomer. Binding to the working artificial receptor indicates that the sample contains the isomer. Substrates comprising working artificial receptors for multiple isomers can be used in a method or system for detecting one, some, or all isomers. Binding to the working artificial receptor for a particular isomer or isomers indicates that the sample contains such isomer or isomers.

  The working artificial receptor or receptor complex can be configured to provide a pattern indicating the presence of one or more isomers. The method includes detecting a binding pattern of a sample and comparing it to a binding pattern from a known sample. FIG. 6 schematically shows the binding pattern on an array of working artificial receptors. Such patterns and schemes can be used to identify various test ligands including isomers.

  In one embodiment, the method can generate an assay or system for characterizing or detecting stereoisomers using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the stereoisomer. The building blocks that make up the artificial receptors can be naive to stereoisomers. Stereoisomers can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize research or clinical samples or stereoisomers Can be used in a method for characterizing or detecting.

  In one embodiment, the method uses an array comprising a significant number of artificial receptors of the invention to assay for characterizing or detecting geometric isomers (eg, cis- and trans-isomers). Or a system can be generated. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to geometric isomers (eg, cis- and trans-isomers). The building blocks that make up the artificial receptors can be naive to geometric isomers. Geometric isomers can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize research or clinical samples, or geometric isomerism Can be used in methods for characterizing or detecting isomers (eg, cis- and trans-isomers).

  In one embodiment, the method can generate an assay or system for characterizing or detecting optical isomers using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to an optical isomer. The building blocks that make up the artificial receptors can be naive to the optical isomers. An optical isomer may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize research or clinical samples or optical isomers Can be used in a method for characterizing or detecting.

  In one embodiment, the method can generate an assay or system for characterizing or detecting enantiomers using an array comprising a significant number of artificial receptors of the invention. The method can include evaluating an array including a significant number of candidate artificial receptors for binding to the enantiomer. The enantiomer may exhibit characteristic binding to one or several of the candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize research or clinical samples, or enantiomeric It can be used in a method for characterizing or detecting the body.

  In one embodiment, the method can be used in an assay or system for characterizing or detecting diastereomers using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the diastereomer. The building blocks that make up the artificial receptors can be naive to diastereomers. Diastereomers can exhibit characteristic binding to one or several of the candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize research or clinical samples or diastereomers Can be used in a method for characterizing or detecting.

Methods for binding or detecting peptides In one embodiment, the present invention may include methods and / or devices for binding or detecting peptides. Methods and systems for detection can include methods and systems for all types of clinical chemistry, environmental analysis, and diagnostic assays. For example, the artificial receptor can be contacted with a sample comprising or suspected of containing at least one peptide. The binding of one or more of the peptides to the artificial receptor can then be detected. The binding can then be interpreted as providing information about the sample. In one embodiment, the present invention includes a method for detecting a peptide in a sample comprising contacting an artificial receptor specific for the peptide with a sample suspected of containing the peptide. The method can also include detecting or quantifying the binding of the peptide to the artificial receptor.

  FIG. 4 schematically illustrates an example of a method for evaluating candidate artificial receptors for binding to a test ligand. This embodiment of the method of the invention can be used to detect test ligands such as peptides. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with a peptide and identifying which receptor binds the peptide. The method can include generating an array or device comprising the working artificial receptor or receptor complex. In one embodiment, the method can include using an array or device to detect or characterize the peptide in a sample, such as a biological, laboratory, or clinical sample.

  FIG. 5 schematically illustrates an embodiment of the method of the invention using an array of candidate artificial receptors. This embodiment of the method can use an array containing a significant number of the artificial receptors of the invention to generate an assay or system for characterizing or detecting peptides. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding peptides. The building blocks that make up the artificial receptors can be naive to the peptide. The peptide can exhibit characteristic binding to one or several of the candidate artificial receptors from the array. One or several artificial receptors characterize biological or environmental samples, or artificial receptors that can be used in methods for characterizing or detecting the peptides (eg, working artificial receptors or working artificial receptor complexes). ) Can be selected.

  FIG. 7 schematically illustrates an example of a method for developing a method and system for detecting a test ligand, such as a peptide or a mixture of peptides. This embodiment of the method of the invention includes evaluating a plurality (eg, an array) of candidate artificial receptors for binding to each of a plurality of peptides. The building blocks that make up the artificial receptors can be naive to one or more peptides. A plurality of peptides may include the peptides found in cells or organisms. The method can include detecting binding of individual peptides to a subset of a plurality or an array of candidate artificial receptors. The method can include detecting binding to a candidate artificial receptor of a subset or all or a plurality of or a subset of peptides found in a cell or organism. This can be expected to develop a working artificial receptor or artificial receptor complex for each peptide or mixture of peptides.

  Thus, each peptide or mixture of peptides can provide a pattern of bound receptors in multiple arrays. The pattern of bound receptors can be characteristic of the peptide or mixture of peptides or a sample containing the peptide or mixture of peptides. The method may include saving the representation of the binding pattern as an image or data structure. The display of the coupling pattern can be evaluated either by the operator or by the data processing system. The method can include such an assessment. A binding pattern from an unknown sample that matches the binding pattern for a particular peptide then characterizes the unknown sample as that peptide. Then, the binding pattern from an unknown sample that matches the binding pattern for a particular mixture of peptides is unknown as including or being a mixture of peptides, or including or being an organism or cell containing a mixture of peptides. Characterize the sample. Multiple binding patterns can be stored as a database.

  Specific examples of the method shown may include creating an array of artificial receptors. This embodiment also includes accumulating a database of binding patterns of specific peptides or peptide mixtures, for example by probing the array with a plurality of individual peptides or peptides found in cells or organisms. May be included. The array can be contacted with an unidentified peptide or mixture of peptides to create a test binding pattern. The method can then compare the test binding pattern with the binding pattern of a known peptide or mixture of peptides in a database to characterize or classify an unidentified peptide, mixture of peptides, or cells or organisms. it can. In one embodiment, a database and receptor array has already been constructed, and the method probes the array with an unknown peptide or mixture of peptides to create a test binding pattern, which is then Comparing this binding pattern with a binding pattern in a database to characterize or classify an unidentified peptide, mixture of peptides, or cells or organisms.

  An array constructed to identify a mixture of peptides can be contacted with a sample from an organism, cell, or tissue of interest. Peptides that bind to the array can characterize or detect an organism, cell, or tissue; can indicate a disorder caused by or affecting the cell or tissue; caused by or affecting the cell or tissue Can indicate successful therapy of the disorder; characterize the disease process; identify therapeutic leads or strategies, etc.

  In one embodiment, the method can include generating or using a selected working artificial receptor or receptor complex on a substrate. The substrate can include an artificial receptor that acts on a single peptide or an artificial receptor that acts on multiple peptides. For example, the method can include contacting the artificial receptor with a sample. Substrates comprising artificial receptors that act on a single peptide can be used in a method or system for detecting the peptide. Binding to the working artificial receptor indicates that the sample contains the peptide. Substrates comprising artificial receptors that act on multiple peptides can be used in a method or system for detecting one, some, or all of the peptides. Binding to the acting artificial receptor for a particular peptide or peptides indicates that the sample contains such a peptide or peptides.

  The working artificial receptor or receptor complex can be configured to provide a pattern that indicates the presence of one or more of the peptides. The method can include detecting a binding pattern of the sample and comparing it to a binding pattern from a known sample. FIG. 6 schematically shows the binding pattern on an array of working artificial receptors. Such patterns and schemes can be used to identify various test ligands including peptides.

  The methods of the invention can develop or use multiple acting receptors that are specific for a particular addictive drug or characteristics of the addictive drug. That is, the acting receptor can be specific for a particular peptide, but different receptors can interact with different distinct ligands, functional groups, or structural features of the peptide. Such a method can provide a robust assay for the presence of peptides. For example, such a robust assay can reduce the chance of false positive or false negative results in comparison to assays that rely on a single unique receptor to detect a given peptide. In addition, this embodiment of the invention can develop or use a working receptor, which is more due to interaction with multiple ligands or features for the same peptide (e.g., multivalent binding). High binding affinity.

  In one embodiment, the method can generate an assay or system for characterizing or detecting peptides using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding peptides. The building blocks that make up the artificial receptors can be naive to the test ligand. Peptides can exhibit characteristic binding to one or several candidate artificial receptors from the array. Select one or several artificial receptors as artificial receptors that can be used in a method for characterizing a biological sample or characterizing or detecting peptides (eg, working artificial receptors or working artificial receptor complexes) can do.

  The method of the present invention can be used as a lead for pharmaceutical development or to modulate the activity of the peptide, binding specific blocks and / or building blocks that create these receptors (eg, bound to a backbone molecule). Selecting as an active agent to do. Select the artificial receptor or building block that creates the artificial receptor and bind to the portion of the peptide that is required for its interaction with other macromolecules (e.g., carbohydrates, proteins, or polynucleotides); Therefore, this interaction can be disturbed.

Methods for Binding or Detecting Proteins or Proteomes In one embodiment, the present invention may include methods and / or devices for binding or detecting proteins, one or more proteins, or proteomes. Methods and systems for detection can include methods and systems for clinical chemistry, environmental analysis, diagnostic assays, and proteomic analysis. For example, the artificial receptor can be contacted with a sample containing at least one protein or one proteome. The building blocks that make up the artificial receptors can be naive to the test ligand. The binding of one or more proteins to the artificial receptor can then be detected. The binding results can then be interpreted to provide information about the sample, eg, the proteome. In one embodiment, the present invention includes a method for detecting a protein in a sample comprising contacting an artificial receptor specific for the protein with a sample suspected of containing the protein. The method can also include detecting or quantifying the binding of the protein to the artificial receptor.

  FIG. 4 schematically illustrates an example of a method for evaluating candidate artificial receptors for binding to a test ligand. This embodiment of the method of the invention can be used to detect test ligands such as one or more proteins. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with a protein and identifying which receptor binds the protein. The method can include generating an array or device comprising the working artificial receptor or receptor complex. In one embodiment, the method can include using an array or device to detect or characterize proteins in a sample, such as a biological, research, or environmental sample.

  In one embodiment, the method can include generating or using a selected working artificial receptor or receptor complex on a substrate. The substrate can include a single protein for multiple proteins or a working artificial receptor for a working artificial receptor. For example, the method can include contacting the artificial receptor with a sample. A substrate comprising a working artificial receptor for a single protein can be used in a method or system for detecting the protein. Binding to the working artificial receptor indicates that the sample contains protein. Substrates comprising working artificial receptors for multiple proteins can be used in a method or system for detecting one, some, or all proteins. Binding to the working artificial receptor for a particular protein or protein indicates that the sample contains such a protein or protein.

  A working artificial receptor or receptor complex can be configured to provide a pattern that indicates the presence of one or more proteins. The method can include detecting the binding pattern of the sample and comparing it to binding patterns from known samples. FIG. 6 schematically shows the binding pattern on an array of working artificial receptors. Such patterns and schemes can be used to identify various test ligands including proteins.

  FIG. 7 schematically illustrates an embodiment of a method for developing a method and system for detecting a test ligand such as a protein or proteome. This embodiment of the method of the invention includes evaluating a plurality of candidate artificial receptors (eg, arrays) for binding to each of a plurality of test ligands. The building blocks that make up the artificial receptors can be naive to the test ligand. The plurality of test ligands can include a plurality of proteins. The plurality of test ligands can include proteins that make up the proteome of a cell or organism. The method can include detecting binding of individual proteins to a subset of an array of multiple or array of candidate artificial receptors. The method can include detecting binding of a protein that creates a proteome to a subset or all of a plurality or array of candidate artificial receptors. This can be expected to develop a working artificial receptor or artificial receptor complex for each protein or for the proteome.

  Thus, each protein or proteome may provide multiple or example binding receptor patterns. The pattern of bound receptors can be characteristic of a protein or proteome or a sample containing a protein or proteome. The method may include saving the display of the binding pattern as an image or data structure. The display of the coupling pattern can be evaluated either by the operator or by the data processing system. The method can include such an assessment. A binding pattern from an unknown sample that matches the binding pattern for a particular protein then characterizes the unknown sample when it contains the protein. A binding pattern from an unknown sample that matches the binding pattern for a particular proteome then characterizes the unknown sample as including or being the proteome or including or being an organism or cell having the proteome. Similarly, binding patterns from unknown samples can be assessed against multiple specific protein or proteome patterns, and samples can be characterized as containing one or more proteins or proteomes. Multiple binding patterns can be stored as a database.

  Specific examples of the method shown may include creating an array of artificial receptors. This embodiment may also include accumulating a database of specific protein or proteome binding patterns, eg, by probing the array with a plurality of individual proteins or proteomes. A test binding pattern can be created by contacting the array with an unidentified protein or proteome. The method can then compare test binding patterns with known protein or proteome binding patterns in a database to characterize or classify unidentified proteins, proteomes, or cells or organisms. In one embodiment, an array of databases and receptors has already been constructed, and the method probes the array with an unknown protein or proteome to create a test binding pattern, and then an unidentified protein, Comparing this binding pattern to the binding pattern in the database to characterize or classify the proteome, or cell or organism.

  A proteome array can be contacted with a sample from an organism, cell, or tissue of interest. Proteins that bind to the proteome array can characterize or detect an organism, cell or tissue; can indicate a disorder caused by or affecting a cell or tissue; caused by or affected by an organism May indicate successful therapy of disorders affecting; characterizing disease processes; identifying therapeutic leads or strategies, etc.

  The methods of the present invention can develop or use multiple working receptors specific for a particular protein or characteristic of the protein. That is, the acting receptor can be specific for a particular protein, but different receptors can interact with different distinct ligands, functional groups, or structural features of the protein. Such a method can provide a robust assay for the presence of protein. For example, such a robust assay can reduce the chance of false positive or false negative results in comparison to assays that rely on a single unique receptor to detect a given protein. Furthermore, this embodiment of the method develops or detects a working receptor that exhibits a higher binding affinity by interacting with multiple ligands or features for the same protein (eg, multivalent binding). be able to.

  In one embodiment, the method can generate an assay or system for characterizing or detecting a protein using an array comprising a significant number of the artificial receptors of the invention. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the protein. The building blocks that make up the artificial receptors can be naive to the test ligand. The protein may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in a method for characterizing a biological sample or characterizing or detecting a protein (eg, working artificial receptor or working artificial receptor complex). Can be selected as

  The methods of the present invention may be used as a lead for drug development or as an activity of a protein that binds a specific protein and / or the building blocks that make up these receptors (eg, bound to a backbone molecule). Selecting as the active agent to modulate. The artificial receptor or building block that makes up the artificial receptor can be selected to bind to or disrupt the activity of the enzyme or the ligand binding site of the receptor. The artificial receptor or building block that creates the artificial receptor is selected to bind to the portion of the protein that is required for its interaction with other macromolecules (e.g., carbohydrates, proteins, or polynucleotides) Therefore, this interaction can be disturbed. The artificial receptor or building block that makes up the artificial receptor can be selected to bind to the binding site of the receptor and act as an agonist of the receptor.

  The methods of the invention can include selecting a working artificial receptor that binds a preselected protein for use in a system for proteome analysis. A working artificial receptor for a preselected protein can be provided on the substrate and the protein bound to the receptor. In one embodiment, selection and binding uses a plurality of different acting artificial receptors for a preselected protein. The plurality of artificial receptors can bind to different features for the preselected protein and leave the different features free for the preselected protein intact. This embodiment of the method involves contacting a working receptor with a preselected protein bound to at least one candidate binding partner for the preselected protein. The method can include detecting the binding or absence of binding of a candidate binding partner to a preselected protein. A candidate binding partner that binds to a preselected protein can be considered a lead binding partner.

  In one embodiment, the method comprises contacting a working receptor with a preselected protein bound to a proteome of a cell or organism that serves as a source of candidate binding partners. The method can then recover one or more lead binding partners from the proteome. It can then characterize the proteome with or without a binding partner for the preselected protein.

  In one embodiment, the present artificial receptors can be used in proteomics studies. In such embodiments, an array of candidate or working artificial receptors can be contacted with a mixture of peptides, polypeptides, and / or proteins. Each mixture can generate a characteristic fingerprint of binding to the array. In addition, identification of a specific receptor environment for a target peptide, polypeptide, and / or protein can be used to isolate and analyze the target. That is, in yet another embodiment, a specific receptor surface can be used in an affinity purification method such as, for example, affinity chromatography.

  In one embodiment, candidate artificial receptors of the present invention can be used to find receptor surfaces that bind proteins in a preferred conformation or orientation. Many proteins (eg, antibodies, enzymes, receptors) are stable and / or active in specific environments. A defined receptor surface can be used to create a binding environment that selectively retains or orients proteins for maximum stability and / or activity. In one embodiment, the artificial receptors of the present invention can be used to form a bioactive surface. For example, the receptor surface can be used to specifically bind the active conformation of an antibody or enzyme.

  In one embodiment, the method can include labeling the protein while remaining bound to the artificial receptor. The resulting protein will be labeled on the portion available for labeling reagent, not the portion bound to the artificial receptor. The method can include releasing the labeled protein from the artificial receptor. Determining the distribution of the label on the protein indicates which part of the protein has bound to the receptor.

  In certain embodiments, the artificial receptors of the present invention can be used to distinguish between two conformations of a single protein. Certain proteins exist in more than one stable conformation. In one embodiment, the working artificial receptor or complex of the invention can bind the first conformation of the protein. In one embodiment, the working artificial receptor or complex of the invention can bind a second conformation of the protein. In one embodiment, the working artificial receptor or complex of the present invention can bind to the first conformation of the protein but not to the second conformation of the same protein. In one embodiment, the working artificial receptor or complex of the present invention can bind to the second conformation of the protein but not to the first conformation of the same protein.

  For example, in one embodiment, a working artificial receptor or complex of the invention can bind a first or non-infectious conformation of a prion, where the second or infectious conformation is Do not combine. For example, in embodiments, the working artificial receptor or complex of the invention can bind a second or infectious conformation of a prion, but not its first or non-infectious conformation. For example, in embodiments, the working artificial receptor or complex of the present invention may bind the first or non-plaque formation of β-amyloid, but not its second or plaque-forming conformation. For example, in embodiments, the working artificial receptor or complex of the invention can bind a second or plaque-forming conformation of β-amyloid, but not its second or non-plaque-forming conformation. .

  In one embodiment, the method can generate an assay or system for characterizing or detecting a desired conformation of a protein using an array comprising a significant number of the artificial receptors of the invention. it can. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the desired conformation of the protein. The building blocks that make up the artificial receptors can be naive to the protein or its desired conformation. The desired conformation of the protein may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as an artificial receptor (eg, working artificial receptor or working artificial receptor complex), which characterizes a biological sample or a desired conformation of a protein. It can be used in a method for characterizing or detecting a conformation.

  In one embodiment, the method comprises an assay or system for characterizing or detecting a first or non-infectious conformation of prions using an array comprising a significant number of artificial receptors of the invention. Can be generated. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the first or non-infectious conformation of the prion. The building blocks that make up the artificial receptors can be naive to the prion. The first or non-infectious conformation of the prion can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as an artificial receptor (eg, working artificial receptor or working artificial receptor complex), which characterizes a biological sample or the first or It can be used in a method for characterizing or detecting non-infectious conformations.

  In one embodiment, the method comprises an assay or system for characterizing or detecting a second or infectious conformation of a prion using an array comprising a significant number of artificial receptors of the invention. Can be generated. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a second or infectious conformation of the prion. The building blocks that make up the artificial receptors can be naive to the test ligand. The second or infectious conformation of the prion can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as an artificial receptor (eg, working artificial receptor or working artificial receptor complex), which characterizes a biological sample or a second of a prion. Or it can be used in a method for characterizing or detecting infectious conformations.

  In one embodiment, the method of the invention provides a receptor or receptor system capable of discriminating between a first or non-infectious conformation of prions and a second or infectious conformation of prions. Including developing. Such a method can include selecting a working artificial receptor, or the complex can bind to the first or non-infectious conformation of the prion, while the second or It does not bind to infectious conformations. This embodiment can include selecting a working artificial receptor, or the complex can bind the second or infectious conformation of the prion, but the first or non-infection of the prion. Sex conformation does not bind. When used together, these two sets of working artificial receptors or systems can characterize a biological sample as containing one or both forms of prions.

  In one embodiment, the method uses an array comprising a significant number of artificial receptors of the invention to assay or characterize or detect a first or non-plaque-forming conformation of β-amyloid or A system can be generated. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a first or non-plaque-forming conformation of β-amyloid. The building blocks that make up the artificial receptors can be naive to β-amyloid. The first or non-plaque forming conformation of β-amyloid may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in a method for characterizing a biological sample or characterizing or detecting a first or non-plaque-forming conformation of β-amyloid.

  In one embodiment, the method comprises an assay for characterizing or detecting a second or plaque-forming conformation of β-amyloid using an array comprising a significant number of artificial receptors of the invention or A system can be generated. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a second or plaque forming conformation of β-amyloid. The building blocks that make up the artificial receptors can be naive to β-amyloid. The second or plaque-forming conformation of β-amyloid can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (e.g., working artificial receptors or working artificial receptor complexes), which characterize a biological sample or β-amyloid It can be used in methods for characterizing or detecting two or plaque-forming conformations.

  In one embodiment, the methods of the invention allow a receptor to discriminate between a first or non-plaque-forming conformation of β-amyloid and a second or plaque-forming conformation of β-amyloid. Or developing a receptor system. Such methods select acting artificial receptors or complexes that can bind to the first or non-plaque-forming conformation of β-amyloid but cannot bind to the second or plaque-forming conformation of β-amyloid. Can include. This embodiment selects a working artificial receptor or complex that can bind to the second or plaque-forming conformation of β-amyloid but cannot bind to the first or non-plaque-forming conformation of β-amyloid. Can be included. When used together, these two sets of working artificial receptors or systems can characterize biological samples as containing one or both forms of β-amyloid.

  In one embodiment, the method can generate an assay or system for characterizing or detecting cholera toxin using an array comprising a significant number of artificial receptors of the invention. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to cholera toxin. The building blocks that make up the artificial receptors can be naive to the test ligand. Cholera toxin can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize biological samples or characterize cholera toxin Or it can be used in a method for detection.

  In one embodiment, the method may generate an assay or system for characterizing or detecting at least one protein of a cancer cell using an array comprising a significant number of the artificial receptors of the invention. it can. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a cancer cell protein. The building blocks that make up the artificial receptors can be naive to the test ligand. Cancer cell proteins can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize biological samples or cancer cell proteins Or it can be used in a method for detection.

  In one embodiment, the methods of the invention can include contacting a working artificial receptor or array with a sample from a cell or tissue suspected of being cancerous or containing a tumor. The sample can be serum. Binding of at least one protein to the working artificial receptor or array can indicate or characterize the presence of a particular cancer or tumor, for example, by characterizing the pattern of protein present.

  Cancers that can be detected or characterized by such methods include, for example, bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer including small cell lung cancer, esophageal cancer, gallbladder cancer, uterine cancer, pancreatic cancer, Skin cancer including gastric cancer, cervical cancer, thyroid cancer, prostate cancer, and squamous cell carcinoma; leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma Hematopoietic tumors of the lymphoid system, including hair cell lymphoma and Burkitt lymphoma; Hematopoietic tumors of the myeloid lineage, including acute and chronic myelogenous leukemia, myelodysplastic syndrome and promyelocytic leukemia; fiber Tumors of mesenchymal origin, including sarcomas and rhabdomyosarcomas; tumors of the central and peripheral nervous systems, including astrocytoma, neuroblastoma, glioma and schwannoma; black Including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, thyroid small cell carcinoma, the other tumors, including Kaposi's sarcoma and the like.

Methods of binding or detecting microorganisms In one embodiment, the present invention may include methods and / or devices for binding or detecting microorganisms such as, for example, cells or viruses. Methods and systems for detection can include methods and systems for clinical chemistry, environmental analysis, and all types of diagnostic assays. For example, the artificial receptor can be contacted with a sample that contains or is suspected of containing at least one microorganism, eg, a cell or virus. The building blocks that make up the artificial receptors can be naive to the test ligand. The binding of one or more microorganisms to the artificial receptor can then be detected. The binding results can then be interpreted to provide information about the sample. In one embodiment, the present invention involves contacting an artificial receptor specific for a microorganism, eg, a cell or virus, with a sample suspected of containing a microorganism, eg, a cell or virus, for example in a sample Includes methods for detecting microorganisms such as cells or viruses. The method can also include detecting or quantifying the binding of a microorganism, such as a cell or virus, to an artificial receptor.

  FIG. 4 schematically illustrates an example of a method for evaluating candidate artificial receptors for binding to a test ligand. This embodiment of the method of the invention can be used to detect test ligands such as microorganisms such as cells or viruses. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with a microorganism, eg, a cell or virus, and identifying which receptor binds the microorganism. The method can include generating an array or device comprising the working artificial receptor or receptor complex. In one embodiment, the method can include using an array or device to detect or characterize microorganisms, such as cells or viruses, in a sample such as a biological, research, or environmental sample.

  FIG. 5 schematically illustrates an embodiment of the method of the invention using an array of candidate artificial receptors. This embodiment of the method can use an array containing a significant number of the artificial receptors of the invention to generate an assay or system for characterizing or detecting a microorganism, eg, a cell or virus. The method can include, for example, evaluating an array that includes a significant number of candidate artificial receptors for binding a microorganism, such as a cell or virus. The building blocks that make up the artificial receptors can be naive to the test ligand. The microorganism may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be selected as artificial receptors (eg, working artificial receptors or working artificial receptor complexes), which characterize a biological sample or, for example, cells or It can be used in a method for characterizing or detecting microorganisms such as viruses.

  FIG. 7 schematically illustrates an example of a method for developing a method and system for detecting a test ligand, such as a disease-causing organism. This embodiment of the method of the present invention includes evaluating a plurality (eg, array) candidate artificial receptors for binding to each of a plurality of test ligands such as disease causing organisms. The building blocks that make up the artificial receptors can be naive to the test ligand. The method can include detecting binding of each test ligand (eg, a disease causing organism) to a plurality or a subset of candidate artificial receptors in an array. This can be expected to develop a working artificial receptor or artificial receptor complex for each of a plurality of test ligands.

  Thus, each test ligand (eg, a disease causing organism) can provide a pattern of multiple or arrayed receptors. The pattern of bound receptors can be characteristic for a test ligand or a sample containing a test ligand. The method may include saving the representation of the binding pattern as an image or data structure. The display of the coupling pattern can be evaluated either by the operator or the data processing system. The method can include such an assessment. A binding pattern from an unknown sample that matches the binding pattern for a particular test ligand (eg, a disease causing organism) then characterizes the unknown sample as containing the test ligand. Similarly, binding patterns from unknown samples can be evaluated against a plurality of specific test ligand patterns, and a sample can be characterized as containing one or more test ligands. Multiple binding patterns can be stored as a database.

  Specific examples of the method shown may include creating an array of artificial receptors. This embodiment may also include accumulating a database of binding patterns of organisms that cause specific diseases, for example by probing multiple individual organisms and arrays. The array can be contacted with an unidentified organism to create a test binding pattern. The method can then compare the test binding pattern to the binding patterns of known organisms in the database to characterize or classify the unidentified organisms. In one embodiment, a receptor database and array have already been constructed and the method probes the array with an unknown organism to create a test binding pattern, which is then identified. Comparing with binding patterns in the database to characterize or classify the living organisms.

  In one embodiment, the method can include generating or using a selected working artificial receptor or receptor complex on a substrate. The substrate can include, for example, a working artificial receptor for a single microorganism such as a cell or virus, or a working artificial receptor for a plurality of microorganisms such as cells or viruses. For example, the method can include contacting an artificial receptor with a sample. For example, a substrate comprising a working artificial receptor for a single microorganism, such as a cell or virus, can be used in a method or system for detecting the microorganism. Binding to the working artificial receptor indicates that the sample contains microorganisms. For example, a substrate comprising a working artificial receptor for a plurality of microorganisms such as cells or viruses can be used in a method or system for detecting one, some, or all microorganisms. Binding to the working artificial receptor for a particular microorganism or microorganisms indicates that the sample contains such microorganisms or microorganisms.

  A working artificial receptor or receptor complex can be configured to provide a pattern that indicates the presence of one or more microorganisms, eg, cells or viruses. The method can include detecting a binding pattern of the sample and comparing it to a binding pattern from a known sample. FIG. 6 schematically shows the binding pattern on an array of working artificial receptors. Such patterns and schemes can be used to identify various test ligands including microorganisms.

  The methods of the present invention can develop or use a specific microorganism or multiple acting receptors specific for the characteristics of the microorganism. That is, the acting receptor can be specific for a particular microorganism, but different receptors interact with different distinct antigens (e.g., proteins or carbohydrates), ligands, or microbial features. Can do. Such a method can provide a robust assay for the presence of microorganisms. For example, such a robust assay can reduce the chance of false positive or false negative results in comparison to assays that rely on a single unique receptor to detect a given microorganism. Furthermore, this embodiment of the method can develop or use acting receptors that exhibit higher binding affinity due to multiple antigens or ligands on the same microorganism (eg, multivalent binding).

  In one embodiment, the method may generate an assay or system for characterizing or detecting bacteria using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to bacteria. The building blocks that make up the artificial receptors can be naive to the test ligand. Bacteria may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in methods for characterizing a biological sample or characterizing or detecting bacteria (eg, working artificial receptors or working artificial receptors complexes). Can be selected as

  In one embodiment, the method can generate an assay or system for characterizing or detecting viral particles using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to viral particles. The building blocks that make up the artificial receptors can be naive to the test ligand. Viral particles may exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in methods for characterizing biological samples or for characterizing or detecting viral particles (eg, working artificial receptors or working artificial receptors). Complex).

  In one embodiment, the method can generate an assay or system for characterizing or detecting biohazards using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a biohazard. The building blocks for creating artificial receptors can be naive to the test ligand. A biohazard can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors characterize a biological sample, or artificial receptors that can be used in a method for characterizing or detecting biohazards (eg, working artificial receptors or working artificial receptor complexes) Body).

  In one embodiment, the method can generate an assay or system for characterizing or detecting Vibrio cholerae using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising significant candidate artificial receptors for binding to Vibrio cholerae. The building blocks that make up the artificial receptors can be naive to Vibrio cholerae. Vibrio cholerae can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in methods for characterizing biological samples or in methods for characterizing or detecting Vibrio cholerae (e.g., working artificial receptors or working artificial receptor complexes). Body).

  In one embodiment, the method can generate an assay or system for characterizing or detecting microorganisms using an array comprising a significant number of the present artificial receptors. The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to the microorganism. The building blocks that make up the artificial receptors can be naive to the test ligand. One or more artificial receptors that bind a microorganism under suitable conditions can be selected for use on an affinity support capable of binding the microorganism. Under appropriate conditions, one or more artificial receptors that are sufficiently rigid to bind microorganisms or cells can be selected, the building blocks of which are incorporated into the scaffold molecule. Embodiments of the method can use a support having one or more artificial or skeletal receptors on its surface for binding or immobilizing microorganisms. Supports having multiple artificial receptors, each on its surface that binds to a different part of the microorganism, can be used for multivalent capture or immobilization of the microorganism.

  The methods of the present invention can be used to lead a specific microorganism and / or an artificial receptor that binds the building blocks that make up these receptors (eg, bound to a backbone molecule) as a lead for pharmaceutical development or the activity of a microorganism. It may include selecting as an active agent to modulate or as an antibiotic against the microorganism.

  In one embodiment, the method can use an array containing a significant number of the artificial receptors of the invention to generate an assay or system for characterizing or detecting a microorganism of clinical or environmental interest. . The method can include evaluating an array comprising a significant number of candidate artificial receptors for binding to a microorganism of clinical or environmental interest. The building blocks for creating artificial receptors can be naive to the test ligand. Microorganisms of clinical or environmental interest can exhibit characteristic binding to one or several candidate artificial receptors from the array. One or several artificial receptors can be used in a method for characterizing biological samples or in methods for characterizing or detecting microorganisms of clinical or environmental interest (eg, working artificial receptors or actions As an artificial receptor complex).

  Suitable microorganisms of clinical or environmental interest include bacteria, mycoplasma, fungi, rickettsia, or viruses. Suitable bacteria or mycoplasma of clinical or environmental interest are Escherichia coli (e.g. E. coli H157: O7), Vibrio cholerae, Acinetobacter caicoaceticus, Haemophilus influenzae, Actinobacillus actinoides, Haemophilus parahaemolyticus, Actinobacillus lignieresoba, enza su Legionella pneumophila, Actinomyces bovis, Leptospira interrogans, Actinomyces israelli, Mima polymorpha, Aeromonas hydrophila, Moraxella lacunata, Arachnia propionica, Burkholderia mallei, Burkholderia pseudomallei, Moraxella osioensis, Arizona , Bartonella bacilliformis, Plesiomonas shigelloides, Bordetella bronchiseptica, Proteus spp, Clostridium difficile, Pseudomonas aeruginosa, Clostridium sordellii, Salmonella cholerasuis, Clostridium tetani, Salmonella enteritidis, Corynebacterium diphne typhi, Edwardsiella tarda, Serratia marcescens, Enterobacter aerogenes, Shigella spp, Staphylococcus epidermidis, Francisella novicida, Vibrio parahaemolyticus, Haemophilus ducreyi, Haemophilus gallinarum, Haemophilus haemolyticus, Bacillusco Borrella spp, Neisseria gonorrhoeae, Campylobacter, Neisseria meningitides, Chlamydia psittaci, Nocardia asteroids, Chlamydia trachomatis, Nocardia brasillensis, Clostridium botulinum, Pasturella haemolytica, Clostridium chauvoei, Pasteurelialos Staphylococcus aureus, Clostridium perfringens, Streptobacillus moniliformis, Clostridium septicum, Cyclospora cayatanensis, Streptococcus agalacetiae, Erysipelothrix insidiosa, Streptococcus pneumoniae, Klebsie Includes lla pneumoniae, Streptococcus pyogenes, Listeria manocytogenes, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, and Francisella tularensis.

  Suitable fungi include Absidia, Piedraia hortae, Aspergillus, Prototheca, Candida, Paecilomyces, Cryptococcus neoformans, Cryptosporidium parvum, Phialaphora, Dermatophilus congolensis, Rhizopus, Epidermophyton, Scopulariopsis, Thophylus , Microsporum, Microsporidia, Wangiella dermatitidis, Mucor, Blastomyces dermatitidis, Giardia lamblia, Entamoeba histolytica, Coccidioides immitis, and Histoplasma capsulatum.

  Appropriate rickettsia or viruses of clinical or environmental interest include coronavirus, hepatitis virus, hepatitis A virus, mucus-paramyxovirus (influenza virus, measles virus, mumps virus, Newcastle disease virus), pico Luna virus (Coxsackie virus, Echo virus, gray leukitis virus), pressure ulcer rickettsia, Rochalimaea Quintana, Rochalimaea vinsonii, Norwalk factor, adenovirus, arenavirus (lymphocytic choriomeningitis, internal orientation ), Herpesvirus group (human herpesvirus, cytomegalovirus, Epstein-Barr virus, calcivirus, pseudorabies virus, varicella virus), human immunodeficiency virus, parainfluenza virus (respiratory syncytial virus, suburb) Subsclerosing panencephalitis virus), picornavirus (gray white pulp virus), smallpox virus (Poxviruses Variola), cowpox virus (contagious molluscum virus, monkeypox virus, orf virus, paravaccinia virus) , Tanapox virus, Vaccinia virus, Yabapox virus, Papova virus (SV 40 virus, BK-virus), Spongiform encephalopathy virus (Kreuzfeld Jacob factor, Kool factor, BSE), Rhabdovirus (rabies virus) , Tobaviruses (rubella virus), Q fever rickettsia, Rickettsia canada, rash typhoid rickettsia, Rocky mountain spotted fever rickettsia, helminth rickettsia, rash fever rickettsia (R. mooseri), spotted fever group factor, blister Stomatitis virus (VSV), and Toga, Arena (eg LCM, Junin, Lassa, Marchupo, Guanarito, etc.), bunya (eg, Hantavirus, Rift Valley fever, etc.), flavivirus (Dengue), and all types of filovirus (eg, Ebola, Marburg, etc.) , Nipah virus, viral encephalitis factor, lacrosse, Kazanur forest disease virus, yellow fever, and West Nile virus.

  Suitable microorganisms of clinical or environmental interest include smallpox virus, Crimea Congo hemorrhagic fever, tick-borne encephalitis virus complex (Absetarov, Hanzalova, Hyple, Kumlinge, Kasanur forest disease virus, Omsk hemorrhagic fever, and Russian spring Summer encephalitis), Marburg, Ebola, Junin, Lassa, Machupo, monkey herpes virus, bluetongue, jumping disease III, Rift Valley fever (Jinga), Wessel's bron, foot-and-mouth disease, Newcastle disease, African swine fever, chicken pox , Swine blister disease, cattle, African horse sickness, avian influenza, and sheep pupae. Other elements of interest include castor bean.

Methods for perturbing binding interactions In one embodiment, the present invention provides methods and / or devices for detecting agents that disrupt molecular binding, eg, a plurality of macromolecules or macromolecules and small molecules. Can be included. Methods and systems for detecting such agents can include methods and systems useful for developing therapeutic agents in clinical chemistry, environmental analysis, and all types of diagnostic assays. In one embodiment, such a method includes reducing binding of a test ligand to one or more working artificial receptors having one or more candidate disruptors. In one embodiment, such a method includes reducing binding partner binding to a test ligand having one or more candidate disruptors, wherein the test ligand binds to one or more working artificial receptors. .

  In one embodiment, the method of the invention comprises selecting a working artificial receptor or a receptor complex that binds a target molecule. This embodiment of the method involves binding the target molecule to acting receptor (s). The method then includes contacting the receptor with the bound target molecule with one or more disruptor candidates. Contact can occur in a high throughput screening format. The method includes selecting one or more disruptor candidates that reduce binding of the target molecule to the acting receptor (s) as the lead disruptor (s).

  Any of the general types of test ligands described herein can be target molecules. In one embodiment, the target molecule may be one that is known to participate in a binding interaction with another molecule. The target molecule may be on the microorganism and the entire microorganism can be used with the target molecule. The target molecule can be a complex of two or more macromolecules, for example a complex of two proteins. The target molecule can be a protein. The target molecule can be a polynucleotide. The target molecule may be on a cell and the whole cell can be used as a target molecule. The target molecule can be a receptor.

  Leading disruptors can be subjected to further evaluation, for example, as candidate therapeutics, candidate vaccines, or candidate antigens. Leading disruptors that disrupt the binding of microorganisms to the artificial receptors of the present invention may be selected for further evaluation as antibiotics against the microorganisms, as candidate immunogens against the microorganisms, or as candidate vaccines against the microorganisms. it can. Leading disrupters that disrupt the binding of macromolecules to the artificial receptors of the present invention are therapeutic agents, as candidate immunogens for the macromolecules or organisms containing the macromolecules, or the macromolecules or macromolecules. Candidate vaccines against the containing organism can be selected for further evaluation.

  FIG. 8 schematically shows a specific example of a method for detecting an agent that disrupts the binding interaction of a target molecule. This embodiment of the method of the invention can be used to detect agents that disrupt the binding interaction of a target molecule, such as a macromolecule or a microorganism. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with a target molecule and identifying which receptor binds the target molecule. The method can include generating an array or device comprising the working artificial receptor or receptor complex. The method can include generating or using a selected working artificial receptor or receptor complex on a substrate such as a slide. The substrate can include a working artificial receptor for a single target molecule or a working artificial receptor for multiple target molecules. The method involves binding of the target molecule (s) to an artificial receptor.

  This illustrated embodiment involves contacting an artificial receptor having a bound target molecule with one or more candidate disruptors. Release of the target molecule from the acting artificial receptor or a decrease in binding of the target molecule to the artificial receptor is a candidate disruptor acting or a leading disruptor and can be selected as such Indicates. A substrate comprising an acting artificial receptor for a single target molecule can be used in a method or system for detecting a disruptor of the binding interaction of the target molecule. Substrates comprising working artificial receptors for multiple target molecules can be used in a method or system for detecting disrupters of binding interactions for one, some, or all target molecules. The method can include washing target molecules that are not bound or released from the support.

  In one embodiment, the method of the invention comprises selecting a working artificial receptor or a receptor complex that binds a protein. This embodiment of the method involves binding the protein to the acting receptor (s). The method then comprises contacting the receptor with a bound protein having one or more disruptor candidates. Contact can occur in a high throughput screening format. The method includes selecting one or more candidate disruptors that reduce binding of the protein to the acting disrupter (s) as the lead disruptor (s).

  Such methods can be used to detect agents that disrupt protein binding interactions. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with a protein and identifying which receptor binds the protein. The method can include generating an array or device comprising the working artificial receptor or receptor complex. The method can include generating or using a selected working artificial receptor or receptor complex on a substrate such as a slide. The substrate can include a working artificial receptor for a single protein or a working artificial receptor for multiple proteins. The method includes binding the protein (s) to an artificial receptor. This embodiment of the method includes contacting an artificial receptor having bound protein with one or more candidate disruptors. Release of the protein from the acting artificial receptor or a decrease in binding of the protein to the artificial receptor indicates that the candidate disruptor is acting or is a leading disruptor and can be selected as such .

  In one embodiment, the disrupter disrupts the protein binding interaction. Such disruptors can be envisioned as mimicking one or more structural features to which the protein binds, eg, on a microorganism, tissue, or cell. Disturbing agents can be evaluated for such mimicry. The mimetic disruptor can then be used as an antigen against structural features on the microorganism, tissue, or cell. The mimetic disruptor can be used as an idiotype or anti-idiotype for structural features on microorganisms, tissues, or cells.

  In one embodiment, the method of the invention comprises selecting a working artificial receptor or receptor complex that binds a microorganism. This embodiment of the method involves binding the microorganism to the acting receptor (s). The method then comprises contacting the receptor with a microorganism that has bound to one or more disruptor candidates. Contact can occur in a high throughput screening format. The method includes selecting one or more disruptor candidates that reduce microbial binding to the acting receptor (s) as the lead disruptor (s).

  Such a method can be used to detect agents that disrupt microbial binding interactions. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. Working artificial receptors can be identified by contacting the array with microorganisms and identifying which receptors bind the microorganisms. The method can include generating an array or device comprising the working artificial receptor or receptor complex. The method can include generating or using a selected working artificial receptor or receptor complex on a substrate, such as a slide. The substrate can include a working artificial receptor for a single microorganism or a working artificial receptor for multiple microorganisms. The method includes binding the microorganism (s) to an artificial receptor. This embodiment of the method includes contacting the artificial receptor with a microorganism associated with one or more candidate disruptors. Release of the microorganism from the acting artificial receptor or binding of the microorganism to the artificial receptor indicates that the candidate disruptor is acting or a leading disruptor and can be selected as such.

  In one embodiment, the disrupter disrupts the binding interaction of the microorganism. Such a disruptor can be envisioned as a mimic of one or more structural features to which the microorganism binds, eg, on a protein, another microorganism, tissue, or cell. Disturbing agents can be evaluated for such mimicry. The mimetic disruptor can then be used as an antigen for structural features on proteins, other microorganisms, tissues, or cells. The mimetic disruptor can be used as an idiotype or anti-idiotype for structural features on proteins, other microorganisms, tissues, or cells.

  In one embodiment, the method of the invention comprises selecting a working artificial receptor or receptor complex that binds cells. This embodiment of the method includes binding a cell to the acting receptor (s). The method then comprises contacting the receptor with cells that have bound to one or more disruptor candidates. Contact can occur in a high throughput screening format. The method includes selecting one or more candidate disruptors that reduce cell binding to the acting receptor (s) as the lead disruptor (s).

  Such methods can be used to detect agents that disrupt cell binding interactions. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. Acting artificial receptors can be identified by contacting the array with cells and identifying which receptors bind the cells. The method can include generating an array or device comprising the working artificial receptor or receptor complex. The method can include generating or using a selected working artificial receptor or receptor complex on a substrate such as a slide. The substrate can include an artificial receptor that acts on a single cell or an artificial receptor that acts on multiple cells. The method involves binding of the cell (s) to an artificial receptor. This embodiment of the method includes contacting the artificial receptor with a bound cell having one or more candidate disruptors. Release of cells from the acting artificial receptor or decreased binding of the cells to the artificial receptor indicates that the candidate disruptor is acting or a leading disruptor and can be selected as such .

  In one embodiment, the disruptor disrupts cellular binding interactions. Such disruptors can be envisioned as mimicking one or more structural features to which the cell binds, eg, on a protein, another cell, tissue, or microorganism. Disturbing agents can be evaluated for such mimicry. The mimetic disruptor can then be used as an antigen for structural features on proteins, microorganisms, tissues, or other cells. Mimic disruptors can be used as idiotypes or anti-idiotypes for structural features on proteins, microorganisms, tissues, or other cells.

  Any of a variety of compounds can be used as a candidate disruptor. For example, the candidate disruptor can include at least one small molecule. For example, candidate disruptors can include a library of small molecules. For example, the candidate disruptor can include at least one peptide. For example, candidate disruptors can include a library of peptides.

Complex Disturbation In one embodiment, the methods of the invention include a method for detecting a disruptor of binding partner binding to a test ligand (eg, a target molecule), wherein the test ligand comprises 1 It binds to the above artificial receptors. In one embodiment, the method of the invention comprises selecting a working artificial receptor or receptor complex that binds a complex comprising a target molecule. Such selection can include evaluating an artificial receptor for binding to the target molecule. The building blocks that make up the artificial receptors can be naive to the test ligand. Among the artificial receptors that bind the target molecule, the method selects those artificial receptors that can bind the complex. A complex that includes a target molecule may also include one or more binding partners for the target molecule. This embodiment of the method involves binding the complex to a selected working receptor (s). The method then includes contacting the receptor with a complex bound to one or more disruptor candidates. Contact can occur in a high throughput screening format. The method includes selecting one or more disruptor candidates that reduce binding of at least one binding partner to the complex as the leading disruptor (s).

  FIG. 9 schematically shows a specific example of a method for detecting an agent that disrupts the binding interaction of a complex containing a target molecule. This embodiment of the method of the present invention includes protein: small molecule complex, protein: protein complex, protein: polynucleotide complex, protein: polysaccharide complex, protein: microbial complex, or protein: cell complex. It can be used to detect agents that disrupt the binding interactions of complex complexes. The method can include creating an array of candidate artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. A working artificial receptor can be identified by contacting the array with a target molecule and identifying which receptor binds the target molecule. The identified working artificial receptor can be contacted with the complex containing the target molecule and the receptor that binds the complex can be selected. The method can include generating an array or device comprising a selected working artificial receptor or receptor complex. The method can include generating or using a selected working artificial receptor or receptor complex on a substrate such as a slide. The method includes binding the complex to an artificial receptor.

  This illustrated embodiment involves contacting an artificial receptor with a complex bound to one or more candidate disruptors. Release the binding partner portion of the complex from the acting artificial receptor but maintain the target molecule on the receptor is a candidate disruptor acting or a leading complex disruptor, such as It can be selected as. Binding of the binding partner portion of the complex to the artificial receptor is reduced, but maintaining the target molecule on the receptor is a candidate disruptor acting or a leading complex disruptor, such as It can be selected as. In one embodiment, an acting or lead complex disruptor can be selected as a lead to develop a therapeutic for a disorder mediated by the complex. The method can include washing unbound or released binding partner from the support.

  In one embodiment, the complex disruptor disrupts a complex comprising at least two proteins, a first protein and a second protein. FIG. 10 schematically illustrates candidate disruptors that disrupt protein: protein complexes. In this embodiment, one protein component remains bound to the receptor while the other separates and leaves the receptor. Specific examples of such disruptors can be envisioned as mimicking one or more structural features of one binding moiety of the proteins contained in the complex. Disturbing agents can be evaluated for such mimicry. For example, the disruptor can mimic the structural features of the first protein that interact with the second protein. The mimetic disruptor can then be used as an antigen for that feature of the first protein. The mimetic disruptor can be used as an idiotype or anti-idiotype for structural features on the first protein.

  In one embodiment, the method of the invention comprises contacting at least one candidate artificial receptor with a complex such as, for example, a protein: protein complex, a protein: polynucleotide complex, and the like. This embodiment of the method may include detecting an artificial receptor that binds only one member of the complex or binds less than a complete complex. Such artificial receptors can be selected as lead complex disruptors.

Methods for Making and Using Affinity Supports In one embodiment, an affinity support for any of the test ligands described herein using a working artificial receptor or receptor complex. Can be used or used as it. For example, the methods of the invention can include a method for generating an affinity support for a test ligand. The method can include a working artificial receptor or a receptor complex that binds to a test ligand. The method can also include coupling of the working artificial receptor or receptor complex to a support. FIG. 11 schematically shows an example of such a method. The support may be suitable for use as an affinity support for, for example, chromatography, membrane filtration, electrophoresis (eg, 1 or 2D electrophoresis) and the like.

  The methods of the present invention provide artificial receptors that bind specific test ligands and / or building blocks that create these receptors for isolation or analysis of specific test ligands (eg, bound to a backbone molecule). Can include selecting. The building blocks that make up the artificial receptors can be naive to the test ligand. For example, an artificial receptor can be used as a receptor surface that can bind a test ligand and remove it (eg, purity) from a mixture or biological sample.

  Such a method can include contacting one or more candidate artificial receptors with a test ligand of interest. The building blocks that make up the artificial receptors can be naive to the test ligand. The method can include selecting one or more candidate artificial receptors that bind the test ligand as the acting artificial receptor (s). The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind a significant amount of the test ligand of interest. The support can be a chromatographic support or medium. The support can be a plate, tube, or membrane. In one embodiment, following the binding of the test ligand of interest to the support, the test ligand of interest elutes from the support. Elution can use pH, buffer, solvent, salt concentration, or wash with ligand concentration effective to elute the test ligand of interest from the support.

  FIG. 12 schematically shows evaluating an array of candidate artificial receptors and binding one or more working artificial receptors for test ligand binding. The building blocks that make up the artificial receptors can be naive to the test ligand. FIG. 12 shows that receptors using such working artificial receptors bind proteins, immobilize antibodies, bind single enantiomers, or structural features on compounds (eg, sensory It can be used to protect the group). In one embodiment, the receptor surface can bind one or more structural features on the protein. In one embodiment, the working artificial receptor can be selected to bind a constant portion rather than a variable portion of an antibody. In one embodiment, the receptor surface can include a catalytic site that can catalyze the reaction of the functional group of the bound test ligand. Such a catalytic site can be a building block, for example an organometallic building block.

  The methods of the present invention allow artificial receptors that bind specific isomers of compounds and / or the building blocks that make these receptors (eg, bound to a backbone molecule) to isolate or analyze specific isomers. Can include selecting for. For example, an artificial receptor can be used as a receptor surface that can bind an isomer and remove it (eg, purity) from a mixture or biological sample.

  Such a method can include contacting one or more candidate artificial receptors with an isomer of interest. The building blocks that make up the artificial receptors can be naive to the test ligand. The method can include selecting one or more candidate artificial receptors that bind isomers as acting artificial receptor (s). The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind a significant amount of the isomer of interest. The support can be a chromatographic support or medium. The support can be a plate, tube, or membrane. In one embodiment, following binding of the isomer of interest to the support, the isomer of interest can be eluted from the support. Elution may use washing with a pH, buffer, salt concentration, or ligand concentration effective to elute the isomer of interest from the support.

  The methods of the present invention include artificial receptors that bind or protect certain structural features of a compound and / or building blocks that create these receptors (eg, bound to a skeletal molecule) that contain structural features. Selecting for isolation or analysis. For example, artificial receptors can be used as receptor surfaces that can bind or protect the structural characteristics of a compound. The binding of structural features can be determined by the lack of binding of similar compounds lacking structural features. Structural feature protection can be assessed by structural features that are not available to, for example, solution phase reactive species when the compound is bound to the receptor surface.

  Such methods can include contacting one or more candidate artificial receptors with a compound of interest. The building blocks that make up the artificial receptors can be naive to the test ligand. The method can include selecting one or more candidate artificial receptors that combine the structural features of the compound as the lead artificial receptor (s). Leading artificial receptors can be evaluated to protect structural features. The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind a significant amount of the compound of interest. In one embodiment, following binding of the compound of interest to the support, a portion of the compound that is not bound or protected structural feature is reacted.

  The methods of the present invention can be used to isolate artificial peptides that bind specific peptides or proteins and / or the building blocks that make these receptors (eg, bound to backbone molecules), or to isolate or analyze specific peptides or proteins. Can include selecting. For example, an artificial receptor can be used as a receptor surface that can bind a peptide or protein, which can be removed (eg, purified) from a mixture or biological sample.

  Such a method can include contacting one or more candidate artificial receptors with a peptide or protein of interest. The building blocks that make up the artificial receptors can be naive to the test ligand. The method can include selecting one or more candidate artificial receptors that bind the peptide or protein as the acting artificial receptor (s). The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind a significant amount of the peptide or protein of interest. The support can be a chromatographic support or medium. The support can be a plate, tube, or membrane. In one embodiment, the peptide or protein of interest elutes from the support following binding of the peptide or protein of interest to the support. Elution can use washing with a pH, buffer, salt concentration, or ligand concentration effective to elute the peptide or protein of interest from the support.

  In one embodiment, the artificial receptors of the present invention can be used to form a selective membrane. Such selective membranes can be based on molecular gates that include artificial receptor surfaces. For example, the artificial receptor surface can align the walls of the holes in the membrane and either allow or block the target molecules from passing through the holes. For example, the artificial receptor surface aligns the walls of the hole in the membrane and acts as a “gate keeper”, for example, on a microcantilever / molecular cantilever, where the gate opens and closes for target binding Can be tolerated. Artificial receptors used in selective membranes can be identified by exposing the target molecule to a number of different artificial receptors and then determining which it binds. For example, binding can be detected through any of the techniques described herein including fluorescence.

  In certain embodiments, the method can include purifying one or more receptor surfaces, wherein each receptor surface includes a building block from a working receptor for a particular test ligand. Such methods can include using the receptor surface for chromatography of the test ligand. Chromatography of the test ligand against a plurality of such receptor surfaces can rate the surface affinity of the test ligand. Under a given set of conditions, the receptor surface that can maintain the longest chromatographed test ligand exhibits the greatest affinity for the test ligand. The method can include selecting a receptor surface with an appropriate (eg, maximal) affinity as an affinity support for the test ligand.

  Any of a variety of supports can be used as the affinity support. In certain embodiments, the affinity support can be a dish, tube, well, bead, chromatography support, microchannel, and the like. Artificial receptor affinity supports can be used in various applications such as chromatography, microchannel devices, as immunoassay supports and the like. Microchannels with artificial receptors on their surface can be used as analytical devices. In one embodiment, the artificial receptors of the present invention can be used to form a bioactive surface. For example, the receptor surface can be used to specifically bind an antibody or enzyme.

Methods for Creating and Using Reaction Supports In one embodiment, a working artificial receptor or receptor complex generates a reaction support for any of the test ligands described herein. Can be used to do or as it is. For example, the methods of the invention can include a method for generating a reaction support for at least one test ligand. The method can include selecting a working artificial receptor or receptor complex that binds to the test ligand under conditions appropriate for the desired reaction with the test ligand. The method can also include coupling the working artificial receptor or receptor complex to a support. FIG. 11 schematically shows an example of such a method. The support may be suitable for use as a reaction support, for example for oxidation, reduction, substitution, or substitution reactions.

  The methods of the present invention include selecting artificial receptors that bind specific test ligands and / or the building blocks that make these receptors (eg, bound to a backbone molecule) for the reaction of a specific test ligand. May be included. For example, an artificial receptor can be used as a receptor surface to which a test ligand can be attached, which can be placed for reaction at a particular prochiral group, functional group, or orientation.

  Such a method can include contacting one or more candidate artificial receptors with a test ligand of interest. The building blocks that make up the artificial receptors can be naive to the test ligand. The method can include selecting one or more candidate artificial receptors that bind the test ligand as the acting artificial receptor (s). The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind the desired amount of the test ligand of interest. The support can be a chromatographic support or medium. The support can be a plate, bead, tube, or membrane.

  The method also includes contacting a support including bound test ligand with the reactants for the desired reaction. Suitable reactants include reducing agents, oxidants, nucleophiles, electrophiles, solvents (eg, aqueous or organic solvents) and the like. The method can include contacting the one or more reactants and selecting an appropriate reactant or reactants to participate in the desired reaction. This embodiment of the method includes reacting a test ligand with a reactant. In one embodiment, after the reaction, the by-product from the reactants or support can be washed. In one embodiment, after the reaction, the product (eg, reacted test ligand) can be eluted from the support. Elution can use washing with a pH, buffer, solvent, salt concentration, or ligand concentration effective to elute the product from the support.

  FIG. 12 schematically illustrates evaluating an array of candidate artificial receptors to select test ligand binding and one or more working artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. FIG. 12 shows that a receptor surface using such a working artificial receptor can be used to bind a test ligand. The test ligand can bind in orientation, leaving a reactive site available for reaction with a reactant placed in contact with the receptor surface. In one embodiment, the test ligand can be bound in an orientation that blocks or protects the second reactive site from reacting with the reactant. This embodiment includes reacting a test ligand and releasing the reacted test ligand from the receptor surface. Specifically, the figure shows the reduction of aldehyde with sodium borohydride to produce alcohol. In one embodiment, the receptor surface includes a catalytic site that can catalyze the reaction of the functional group of the bound test ligand, and the catalytic reaction can also use a reactant. Such catalytic sites can be, for example, building blocks such as organometallic building blocks.

  The methods of the present invention include artificial receptors that bind or protect certain structural features of a compound and / or building blocks that create these receptors (eg, bound to a backbone molecule) that contain the structural features. Can be selected to react. For example, an artificial receptor can be used as a receptor surface that can bind and protect the structural characteristics of a compound while another characteristic of the compound reacts with a reactant. The protection of structural features can be assessed by the structural features that do not react with the reactants. For example, a substrate (eg, a steroid) can bind stereospecifically to an artificial receptor and presents a specific site / substructure / “face” for reaction with a reactant in solution. .

  In one embodiment, the first side of the molecule (or functional group) binds to the receptor surface, while the second side is exposed to the left. A reagent that can react with either side (or group) is then added, which binds to the receptor surface, so that the reagent only reacts with the second side of the molecule, so that the first of the molecule Prevents reaction with the sides.

  Such methods can include contacting one or more candidate artificial receptors with a compound of interest. The method can include selecting one or more candidate artificial receptors that combine the structural features of the compound as the lead artificial receptor (s). Leading artificial receptors can be evaluated to protect structural features. The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind the desired amount of the compound of interest. In one embodiment, after binding of the compound of interest to the support, a portion of the compound that is not a bound or protected structural feature can be reacted.

  In general, conventional synthesis of chiral compounds requires complex procedures. In one embodiment, the candidate artificial receptors of the present invention can be used to discover receptor surfaces that provide a spatially oriented binding surface for stereospecific reactions. For example, artificial receptor surfaces can bind small molecules such that certain functional groups are exposed to the environment and others are obscured by the receptor. Thus, the stereospecificity of the reaction can be controlled. Thus, the artificial receptor surface can be used in synthesis including chiral introduction. Similarly, region specificity can also be controlled using the receptors of the present invention.

  The methods of the present invention comprise first and second reactive ligands or artificial receptors that bind the building blocks that make up these receptors (eg, bound to a backbone molecule). Selecting for reactions involving reactive ligands. For example, an artificial receptor can be used as a receptor surface that can bind a first reactive ligand and a second reactive ligand at a distance or orientation at which these ligands can react with each other. The reaction can optionally include one or more reactants that do not bind to the receptor surface.

  Such a method can include contacting one or more candidate artificial receptors with a first reactive ligand and a second reactive ligand. The building blocks that make up the artificial receptors can be naive to the ligand. The method can include selecting one or more candidate artificial receptors that bind both reactive ligands as the acting artificial receptor (s). The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind the desired amount of the first and second reactive ligands. The support can be a chromatographic support or medium. The support can be a plate, bead, tube, or membrane. The first and second reactive ligands can be bound to the support in one or several molar ratios, the evaluated reaction, and the molar ratio selected to carry out the reaction.

  The method can also include contacting a support comprising bound reaction ligand with a reactant for the desired reaction. Suitable reactants include reducing agents, oxidants, nucleophiles, electrophiles and the like. This embodiment of the method includes reacting a test ligand with a reactant. In one embodiment, after the reaction, the reactants or by-products from the support can be washed. In one embodiment, the first and second reactive ligands react without reactants. In one embodiment, after the reaction, the product (eg, reacted test ligand) can be eluted from the support. Elution may use washing with a pH, buffer, solvent, salt concentration, or ligand concentration effective to elute the product from the support.

  In one embodiment, the candidate artificial receptors of the present invention can be used to discover receptor surfaces that provide a spatially oriented binding surface for stereospecific reactions. For example, an artificial receptor surface can bind certain functional groups exposed to the environment, and small molecules that are obscured by the receptor. Such artificial receptor surfaces can be used in syntheses involving chiral introduction. For example, substrates (eg, steroids) bind stereospecifically to artificial receptors and present specific sites / substructures / “faces” for reaction with reagents in solution. Similarly, an artificial receptor surface can act as a protecting group that is “protected” by binding to the receptor surface so that the reactive site of the molecule can be transformed with a different site with similar reactivity. .

  In one embodiment, one or more working artificial receptors that bind multiple (eg, 2) reactants can be generated on the substrate and the bound reactants. Each receptor having a plurality of bound reactants can then be screened against one or more reagents or conditions (eg, various molar ratios of reactants or various solvents). Artificial receptors that allow or facilitate a reaction between two or more reactants can be identified. Artificial receptors can then be generated on the substrate to provide a reactor for the reaction of interest.

  Any of a variety of supports can be used as the reaction support. In certain embodiments, the reaction support can be a dish, tube, well, bead, chromatography support, microchannel, and the like. Artificial receptor reaction supports can be used in various applications such as microchannel devices. A microchannel with artificial receptors on its surface can be used as a reactor.

Methods for Making and Using Supported Catalysts In one embodiment, a working artificial receptor or receptor complex is used as a supported catalyst for any of the test ligands described herein or Can be used to generate it. For example, the method of the present invention can include a method for producing a supported catalyst for at least one test ligand, the method being tested under conditions suitable to catalyze a reaction with the test ligand. It may include selecting a working artificial receptor or receptor complex that binds to the ligand. The method can also include coupling the working artificial receptor or receptor complex to a support. FIG. 11 schematically shows an example of such a method. The support is used as a supported catalyst for any of various catalytic sites or building blocks such as organometallic sites, coenzymes, redox active sites, nucleophilic sites, acid sites, base sites, etc. May be appropriate.

  The methods of the present invention may be used to generate artificial receptors that bind and catalyze specific test ligand reactions and / or building blocks that create these receptors (eg, bound to backbone molecules) to react specific test ligand reactions. Selecting to catalyze may be included. For example, an artificial receptor can be used with a receptor surface that can bind a test ligand and place it for reaction with a catalytic site in a specific prochiral group, functional group, or orientation. Can do.

  Such a method can include contacting one or more candidate artificial receptors with a test ligand of interest. The building blocks that make up the artificial receptors can be naive to the test ligand. The method may include selecting one or more candidate artificial receptors that bind and catalyze the desired reaction of the test ligand as the acting artificial receptor (s). The method may then include creating a receptor surface using the working artificial receptor (s). Creating the receptor surface can include coupling a building block that creates the working artificial receptor (s) to a support. The support can have sufficient area to bind the desired amount of the test ligand of interest. The support can be a chromatographic support or medium. The support can be a plate, bead, tube, or membrane.

  The method also includes contacting a support containing bound test ligand with a reactant or cofactor for the desired reaction. Suitable reactants include reducing agents, oxidants, nucleophiles, electrophiles, solvents (eg, aqueous or organic solvents) and the like. The method can include contacting one or more selected reactants and selecting an appropriate reactant or reactants to participate in the desired reaction. This embodiment of the method includes reacting a test ligand with a reactant. In one embodiment, after the reaction, the reactants or by-products from the support can be washed. In one embodiment, after the reaction, the product (eg, reacted test ligand) can be eluted from the support. Elution can use washing with a pH, buffer, solvent, salt concentration, or ligand concentration effective to elute the product from the support.

  FIG. 12 schematically illustrates evaluating an array of candidate artificial receptors for binding and catalysis of test ligand reactions and selecting one or more working artificial receptors. The building blocks that make up the artificial receptors can be naive to the test ligand. FIG. 12 shows that a receptor surface using such working artificial receptors can be used for binding and catalysis of the test ligand reaction. The test ligand can be bound in an orientation that makes the reactive site available for reaction with the catalytic site of the receptor. In one embodiment, the test ligand can be bound in an orientation that blocks or protects the second reactive site from reacting with the catalytic site. This embodiment includes reacting the test ligand and releasing the reacted test ligand from the receptor surface. Specifically, the figure shows the reduction of aldehydes at the catalytic site (MC) on the catalyst support to produce alcohol. In one embodiment, the receptor surface includes a catalytic site that can catalyze the reaction of a functional group of the bound test ligand, and the catalytic reaction can also use a reactant. . Such catalytic sites can be building blocks, such as, for example, organometallic building blocks.

  In one embodiment, the invention includes a catalyst comprising binding a first reactive ligand to an artificial receptor array, contacting the array with a reactant, and then identifying the artificial receptor that promoted the reaction. A method for identifying. The array can be screened for artificial receptors that produce the desired product (eg, catalyze conversion of the reactants thereto).

  Any of a variety of supports can be used as the catalyst support. In certain embodiments, the catalyst support can be a dish, tube, well, bead, chromatography support, microchannel, and the like. Artificial receptor reaction supports can be used in various applications such as microchannel devices. A microchannel with artificial receptors on its surface can be used as a reactor.

Methods for Creating or Detecting Unbound Surfaces or Substances In one embodiment, the present invention can include methods and / or devices for not binding test ligands. The methods and systems for not binding test ligands can be used in systems useful in clinical chemistry, environmental analysis, and all types of diagnostic assays. In one embodiment, the present invention includes a method for making a substrate that does not bind a test ligand. The method comprises contacting at least one candidate artificial receptor with a test ligand, detecting binding of the test ligand to one or more artificial receptors or detecting lack of binding, and then as a non-binding surface acting on the test ligand. It may include selecting artificial receptors that do not bind. Artificial receptors or receptors that do not bind the first test ligand can be tested against one or more additional test ligands. In such a manner, unbound artificial receptors can be screened for artificial receptors that do not bind any of the plurality of test ligands. The surface covered with building blocks that create such unbound artificial receptors can then be used as a working unbound surface.

  In one embodiment, the methods of the invention can use an array of candidate artificial receptors. This embodiment of the method can generate an unbound surface for one or more working artificial receptors or one or more test ligands using an array containing a significant number of the present artificial receptors. . The method can include evaluating an array that includes a significant number of candidate artificial receptors for not binding to at least one test ligand. Candidate artificial receptors that do not bind to any of the one or more test ligands can be selected as non-binding surfaces for the one or more test ligands.

  In one embodiment, the method of the present invention can evaluate an array of candidate artificial receptors to develop a surface that does not bind one or more proteins (eg, plasma proteins). This embodiment of the method uses an array comprising a significant number of artificial receptors of the invention to produce a non-binding surface for one or more working artificial receptors or one or more proteins (eg, plasma proteins). Can be generated. The method can include evaluating an array that includes a significant number of candidate artificial receptors for not binding to at least one protein (eg, a plasma protein). Candidate artificial receptors that do not bind to any of the one or more proteins (eg, plasma proteins) can be selected as non-binding surfaces for the protein. Such a surface can be used for implantable medical devices.

  In one embodiment, the method of the present invention can evaluate an array of candidate artificial receptors to develop a surface that does not bind to one or more cells. This embodiment of the method can generate an unbound surface for one or more working artificial receptors or one or more cells using an array containing a significant number of the artificial receptors of the present invention. The method can include evaluating an array that includes a significant number of candidate artificial receptors for not binding to at least one cell. Candidate artificial receptors that do not bind to any of the one or more cells can be selected as non-binding surfaces for those cells. Such a surface can be used for implantable medical devices.

  In one embodiment, the method of the present invention can evaluate an array of candidate artificial receptors to develop a surface that does not bind one or more microorganisms. This embodiment of the method can generate an unbound surface for one or more working artificial receptors or one or more microorganisms using an array comprising a significant number of the artificial receptors of the present invention. The method can include evaluating an array comprising a significant number of candidate artificial receptors for not binding to at least one microorganism. Candidate artificial receptors that do not bind to any of the one or more microorganisms can be selected as non-binding surfaces for the microorganism. Such a surface can be used for implantable medical devices. Such surfaces can be used in systems such as water pipes, storage or waterways in food processing plants, cooling towers, ship bottoms that would otherwise be subjected to biofouling.

Methods for Detection Contacting an array comprising a plurality of artificial receptors with a test ligand can identify one or more lead or working artificial receptors. Binding of the test ligand to the lead or acting artificial receptor or complex can produce a detectable signal. The detectable signal can be generated through mechanisms and properties such as, for example, scattering, absorbing or emitting light, generating or quenching fluorescence or luminescence, generating or quenching an electronic signal, and the like. The spectroscopic detection method includes generating light for detection by an optical sensor or optical sensor array using a label or enzyme. The light can be ultraviolet, visible, or infrared that can be generated and / or detected through fluorescence, fluorescence polarization, chemiluminescence, bioluminescence, or chemibioluminescence.

  Systems and methods for detecting electrical conduction and changes in electrical conduction include ellipsometry, surface plasmon resonance, capacitance, conductivity measurement, surface acoustic wave, quartz crystal microbalance, love wave, infrared evanescent wave Including enzyme labeling by electrochemical detection, nanowire field effect transistor, MOSFETS-metal oxide semiconductor field effect transistor, CHEMFETS-organic film metal oxide semiconductor field effect transistor, ICP-intrinsic conducting polymer, FRET-fluorescence resonance energy transfer.

  In one embodiment, the working artificial receptor or complex can be constructed on the surface of an optical fiber, such as, for example, successive distinct areas, spots, zones, and the like. A working artificial receptor or complex can be contacted with a test ligand or a sample suspected of containing the test ligand. The test ligand sample can be in the form of air, aerosol, or liquid (eg, solution or suspension). Detectable colorimetric, fluorescent, etc. signals can be generated by labels incorporated into the optical fiber surface. A colorimetric or fluorometric signal can be endogenous to the ligand or can be generated upon binding of the ligand to the working artificial receptor.

  Devices capable of detecting binding to or acting on working artificial receptors or complexes include UV, visible, or infrared spectrometers, fluorescence or luminescence spectrometers, surface plasmon resonances, surface acoustic waves or Includes quartz crystal microbalance detector, pH, voltammetry or amperometry meter, radioisotope detector and the like.

  In such a device, the working artificial receptor or complex can be placed on an optical fiber to provide a detectable signal such as an increase or decrease in transmitted light, reflected light, fluorescence, luminescence, etc. The detectable signal can arise, for example, from a signaling site incorporated into the working artificial receptor or complex or a signaling site added to the working artificial receptor. The signal can also be endogenous to the acting artificial receptor or test ligand. The signal can arise, for example, from the interaction of the test ligand with the working artificial receptor, the interaction of the test ligand with a signaling site incorporated into or into the working artificial receptor. In one embodiment, the methods of the invention can include selecting an artificial receptor whose binding induces a change in signal from a signaling moiety, eg, a fluorescent moiety. Such a change may indicate binding to the artificial receptor.

  In one embodiment, the working artificial receptor can be on a support such as a surface of a test tube, microwell, capillary, microchannel, or the like. A test ligand or sample suspected of containing a test ligand can be contacted with the acting artificial receptor or complex by addition of a solution containing the test ligand or a sample suspected of containing the test ligand. A detectable signal can be generated by the conjugate of the labeled compound or test ligand. This labeled site can react with the acting artificial receptor or complex in competition with a solution containing the test ligand or a sample suspected of containing the test ligand.

  In an embodiment of the system, the working artificial receptor is on a support such as the surface of a surface acoustic wave or quartz crystal microbalance or surface plasmon resonance detector. A test ligand or a sample suspected of containing a test ligand is produced by exposure to an air stream, to an aerosol, or to a solution containing a test ligand or a sample suspected of containing a test ligand. Can be in contact with the body. A detectable electronic signal can be generated by the interaction of the test ligand with the working artificial receptor or complex on the active surface of a surface acoustic wave or quartz crystal microbalance or surface plasmon resonance detector.

  In an embodiment of the system, one or more working artificial receptors arranged as regions or spots in an array on a support, such as a glass or plastic surface, are placed on the signaling surface of one or more surface plasmon resonance detectors. Can be incorporated. A working artificial receptor or array of a ligand of interest or a sample suspected of containing a ligand of interest (eg, a sample containing a DNA segment or fragment, protein or protein fragment, carbohydrate or carbohydrate fragment, etc.) Contact with. Contact can be achieved by the addition of a solution of a sample of interest or suspected of containing a ligand of interest. A detectable electronic signal can be generated by binding a ligand of interest to the working artificial receptor array on the surface of a surface plasmon resonance detector. Such a detector generates an array of signals for each working artificial receptor, which generates a pattern of signal response, which is characteristic of the sample composition of interest.

  Artificial receptors of the present invention may include genomic and / or proteomic analysis; drug development; detectors for any of the test ligands; addictive drug diagnostics or therapy; hazardous waste analysis or improvement; chemical warfare alerts or interventions; Can be part of a product used in disease diagnosis or therapy; cancer diagnosis or therapy; biological war alert or intervention; food chain contamination analysis or improvement.

  More specifically, the artificial receptors of the present invention provide sequence-specific small molecule lead identification; protein isolation and identification; identification of protein-protein interactions; detection of food or contaminants in food; food contaminants Clinical analysis of prostate specific antigen; clinical and on-site or clinical analysis of cocaine; clinical and on-site or clinical analysis of other addictive drugs; other clinical analysis systems, home test systems, or electric field analysis systems; It can be used in products for monitoring or alerting systems for chemical weapons.

Specific Examples of Methods The present invention includes a method for making an artificial receptor for binding a test ligand. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with a test ligand, detects binding of the test ligand to at least one candidate artificial receptor, and then detects test ligand binding. Selecting the selected at least one candidate artificial receptor as a working artificial receptor for the test ligand. The candidate artificial receptor can include a plurality of building blocks coupled to a region of the support.

  In one embodiment, contacting at least one artificial receptor with a test ligand can include contacting an array of candidate artificial receptors with the test ligand. In one embodiment, the array can include at least about 100 candidate artificial receptors. In one embodiment, the array can include at least about 10,000 candidate artificial receptors. In one embodiment, the array can include at least about 1,000,000 candidate artificial receptors.

  In one embodiment, the test ligand can include at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism. In one embodiment, the test ligand comprises at least one of an isomer, a protein conformation, and a proteome.

  In one embodiment, the method of the invention contacts at least one candidate artificial receptor with a first protein to produce at least one protein-treated candidate artificial receptor; a proteome comprising a proteome protein; Contacting a protein-treated candidate artificial receptor; detecting binding of at least one proteome protein to at least one protein-treated candidate artificial receptor; and detecting both the first protein and at least one proteome protein Candidate artificial receptors to bind may be selected as working artificial receptors, or may include selecting at least one proteome protein for which binding has been detected and a first protein for analysis.

  In one embodiment, the method of the present invention contacts at least one candidate artificial receptor with a mixture comprising a test ligand and a candidate disruptor; to the at least one candidate artificial receptor in the presence of the candidate disruptor. Then binding at least one candidate artificial receptor to the test ligand, wherein test ligand binding occurs in the absence of the candidate disruptor, but test ligand binding is disrupted by the candidate disruptor It may include selecting as a working artificial receptor.

  The present invention includes a method for detecting a test ligand. In one embodiment, the method of the invention contacts at least one working artificial receptor with a sample suspected of containing a test ligand and then monitoring for detection to the at least one working artificial receptor. Wherein binding to the at least one working artificial receptor is indicative of the presence of the test ligand in the sample. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the at least one working artificial receptor has been found to bind the test ligand. In one embodiment, the at least one working artificial receptor can include a plurality of artificial receptors.

  In one embodiment, test ligand binding produces a detectable pattern between multiple artificial receptors. In one embodiment, the test ligand can include at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism. In one embodiment, the test ligand may comprise at least one of an isomer, a protein conformation, and a proteome.

  In one embodiment, the method of the invention contacts a plurality of candidate artificial receptors with a first test ligand; detects binding of the first test ligand to at least one candidate artificial receptor; Classifying the location or composition of at least one artificial receptor bound to the first test ligand may be included. Each candidate artificial receptor can independently comprise a plurality of building blocks coupled to a region of the support.

  The method of the invention can detect one or more test ligands. In one embodiment, the method comprises contacting one or more candidate artificial receptors with a second test ligand; detecting binding of the second test ligand to at least one candidate artificial receptor; Categorizing the location or composition of at least one artificial receptor bound to the second test ligand.

  In one embodiment, the position or composition of at least one artificial receptor bound to a first test ligand is compared to the position or composition of the at least one artificial receptor bound to a second test ligand; Categorizing the position or composition comparison. In one embodiment, the method can include identifying the first test ligand from the second test ligand based on the composition.

  The methods of the invention include methods for detecting compounds that disrupt test ligand interactions. The method can include contacting at least one working artificial receptor with a test ligand and a candidate disruptor. Contact may be simultaneous, contact with the test ligand may be prior to contact with the candidate disruptor, or contact with the candidate disruptor ligand may be prior to contact with the test ligand. In one embodiment, the method can include contacting at least one working artificial receptor with a mixture comprising a test ligand and a candidate disruptor. The method can also include monitoring for a candidate disruptor that reduces binding of the test ligand to the at least one working artificial receptor, wherein the decrease in binding is determined by the candidate disruptor being led. It shows that it is a disturbing substance. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the at least one working artificial receptor has been found to bind the test ligand.

  In one embodiment, the test ligand can include at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism. In one embodiment, the test ligand may comprise at least one of an isomer, a protein conformation, and a proteome.

  In one embodiment, the candidate disruptor can comprise a compound having a molecular weight of less than 500. In one embodiment, the candidate disruptor can comprise a polypeptide.

  The present invention includes a method for making an affinity support for a test ligand. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with the test ligand; detects binding of the test ligand to at least one candidate artificial receptor; test ligand binding is detected Selecting the at least one candidate artificial receptor as a working artificial receptor for the test ligand; and then coupling the working artificial receptor to a second support to obtain an affinity support Forming. Candidate artificial receptors can include a plurality of building blocks coupled to a region of the first support.

  In one embodiment, the test ligand can include at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism. In one embodiment, the test ligand may comprise at least one of an isomer, a protein conformation, and a proteome.

  The present invention includes a method for isolating a test ligand. In one embodiment, the methods of the invention can include contacting a sample containing a test ligand with a working artificial receptor. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the working artificial receptor has been found to bind a test ligand.

  The present invention includes a method for making a reaction support for a reactant. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with a reactant; detects binding of the test ligand to at least one candidate artificial receptor; test ligand binding is detected Selecting at least one candidate artificial receptor as a working artificial receptor for the test ligand; contacting at least one working artificial receptor with the reactant and monitoring for the reaction of the reactant; The selected working artificial receptor may be selected as a reactive artificial receptor; then, the reactive artificial receptor may be coupled to a second support to form a reactive support. Candidate artificial receptors can include a plurality of building blocks coupled to a region of the first support. In one embodiment, the method can include contacting at least one working artificial receptor with a second reactant.

  In one embodiment, the reactant may include at least one of a molecule having a molecular weight of less than 500, a protein, a polynucleotide, an organic polymer, or a mixture thereof. In one embodiment, the reactants may include molecular isomers having a molecular weight of less than 500.

  The present invention includes a method for reacting reactants. In one embodiment, the method of the present invention can include contacting a sample containing a reactant with a working artificial receptor. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the working artificial receptor has been found to bind a test ligand. In one embodiment, the method can include contacting the working artificial receptor with a second reactant.

  The present invention includes a method of making a catalyst support for a reactant. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with a reactant; detects a catalyst for the reaction of the reactant in the presence of the at least one candidate artificial receptor; At least one candidate artificial receptor for which is detected is selected as a working artificial receptor for the test ligand; the working receptor is then coupled to a second support to form a catalytic support. Can include. Candidate artificial receptors can include a plurality of building blocks coupled to a region of the first support. In one embodiment, the method can include contacting at least one working artificial receptor with a second reactant.

  In one embodiment, the reactant can include at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism. In one embodiment, the reactants may include at least one of isomers, protein conformations, and proteomes.

  The present invention includes a method of catalyzing a reaction. In one embodiment, the method of the invention can include contacting a sample containing a reactant with a working artificial receptor. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the working artificial receptor has been found to bind the test ligand and catalyze the reaction.

  The present invention includes a method of making a surface that does not bind a test ligand. In one embodiment, the method of the invention contacts at least one candidate artificial receptor with the test ligand; detects a loss of binding of the test ligand to at least one candidate artificial receptor; Selecting at least one candidate artificial receptor in which a test deficiency of ligand binding has been detected can be selected as a non-binding surface for the test ligand. The candidate artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the test ligand includes a plurality of test ligands.

  The present invention includes a method for detecting a compound that disrupts the interaction of a test ligand with a second ligand. In one embodiment, the method of the invention may comprise binding the test ligand to at least one working artificial receptor.

  The method can include contacting at least one working artificial receptor with a second ligand and a candidate disruptor. Contact may be simultaneous and contact with the second ligand may come before contact with the candidate disruptor, or contact with the candidate disruptor may occur before contact with the second ligand. You may come. In one embodiment, the method can include monitoring a candidate disruptor that reduces binding of the second ligand to the test ligand, wherein the decreased binding is due to the candidate disruptor being predominant. It shows that it is a disturbing substance. The working artificial receptor may comprise a plurality of building blocks coupled to a region of the support. In one embodiment, the at least one working artificial receptor has been found to bind the test ligand.

  In one embodiment, the test ligand can comprise at least one of a peptide, polypeptide, oligonucleotide, polynucleotide, and microorganism. In one embodiment, the test ligand can comprise a first protein and the second ligand can comprise a second protein; wherein the first protein and the second protein comprise a complex. Form. In one embodiment, the test ligand can comprise a protein and the second ligand comprises a polynucleotide; wherein the protein and the polynucleotide form a complex.

  In one embodiment, the candidate disruptor can comprise a compound having a molecular weight of less than 500. In one embodiment, the candidate disruptor can comprise a polypeptide.

  In one embodiment, in this method, contacting with the first ligand comprises contacting with a complex; then the at least one working artificial receptor having a bound test ligand is added to the first ligand. Contacting the two ligands and the candidate disruptor includes contacting the candidate disruptor.

Test Ligand The test ligand can be any ligand whose binding to the array or surface can be detected. The test ligand may be a pure compound, a mixture, or a “contamination” mixture containing natural products or contaminants. Such a contamination mixture can be a tissue homogenate, body fluid, soil sample, water sample, and the like.

  Test ligands include prostate specific antigen, other cancer markers, insulin, warfarin, other anticoagulants, cocaine, other addictive drugs, markers for E. coli, markers for Salmonella sp., Other food-derived toxins, food Derived toxins, markers for smallpox virus, markers for Bacillus anthracis, other potentially toxic biological agents, pharmaceuticals and pharmaceuticals, pollutants and chemicals in hazardous waste, toxic chemicals, markers for diseases, pharmaceuticals, Contaminants, biologically important cations (eg potassium or calcium ions), peptides, carbohydrates, enzymes, microorganisms, viruses, mixtures thereof, etc. In certain embodiments, the test ligand can be at least one of small organic molecules, inorganic / organic complexes, metal ions, protein mixtures, proteins, nucleic acids, nucleic acid mixtures, mixtures thereof, and the like.

  Suitable test ligands include any compound or category described elsewhere in this document as being a test ligand, including, for example, the above-described microorganisms, proteins, cancer cells, addictive drugs, and the like. Of the compound.

TECHNICAL FIELD The present invention relates to a method for creating an artificial receptor or a candidate artificial receptor. In one embodiment, the method includes preparing a spot or region on a support, wherein the spot or region includes a plurality of building blocks immobilized on the support. The method forms a plurality of spots, each spot comprising a plurality of building blocks on a solid support, and then fixes (eg, reversibly) the plurality of building blocks on the solid support at each spot. Can include. In one embodiment, such an array of spots is referred to as a heterogeneous building block array.

  The method can include mixing a plurality of building blocks and using the mixture in forming the spot (s). Alternatively, the method can include spotting individual building blocks on the support. Coupling the building block to the support can use covalent bonds or non-covalent interactions. Suitable non-covalent interactions include ionic interactions, hydrogen bonds, van der Waals interactions, and the like. In one embodiment, the support can be functionalized with sites that can participate in covalent bonds or non-covalent interactions. Forming spots can yield a microarray of heterogeneous spots of building blocks, each of which can be a candidate artificial receptor. The method can apply or spot a building block on a support in a combination of 2, 3, 4, or more building blocks.

  In one embodiment, the method of the present invention can be used to produce a solid support having a plurality of regions or spots on its surface, where each region or spot includes a plurality of building blocks. . For example, the method includes spotting a glass slide having a plurality of spots, where each spot includes a plurality of building blocks. Such spots can be said to include heterogeneous building blocks. The multiple spots of the building block can be referred to as an array of spots.

  In one embodiment, the method includes creating a receptor surface. Creating a receptor surface forms a region on a solid support (where the region includes a plurality of building blocks), and then the plurality of building blocks are applied to the solid support in the region (e.g., Reversibly). The method can include mixing a plurality of building blocks and using the mixture in forming a region or regions. Alternatively, the method can include applying individual building blocks in regions on the support. Forming the region on the support can be accomplished, for example, by immersing a portion of the support with a building block solution. The resulting coating comprising building blocks can be said to comprise heterogeneous building blocks.

  The region including a plurality of building blocks may be independent and different from other regions including a plurality of building blocks. In one embodiment, one or more regions that include a plurality of building blocks may overlap to produce a region that includes a plurality of combined building blocks. In one embodiment, two or more regions that include a single building block may overlap to form one or more regions that each include a plurality of building blocks. Overlapping regions can be expected, for example, as overlapping portions in a Ven diagram or as overlapping portions in a pattern such as plaid or tweed.

  In one embodiment, the method produces a spot or surface having a density of building blocks sufficient to provide one or more building block interactions with the ligand. That is, the building blocks can be close to each other. The proximity of different building blocks determines the different (eg, greater) binding of a test ligand to a spot or surface containing multiple building blocks compared to a spot or surface containing only one of the building blocks. Can be detected.

  In one embodiment, the method includes forming an array of heterogeneous spots created from a subset of the total building blocks in each spot and / or a combination of smaller groups of building blocks. That is, the method forms a spot containing only 2 or 3 building blocks, for example, rather than 4 or 5. For example, the method can form spots from combinations of a full set of building blocks (eg, 81 of 81 sets) in groups of 2 and / or 3. For example, the method can form spots from combinations of subsets of building blocks (eg, 25 of 81 sets) in groups of 4 or 5. For example, the method can form spots from combinations of subsets of building blocks (eg, 25 of 81 sets) in groups of two or three. The method can include forming a further array incorporating a building block, a lead artificial receptor, or a structurally similar building block.

  In one embodiment, the method includes forming an array comprising one or more spots that serve as controls for authenticating or assessing binding to the present artificial receptors. In one embodiment, the method includes forming one or more regions, tubes, or wells that serve as controls for authenticating or evaluating binding to the present artificial receptors. Such control spots, regions, tubes, or wells can include no building blocks, only a single building block, only a functionalized loan, or a combination thereof.

  The method can immobilize (eg, reversibly) a building block on a support using known methods for immobilizing compounds of the type used as the building block. Coupling the building block to the support can use covalent bonds or non-covalent interactions. Suitable non-covalent interactions include ionic interactions, hydrogen bonds, van der Waals interactions, and the like. In one embodiment, the support can be functionalized with sites that can participate in reversible covalent bonds, sites that can participate in non-covalent interactions, mixtures of these sites, and the like.

  In one embodiment, the support can be functionalized with a site that can participate in a covalent bond, eg, a reversible covalent bond. The present invention can use any of a variety of many known functional groups, reagents, and reactions to form a reversible covalent bond. Suitable reagents for forming reversible covalent bonds include those described in Green, TW; Wuts, PGM (1999), Protective Groups in Organic Synthesis Third Edition, Wiley-Interscience, New York, 779 pp. For example, the support may be a carbonyl group, carboxyl group, silane group, boric acid or ester, amine group (e.g., primary, secondary, or tertiary amine, hydroxylamine, hydrazine, etc.), thiol group, alcohol group (e.g., Primary, secondary, or tertiary alcohols), diol groups (eg, 1,2 diols or 1,3 diols), phenol groups, catechol groups, and the like. These functional groups are ethers (e.g., alkyl ethers, silyl ethers, thioethers, etc.), esters (e.g., alkyl esters, phenol esters, cyclic esters, thioesters, etc.), acetals (e.g., cyclic acetals), ketals (e.g., cyclics). Ketal), silyl derivatives (eg silyl ethers), boronic acids (eg cyclic boronic acids), amides, hydrazines, imines, carbamic acids and the like can be formed with groups having reversible covalent bonds. Such a functional group can be referred to as a covalent bond site such as, for example, a first covalent bond site.

  The carbonyl group on the support and the amine group on the building block can form an imine or Schiff base. The same is true for amine groups on the support and carbonyl groups on building blocks. The carbonyl group on the support and the alcohol group on the building block can form an acetal or ketal. The same is true for the alcohol groups on the support and the carbonyl groups on the building blocks. A thiol (eg, a first thiol) on the support and a thiol (eg, a second thiol) on the building block can form a disulfide.

The carboxyl group on the support and the alcohol group on the building block can form an ester. The same is true for the alcohol group on the support and the carboxyl group on the building block. Any of a variety of alcohols and carboxylic acids can form esters that provide covalent bonds that can be reversed in the context of the present invention. For example, the reversible ester bond is an alcohol such as a phenol having an electron withdrawing group on the aryl ring, other alcohols having an electron withdrawing group acting on a hydroxyl related carbon, other alcohols, etc .; and / or Or from a carboxyl group such as one having an electron withdrawing group acting on an acyl carbon (for example, nitrobenzylic acid, R-CF ₂ -COOH, R-CCl ₂ -COOH, etc.), other carboxylic acids, etc. Can be formed.

  In one embodiment, the support, matrix, or lawn can be functionalized with sites that can participate in non-covalent interactions. For example, the support can include functional groups such as ionic groups, groups capable of hydrogen bonding, or groups that can participate in van der Waals or other hydrophobic interactions. Such functional groups can include positive groups, negative groups, lipophilic groups, amphiphilic groups, and the like.

  In one embodiment, the support, matrix, or lawn includes a charged site (eg, a first charged site). Suitable charged sites include positively charged sites and negatively charged sites. Suitable positively charged sites (eg, at neutral pH in aqueous compositions) include amines, quaternary ammonium sites, ferrocene and the like. Suitable negatively charged sites (eg at neutral pH in aqueous compositions) carboxylates, phenols, phosphates, phosphonates, phosphines substituted by strong electron withdrawing groups (eg tetrachlorophenol) Acid salts, sulfates, sulfonates, thiocarboxylic acids, hydroxamic acids and the like.

  In one embodiment, the support, matrix, or lawn includes a group that can hydrogen bond either as a donor or acceptor (eg, a first hydrogen bonding group). The support, matrix, or lawn can include a surface or region having groups capable of hydrogen bonding. For example, a support, matrix, or lawn can include a surface or region that includes one or more carboxyl groups, amine groups, hydroxyl groups, carbonyl groups, and the like. In addition, the ionic group can participate in hydrogen bonding.

In one embodiment, the support, matrix, or lawn includes a lipophilic site (eg, a first lipophilic site). Suitable lipophilic moieties are, for example, branched or straight chain C _6-36 alkyl having 1 to 4 double bonds, C _8-24 alkyl, C _12-24 alkyl, C _12-18 alkyl, etc .; C _{6- 36} alkenyl, C _8-24 alkenyl, C _12-24 alkenyl, C _12-18 alkenyl and the like; for example, C _6-36 alkynyl, C _8-24 alkynyl, C _12-24 alkynyl having 1 to 4 triple bonds, C _12-18 alkynyl and the like; chains having 1-4 double or triple bonds; chains containing aryl or substituted aryl moieties (eg, phenyl or naphthyl moieties at the end or middle of the chain); aromatic polycyclic hydrocarbons A site; a cycloalkane or substituted alkane site having the number of carbons as described for the chain; combinations or mixtures thereof, and the like. Alkyl, alkenyl, or alkynyl groups may contain terminal functionality such as branched alcohols, amides, carboxylates, etc. within chain functionality such as ether groups. Also, a lipophilic moiety, such as a quaternary ammonium lipophilic moiety, can contain a positive charge.

Artificial Receptor Candidate artificial receptors, lead artificial receptors, or working artificial receptors include, for example, a combination of building blocks immobilized (eg, reversibly) on a support. Individual artificial receptors can be slides, tubes, or slides on wells or heterogeneous building block spots on multiple building blocks. The building blocks can be immobilized via any of a variety of interactions such as covalent, electrostatic, or hydrophobic interactions. For example, the building block and the support or lawn can each include one or more functional groups or sites that can form covalent, electrostatic, hydrogen bonding, van der Waals, etc. interactions.

  The array of candidate artificial receptors can be a commercial product sold to organizations interested in using the candidate artificial receptors as a tool in developing receptors for the test ligand of interest. In one embodiment, a useful array of candidate artificial receptors includes at least one glass slide, wherein the at least one glass slide contains a predetermined number of combination spots of members of a set of building blocks. Where each combination includes a predetermined number of building blocks.

  One or more lead artificial receptors can be developed from a plurality of candidate artificial receptors. In one embodiment, the lead artificial receptor comprises a combination of building blocks such as several picomoles of test ligand at a concentration of 1, 0.1, or 0.01 μg / ml, or 1, 0.1, or 0.01 ng / ml. ml test ligand; 0.01 μg / ml concentration, or 1, 0.1, or 0.01 ng / ml test ligand; or detectable upon exposure to 1, 0.1, or 0.01 ng / ml concentration of test ligand Bind an amount of test ligand.

  Artificial receptors, particularly candidate or lead artificial receptors, may be in the form of an array of artificial receptors. Such an array may include, for example, 1,660,000 spots, where each spot includes one combination of four building blocks from a set of 81 building blocks. Such an array can include, for example, 28,000 spots, where each spot includes one combination of 2, 3, or 4 building blocks from a set of 29 building blocks. Each spot is a combination of candidate artificial receptors and building blocks. The array can also be constructed to include leading artificial receptors. For example, an array of artificial receptors may include a smaller number of building block combinations and / or subsets of building blocks.

In one embodiment, the array of candidate artificial receptors is that RE ₁ is B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9 (shown herein below) and RE Includes building blocks of general formula 2 (shown herein below), where ₂ is A1, A2, A3, A3a, A4, A5, A6, A7, A8, or A9 (shown below) . In one embodiment, the framework is tyrosine.

  One or more working artificial receptors can be developed from one or more leading artificial receptors. In one embodiment, the working artificial receptor comprises a combination of building blocks, for example, at a concentration of 100, 10, 1, 0.1, 0.01, or 0.001 ng / ml test ligand; 10, 1, 0.1, 0.01 , Or 0.001 ng / ml of test ligand concentration; or 1, 0.1, 0.01, or 0.001 ng / ml of test ligand concentration upon exposure to several picomoles of test ligand, Classify or identify.

  In one embodiment, the artificial receptor of the present invention includes a plurality of building blocks attached to a support. In one embodiment, the plurality of building blocks may comprise or be a building block of Formula 2 (shown below). The abbreviation for building block containing the linker, tyrosine framework, and recognition element AxBy is TyrAxBy. In one embodiment, the candidate artificial receptors are the formulas TyrA1B1, TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B6, TyrA6B5, TyrA6B5, TyrA6B5, TyrA6B5, TyrA6B5 Or a combination of building blocks of TyrA8B8.

  The artificial receptors of the present invention can use any of a variety of supports that can couple building blocks or other arrays. For example, the support can be glass or plastic; slide, tube, or well; optical fiber; nanotube or buckyball, nanodevice; dendrimer, or skeleton.

TECHNICAL FIELD The present invention relates to a building block for creating or forming a candidate artificial receptor. Building blocks can be designed, created and selected to provide various structural features among a small number of compounds. The building blocks are positive charge, negative charge, acid, base, electron acceptor, electron donor, hydrogen bond donor, hydrogen bond acceptor, free electron pair, π electron, charge polarization, hydrophilicity, hydrophobicity, etc. One or more structural features. The building block may be large or it may be small.

  A building block can be visualized as including several elements, such as one or more frameworks, one or more linkers, and / or one or more recognition elements. The framework can be covalently coupled to each of the other building block elements. The linker can be covalently linked to the framework. The linker may be attached to the support through one or more covalent, electrostatic, hydrogen bonding, van der Waals interactions and the like. The recognition element can be covalently coupled to the framework. In one embodiment, the building block includes a framework, a linker, and a recognition element. In one embodiment, the building block includes a framework, a linker, and two recognition elements.

  A description of the general and specific features and functions of the various building blocks and their synthesis can be found in the co-pending US filed on September 16, 2002, entitled “ARTIFICIAL RECEPTORS, BUILDING BLOCKS, AND METHODS”, respectively. Patent application 10 / 244,727, 10 / 813,568 filed March 29, 2004, and PCT application PCT / US03 / 05328 filed February 19, 2003; each “ARTIFICIAL RECEPTORS INCLUDING REVERSIBLY IMMOBILIZED BUILDING BLOCKS, THE US patent applications 10 / 812,850 and 10 / 813,612, each filed March 29, 2004, and PCT application PCT / US2004 / 009649, each of the titles of the invention named “BUILDING BLOCKS, AND METHODS”, and BUILDING BLOCKS FOR ARTIFICIAL RECEPTORS The titles of the invention can be found in US Provisional Application 60 / 499,965 filed September 3, 2003 and 60 / 526,699 filed December 2, 2003. These patent documents specifically describe: function, structure, and building block composition, framework sites, recognition elements, building block synthesis, specific examples of building blocks, specific examples of recognition elements, and building blocks. Includes a detailed description of the set.

Frameworks Frameworks can be selected for coupling to a recognition site and coupling to a binding site or a functional group that provides it. The framework can interact with the ligand as part of the artificial receptor. In one embodiment, the framework includes a plurality of reactive sites with orthogonal and reliable functional groups and controlled stereochemistry. Suitable functional groups with orthogonal and reliable chemistry include, for example, carboxyl, amine, hydroxyl, phenol, carbonyl, and thiol groups, which can be individually protected, deprotected, and derivatized. it can. In one embodiment, the framework has 2, 3, or 4 functional groups with orthogonal and reliable chemistry. In one embodiment, the framework has three functional groups. In such embodiments, the three functional groups can be individually selected from, for example, carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol groups. The framework can include moieties such as alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroarylalkyl, and the like.

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４つの官能基を有するフレームワークに対する一般的な構造は、式1b:

によって表すことができる。
これらの一般的な構造において: R₁は、1-12、1-6、または1-4炭素アルキル、置換アルキル、シクロアルキル、複素環、置換複素環、アリールアルキル、アリール、ヘテロアリール、ヘテロアリールアルキル等の基であり得る;およびF₁、F₂、F₃、またはF₄は、独立して、カルボキシル、アミン、ヒドロキシル、フェノール、カルボニル、またはチオール基であり得る。F₁、F₂、F₃、またはF₄は、独立して、1-12、1-6、1-4炭素アルキル、置換アルキル、シクロアルキル、複素環、置換複素環、アリールアルキル、アリール、ヘテロアリール、ヘテロアリールアルキル、またはカルボキシルで置換された無機基、アミン、ヒドロキシル、フェノール、カルボニル、またはチオール基であり得る。F₃および／またはF₄は、不存在であってもよい。 The general structure for a framework with three functional groups is the formula 1a:

Can be represented by
The general structure for a framework with four functional groups is the formula 1b:

Can be represented by
In these general structures: R ₁ is 1-12, 1-6, or 1-4 carbon alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroaryl It can be a group such as alkyl; and F ₁ , F ₂ , F ₃ , or F ₄ can independently be a carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol group. F ₁ , F ₂ , F ₃ , or F ₄ is independently 1-12, 1-6, 1-4 carbon alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, It can be a heteroaryl, heteroarylalkyl, or carboxyl substituted inorganic group, an amine, hydroxyl, phenol, carbonyl, or thiol group. F ₃ and / or F ₄ may be absent.

  The various compounds are compatible with a framework that includes formulas and amino acids, and texts that describe naturally occurring or synthetic compounds that contain, for example, oxygen and sulfur functional groups. The compound can be racemic, optically active, or achiral. For example, the compound can be a natural or synthetic amino acid, α-hydroxy acid, thioic acid, and the like.

  Molecules suitable for use as a framework include natural or synthetic amino acids, particularly amino acids that have a functional group (eg, a third functional group) on their side chains. Amino acids contain carboxyl and amine functional groups. Side chain functional groups can include natural amino acids, amines (eg, alkylamines, heteroarylamines), hydroxyl, phenol, carboxyl, thiol, thioether, or amidino groups.

  Natural amino acids suitable for use as a framework include, for example, serine, threonine, tyrosine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine, lysine, arginine, histidine. Synthetic amino acids can be used as frameworks for alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroarylalkyl, etc., and carboxyl, amine, hydroxyl, phenol, carbonyl Or may include naturally occurring or synthetic side chain functional groups that modify or extend natural amino acids by thiol functional groups. Suitable synthetic amino acids include β-amino acids and homo- or β analogs of natural amino acids. In one embodiment, the framework amino acid can be serine, threonine, or tyrosine, such as serine or tyrosine, such as tyrosine.

  Without being limited to the present invention, framework amino acids such as serine, threonine, or tyrosine with a linker and two recognition elements are pendant and infrared relative to the extended carbon chain of the framework. Besides orientation, it can be visualized by one of the recognition elements.

  All naturally occurring and many synthetic amino acids are commercially available. In addition, forms of these amino acids that are derivatized or protected to be suitable for reaction to coupling to the recognition element (s) and / or linker (s) can be purchased or made by known methods (e.g. , Green, TW; Wuts, PGM (1999), Protective Groups in Organic Synthesis Third Edition, Wiley-Interscience, New York, 779 pp .; Bodanszky, M .; Bodanszky, A. (1994), The Practice of Peptide Synthesis Second Edition, Springer-Verlag, New York, 217 pp.).

Recognition Elements A recognition element can be selected to provide one or more structural features to the building block. The recognition element can interact with the ligand as part of the artificial receptor. For example, the recognition element can be positive charge, negative charge, acid, base, electron acceptor, electron donor, hydrogen bond donor, hydrogen bond acceptor, free electron pair, π electron, charge polarization, hydrophilicity, hydrophobicity, etc. One or more structural features can be provided. The recognition element can be a small group or it can be large.

  In one embodiment, the recognition element is a 1-12, 1-6, or 1-4 carbon alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroaryl It may be a group such as alkyl. Recognition elements include positive charges, negative charges, acids, bases, electron acceptors, electron donors, hydrogen bond donors, hydrogen bond acceptors, free electron pairs, π electrons, charge polarization, hydrophilicity, hydrophobicity, etc. Alternatively, it can be substituted by a conferring group.

  Recognition elements that have a positive charge (eg, in aqueous compositions at neutral pH) include amines, quaternary ammonium moieties, sulfonium, phosphonium, ferrocene, and the like. Suitable amines include alkylamines, alkyldiamines, heteroalkylamines, arylamines, heteroarylamines, arylalkylamines, pyridines, heterocyclic amines (saturated or unsaturated, with or without nitrogen in the ring), amidines, hydrazines, etc. including. Alkylamines generally have 1 to 12 carbons, for example 1-8, and the ring may have 3-12 carbons, for example 3-8. Suitable alkylamines include those of formula B9. Suitable heterocyclic or alkyl heterocyclic amines include those of formula A9. Suitable pyridines include those of formulas A5 and B5. Any of the amines can be used as a quaternary ammonium compound. Further suitable quaternary ammonium moieties include trimethylalkyl quaternary ammonium moieties, dimethylethylalkyl quaternary ammonium moieties, dimethylalkyl quaternary ammonium moieties, arylalkyl quaternary ammonium moieties, pyridinium quaternary ammonium moieties, and the like.

  Negatively charged recognition elements (eg, at neutral pH in aqueous compositions) are carboxylates, phenols substituted by strong electron withdrawing groups (eg, substituted tetrachlorophenol), phosphates , Phosphonates, phosphinates, sulfates, sulfonates, thiocarboxylates, and hydroxamic acids. Suitable carboxylates include alkyl carboxylates, aryl carboxylates, and arylalkyl carboxylates. Suitable phosphates include phosphate mono-, di-, and tri-esters, and phosphate mono-, di-, and tri-amides. Suitable phosphonates include phosphonic acid mono- and di-esters, and phosphonic acid mono- and di-amides (eg, phosphonamides). Suitable phosphinic acid salts include phosphinic acid esters and amides.

  Recognition elements having a negative charge and a positive charge (at neutral pH in an aqueous composition) include sulfoxides, betaines, and amine oxides.

  Acid recognition elements can include carboxylates, phosphates, sulfates, and phenols. Suitable acidic carboxylates include thiocarboxylates. Suitable acidic phosphates include the phosphates listed above herein.

  Basic recognition elements include amines. Suitable basic amines include alkylamines, arylamines, arylalkylamines, pyridines, heterocyclic amines (saturated or unsaturated, with or without nitrogen in the ring), amidines, and any of those listed herein above. Additional amines. Suitable alkylamines include those of formula B9. Suitable heterocyclic or alkyl heterocyclic amines include those of formula A9. Suitable pyridines include those of formulas A5 and B5.

  Recognition elements including hydrogen bond donors include amines, amides, carboxyls, protonated phosphates, protonated phosphonates, protonated phosphinates, protonated sulfates, protonated sulfinates, alcohols, and thiols. Including. Suitable amines include alkylamines, arylamines, arylalkylamines, pyridines, heterocyclic amines (saturated or unsaturated, with or without nitrogen in the ring), amidines, ureas, and any of those listed herein above. Other amines. Suitable alkylamines include those of formula B9. Suitable heterocyclic or alkyl heterocyclic amines include those of formula A9. Suitable pyridines include those of formulas A5 and B5. Suitable protonated carboxylates, protonated phosphates include those listed herein above. Suitable amides include those of formulas A8 and B8. Suitable alcohols include primary alcohols, secondary alcohols, tertiary alcohols, aromatic alcohols (eg phenol). Suitable alcohols include those of formula A7 (primary alcohol) and B7 (secondary alcohol).

  Recognition elements comprising a hydrogen bond acceptor or one or more free electron pairs are amines, amides, carboxylates, carboxyl groups, phosphates, phosphonoates, phosphinates, sulfates, sulfonates, alcohols, ethers , Thiols, and thioethers. Suitable amines are alkylamines, arylamines, arylalkylamines, pyridines, heterocyclic amines (saturated or unsaturated, with or without nitrogen in the ring), amidines, ureas, as listed herein above. And amines. Suitable alkylamines include those of formula B9. Suitable heterocyclic or alkyl heterocyclic amines include those of formula A9. Suitable pyridines include those of formulas A5 and A6. Suitable carboxylate salts include those listed herein above. Suitable amides include those of formulas A8 and B8. Suitable phosphates, phosphonoates and phosphinoates include those listed herein above. Suitable alcohols include primary alcohols, secondary alcohols, tertiary alcohols, aromatic alcohols, and those listed herein above. Suitable alcohols include those of formulas A7 (primary alcohol) and B7 (secondary alcohol). Suitable ethers include alkyl ethers and arylalkyl ethers. Suitable alkyl ethers include those of formula A6. Suitable arylalkyl ethers include those of formula A4. Suitable thioethers include those of formula B6.

  Recognition elements that contain uncharged polar or hydrophilic groups include amides, alcohols, ethers, thiols, thioethers, esters, thioesters, boranes, borates, and complex salts. Suitable amides include those of formulas A8 and B8. Suitable alcohols include primary alcohols, secondary alcohols, tertiary alcohols, aromatic alcohols, and those listed herein above. Suitable alcohols include those of formulas A7 (primary alcohol) and B7 (secondary alcohol). Suitable ethers include those listed herein above. Suitable ethers include those of formula A6. Suitable arylalkyl ethers include those of formula A4.

  Recognition elements containing uncharged hydrophobic groups are alkyl (substituted and unsubstituted), alkenes (conjugated and unconjugated), alkynes (conjugated and unconjugated), Contains aromatics. Suitable alkyl groups include lower alkyl, substituted alkyl, cycloalkyl, arylalkyl, and heteroarylalkyl. Suitable lower alkyl groups include those of formulas A1, A3, A3a, and B1. Suitable arylalkyl groups include those of formula A3, A3a, A4, B3, B3a, and B4. Suitable alkylcycloalkyl groups include those of formula B2. Suitable alkene groups include lower alkenes and arylalkenes. Suitable arylalkene groups include those of formula B4. Suitable aromatic groups include unsubstituted aryl, heteroaryl, substituted aryl, arylalkyl, heteroarylalkyl, alkyl-substituted aryl, and polycyclic aromatic hydrocarbons. Suitable arylalkyl groups include those of formulas A3, A3a and B4. Suitable alkylheteroaryl groups include those of formulas A5 and B5.

  Spacer (eg, small) recognition elements include hydrogen, methyl, ethyl, and the like. Large recognition elements contain 7 or more carbon or heteroatoms.

である。 Formulas A1-A9 and B1-B9 are:

It is.

  These A and B recognition elements are according to the standards: A1, ethylamine; A2, isobutylamine; A3, phenethylamine; A4, 4-methoxyphenethylamine; A5, 2- (2-aminoethyl) pyridine; A6, 2-methoxy Ethylamine; A7, Ethanolamine; A8, N-Acetylethylenediamine; A9, 1- (2-Aminoethyl) pyrrolidine; B1, Acetic acid, B2, Cyclopentylpropionic acid; B3, 3-Chlorophenylacetic acid; B4, Cinnamic acid; B5 , 3-pyridinepropionic acid; B6, (methylthio) acetic acid; B7, 3-hydroxybutyric acid; B8, succinamic acid; and B9, 4- (dimethylamino) butyric acid.

  In one embodiment, the recognition element is of formula A1, A2, A3, A3a, A4, A5, A6, A7, A8, and / or A9 (A recognition element) and / or B1, B2, B3, B3a, Includes one or more structures represented by B4, B5, B6, B7, B8, and / or B9 (B recognition element). In one embodiment, each building block includes an A recognition element and a B recognition element. In one embodiment, a group of 81 such building blocks includes each of 81 unique combinations of 81 A and B recognition elements. In one embodiment, the A recognition element binds to the framework at the pendant position. In one embodiment, the B recognition element binds to the framework at the infrared position. In one embodiment, the A recognition element is coupled to the framework at the pendant position and the B recognition element is coupled to the framework at the infrared position.

  Without being limited to the present invention, the A and B recognition elements are believed to represent the type of functional group and the geometry used by the polypeptide receptor. While not limited to the present invention, the A recognition element represents six advantageous functional groups or configurations, and the addition of functional groups to several aryl groups is believed to increase the range of possible binding interactions. It is done. Without being limited to the present invention, it is believed that the B recognition element represents six advantageous functional groups in a different arrangement than that used for the A recognition element. Without being limited to the present invention, this is further believed to increase the range of binding interactions and further extend the range of functional groups and configurations sought by the molecular configuration of the building blocks.

  In one embodiment, the building blocks containing A and B recognition elements can be visualized as occupying a binding space defined by lipophilicity / hydrophilicity and capacity. Capacity can be calculated (using known methods) for each building block containing various A and B recognition elements. A lipophilic / hydrophilic (logP) measurement can be calculated (using known methods) for each building block containing various A and B recognition elements. A negative value for logP shows more affinity for water than a nonpolar organic solvent and a hydrophilic property. The plot of capacity versus logP can then show the distribution of building blocks through the binding space defined by size and lipophilicity / hydrophilicity.

  Reagents that form many of the recognition elements are commercially available. For example, the reagents for forming the recognition elements A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9 B1, B2, B3, B3a, B4, B5, B6, B7, B8, and B9 are Are commercially available.

Linker The linker is selected to provide suitable coupling of the building block to the support. The framework can interact with the ligand as part of the artificial receptor. The linker can provide bulk, distance from support, hydrophobicity, hydrophilicity, and similar structural features to the building block. Coupling the building block to the support can use covalent or non-covalent bonds. Suitable non-covalent interactions include ionic interactions, hydrogen bonds, van der Waals interactions, and the like. In one embodiment, the linker comprises a site that can participate in a covalent bond or a non-covalent interaction. In one embodiment, the linker includes a site that can participate in a covalent bond. Suitable groups for forming covalent and reversible covalent bonds are described herein above.

Linker to the reversibly immobilizable building block The linker can be selected to provide suitable reversible immobilization of the building block on the support or lawn. In one embodiment, the linker forms a covalent bond with the functional group on the framework. In one embodiment, the linker also includes a functional group that can reversibly interact with the support or lawn, for example, via reversible covalent bonding or non-covalent interaction.

  In one embodiment, the linker comprises one or more sites that can participate in reversible covalent bonding. Suitable groups for reversible covalent bonding include those described hereinabove. Artificial receptors can include building blocks reversibly immobilized on a lawn or support, for example, through bonds such as imines, acetals, ketals, disulfides, esters, and the like. Such functional groups can participate in reversible covalent bonds. Such a functional group can be referred to as a covalent bond site such as, for example, a second covalent bond site.

  In one embodiment, the linker can be functionalized at sites that can participate in non-covalent interactions. For example, the linker may include functional groups such as ionic groups, groups capable of hydrogen bonding, or groups that may participate in van der Waals or other hydrophobic interactions. Such functional groups can include positive groups, negative groups, lipophilic groups, amphiphilic groups, and the like.

  In one embodiment, the methods and compositions of the invention can use a linker that includes a charged site (eg, a second charged site). Suitable charged sites include positively charged sites and negatively charged sites. Suitable positively charged moieties include amines, quaternary ammonium moieties, sulfonium, phosphonium, ferrocene, and the like. Suitable negatively charged sites (eg, in aqueous compositions at neutral pH) include carboxylates, phenols substituted with strong electron withdrawing groups (eg, tetrachlorophenol), phosphates, Includes phosphonates, phosphinoates, sulfates, sulfonates, thiocarboxylates, and hydroxamic acids.

  In one embodiment, the methods and compositions of the present invention can use a linker containing a group capable of hydrogen bonding as either a donor or acceptor (eg, a second hydrogen bonding group). For example, the linker can include one or more carboxyl groups, amine groups, hydroxyl groups, carbonyl groups, and the like. In addition, the ionic group can participate in hydrogen bonding.

In one embodiment, the methods and compositions of the present invention can employ a linker that includes a lipophilic moiety (eg, a second lipophilic moiety). Suitable lipophilic moieties are, for example, one or more branched or straight chain C _6-36 alkyl, C _8-24 alkyl, C _12-24 alkyl, C _12-18 alkyl, etc. having 1 to 4 double bonds; C _6-36 alkenyl, C _8-24 alkenyl, C _12-24 alkenyl, C _12-18 alkenyl and the like; for example, C _6-36 alkynyl having 1 to 4 triple bonds, C _8-24 alkynyl, C _{12- 24} alkynyl, C _12-18 alkynyl, etc .; chains with 1 to 4 double or triple bonds; chains containing aryl or substituted aryl moieties (eg chain end or intermediate phenyl or naphthyl moiety); aromatic polycycles Formula hydrocarbon moiety; as described for the chain, including cycloalkanes or substituted alkanes having multiple carbons; combinations or mixtures thereof, and the like. Alkyl, alkenyl, or alkynyl groups can include branching; within chain functionality such as ether groups; In one embodiment, the linker comprises or is a lipid such as a phospholipid. In one embodiment, the lipophilic moiety comprises or is a 12 carbon amphiphilic moiety.

  In one embodiment, the linker comprises a lipophilic group (eg, a second lipophilic group) and a covalent bond site (eg, a second covalent bond site). In one embodiment, the linker comprises a lipophilic group (eg, a second lipophilic group) and a charged site (eg, a second charged site).

  In one embodiment, the linker can be visualized to form or form a covalent bond with a group such as alcohol, phenol, thiol, amine, carbonyl, etc. on the framework. During attachment to a group that participates in or is formed by a reversible interaction with the framework and support or lawn, the linker is alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl , Aryl, heteroaryl, heteroarylalkyl, ethoxy or propoxy oligomers, glycosides and the like.

  For example, a suitable linker is: involved in or formed by a reversible interaction with a functional group involved in the framework or a functional group formed by binding to it, a support or lawn, and a linker backbone site Functional groups or groups may be included. The linker backbone moiety is about 4 to about 48 carbons or heteroatoms, about 8 to about 14 carbons or heteroatoms, about 12 to about 24 carbons or heteroatoms, about 16 to about 18 carbons or heteroatoms, about It may contain 4 to about 12 carbon or heteroatoms, about 4 to about 8 carbon or heteroatoms, and the like. The linker backbone can include moieties such as alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroarylalkyl, ethoxy or propoxy oligomers, glycosides, mixtures thereof, and the like.

In one embodiment, the linker comprises one or more lipophilic moieties, functional groups involved in or formed by the framework, and optionally a reversible covalent hydrogen bond, or ionic interaction to form an ionic interaction. Including sites. In such embodiments, the lipophilic moiety can have about 4 to about 48 carbons, about 8 to about 14 carbons, about 12 to about 24 carbons, about 16 to about 18 carbons, and the like. In such embodiments, the linker may comprise about 1 to about 8 reversible binding / interaction sites or about 2 to about 4 reversible binding / interaction sites. Suitable linkers have a structure such as (CH ₂ ) _n COOH with n = 12-24, n = 17-24, or n = 16-18.

Further Specific Examples of Linkers Linkers can be selected to provide suitable covalent coupling of the building block to the support. The framework can interact with the ligand as part of the artificial receptor. The linker can also provide structural features such as bulk, hydrophobicity, hydrophilicity, etc. to the building blocks away from the support. In one embodiment, the linker forms a covalent bond with the functional group on the framework. In one embodiment, prior to attachment to the support, the linker comprises a functional group that can be activated or will react with a functional group on the support. In one embodiment, once attached to the support, the linker forms a covalent bond with the support and with the framework.

  In one embodiment, the linker can be visualized as forming or forming a covalent bond with a group such as alcohol, phenol, thiol, amine, carbonyl, etc. on the framework. The linker can include groups such as carboxyl, alcohol, phenol, thiol, amine, carbonyl, maleimide, etc. that can react with or be activated to react with the support. Between the group formed by the bond to the framework and the bond to the support, the linker is alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroarylalkyl, It may contain sites such as ethoxy or propoxy oligomers, glycosides and the like.

  The linker may include a good leaving group attached to, for example, an alkyl or aryl group. The leaving group is “good” enough to be substituted on the framework by groups such as alcohol, phenol, thiol, amine, carbonyl and the like. Such linkers are of the formula: RX, wherein X is a leaving group such as halogen (eg -Cl, -Br or -I), tosylate, mesylate, triflate, R is alkyl, Substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroarylalkyl, ethoxy or propoxy oligomers, glycosides, etc.].

Suitable linker groups include those of the formula: (CH ₂ ) _n COOH where n = 1-16, n = 2-8, n = 2-6, or n = 3. Reagents that form suitable linkers are commercially available and include any of a variety of reagents with orthogonal functionality.

によって表すことができる。式中: RE₁は認識要素 1であり、RE₂は認識要素 2であり、Lはリンカーである。Xは存在せず、C=O、CH₂、NR、NR₂、NH、NHCONH、SCONH、CH=N、またはOCH₂NHである。特定の具体例において、Xは不存在またはC=Oである。Yは不存在、 NH、O、CH₂、またはNRCOである。特定の具体例において、YはNHまたはOである。１つの具体例において、YはNHである。Z₁およびZ₂は、独立して、CH₂、O、NH、S、CO、NR、NR₂、NHCONH、SCONH、CH=N、またはOCH₂NHであってもよい。１つの具体例において、Z₁および／またはZ₂は、独立して、Oであってもよい。Z₂は任意である。R₂はH、CH3、または形成ブロックに対してキラル性を授与し、メチル基と同様またはそれより小さいサイズを有するもう１つの基である。R₃は、CH₂; CH₂-フェニル; CHCH₃; n=2-3を有する(CH₂)n;または、例えば、5-6の炭素といった3-8の炭素を有する環状アルキル、フェニル、ナフチルである。特定の具体例において、R₃は、CH₂またはCH₂-フェニルである。 Specific Example of Building Block In one specific example, the building block is represented by the formula 2:

Can be represented by Where: RE ₁ is recognition element 1, RE ₂ is recognition element 2, and L is a linker. X is absent, a _{C = O, CH 2, NR} , NR 2, NH, NHCONH, SCONH, CH = N or OCH ₂ NH,. In certain embodiments, X is absent or C = O. Y is absent, NH, O, CH ₂ , or NRCO. In certain embodiments, Y is NH or O. In one embodiment, Y is NH. Z ₁ and Z ₂ may independently be CH ₂ , O, NH, S, CO, NR, NR ₂ , NHCONH, SCONH, CH═N, or OCH ₂ NH. In one embodiment, Z ₁ and / or Z ₂ may independently be O. Z ₂ is optional. R ₂ is H, CH 3, or another group that imparts chirality to the building block and has a size similar to or smaller than the methyl group. R ₃ is CH ₂ ; CH ₂ -phenyl; CHCH ₃ ; (CH ₂ ) n with n = 2-3; or cyclic alkyl having 3-8 carbons, for example 5-6 carbons, phenyl, Naphthyl. In certain embodiments, R ₃ is CH ₂ or CH ₂ -phenyl.

RE ₁ is B1, B2, B3, B3a, B4, B5, B6, B7, B8, B9, A1, A2, A3, A3a, A4, A5, A6, A7, A8, or A9. In certain embodiments, RE ₁ is B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9. RE ₂ is A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9, B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9. In certain embodiments, RE ₂ is A1, A2, A3, A3a, A4, A5, A6, A7, A8, or A9. In one embodiment, RE ₁ may be B2, B3a, B4, B5, B6, B7, or B8. In one embodiment, RE ₂ may be A2, A3a, A4, A5, A6, A7, or A8.

  In one embodiment, L is a functional group involved in or formed by binding to a framework (such groups are described herein), reversible interaction with a support or lawn. A functional group or group that participates in or is formed by binding to it (such groups are described herein), and a linker backbone moiety. In one embodiment, the linker backbone moiety is from about 4 to about 48 carbon or heteroatom alkyl, substituted alkyl, cycloalkyl, heterocycle, substituted heterocycle, arylalkyl, aryl, heteroaryl, heteroarylalkyl, ethoxy or Propoxy oligomers, glycosides, or mixtures thereof; or about 8 to about 14 carbons or heteroatoms, about 12 to about 24 carbons or heteroatoms, about 16 to about 18 carbons or heteroatoms, about 4 to about 12 carbons Or a heteroatom, about 4 to about 8 carbons or a heteroatom.

In one embodiment, L is a functional group involved in or formed by binding to the framework (such groups are described herein) and about 4 to about 48 carbons, about 8 A lipophilic group having from about 14 to about 14 carbons, from about 12 to about 24 carbons, from about 16 to about 18 carbons (such groups are described herein). In one embodiment, the L also has from about 1 to about 8 reversible binding / interaction sites (such groups are described herein) or from about 2 to about 4 reversible binding / interaction sites. Including. In one embodiment, L is (CH ₂ ) _n COOH where n = 12-24, n = 17-24, or n = 16-18.

In one embodiment, L is (CH ₂ ) _n COOH where n = 1-16, n = 2-8, n = 4-6, or n = 3.

  Building blocks containing A and / or B recognition elements, linkers, and amino acid frameworks can be made by the method shown in general scheme 1.

Techniques for Using Artificial Receptors The present invention includes methods of using artificial receptors. The present invention includes a method of screening candidate artificial receptors to find lead artificial receptors that bind a particular test ligand. Detection of a test ligand bound to a candidate artificial receptor can be accomplished using known methods for detecting binding to an array on a slide or a coated tube or well. For example, the method can use a test ligand labeled with a detectable label such as a fluorophore or enzyme that produces a detectable product. Alternatively, the method can use an antibody (or other binding agent) that is specific for the test ligand and that contains a detectable label. One or more spots labeled with a test ligand or more or most strongly labeled with a test ligand are selected as lead artificial receptors. The degree of labeling can be assessed by evaluating the signal intensity from the label. The amount of signal can be directly proportional to the amount of label and binding. FIG. 13 provides a schematic diagram of an example of this process.

  According to the method of the present invention, candidate artificial receptors can be screened against a test ligand to obtain one or more lead artificial receptors. The one or more lead artificial receptors can be working artificial receptors. That is, the one or more lead artificial receptors can be useful as is to detect the ligand of interest. The method can then use one or more artificial receptors as a working artificial receptor for monitoring or detecting a test ligand. Alternatively, the one or more lead artificial receptors can be used in a method for developing working artificial receptors. For example, one or more lead artificial receptors may provide structural or other information useful for designing or screening improved lead artificial receptors or working artificial receptors. Such design or screening can include creating and testing additional candidate artificial receptors that include subsets of building blocks, different sets of building blocks, and different numbers of combinations of building blocks.

  The present invention includes a method of screening candidate artificial receptors to find lead artificial receptors that bind a particular test ligand. The method may include allowing movement of the building block that creates the artificial receptor. The movement of the building block includes driving the building block to move along or over the support and / or leave the support and enter a fluid (e.g., liquid) phase separate from the support or lawn. obtain.

  In one embodiment, the building block can be driven to move along or on the support (translation or shuffle). Such translation can be used, for example, to realign building blocks that are already bound to the test ligand to lower energy or tighter binding configurations that are still bound to the test ligand. Such translation can be used, for example, to allow the ligand to access building blocks that are on the support but not bound to the ligand. These building blocks can translate to binding near or to the test ligand.

  The building block can be induced to move along or on the support or reversibly fix on the support via any of a variety of mechanisms. For example, inducing the mobility of the building block may include modifying the support or loan situation. That is, changing the situation can reverse the immobilization of building blocks, ie move them. After they move, reversibly immobilizing the building blocks can include, for example, returning to the previous situation. Appropriate modification of the situation changes pH, changes temperature, changes polarity or hydrophobicity, changes ionic strength, changes nucleophilicity or electrophilicity (eg of solvent or solute), etc. Including that.

  Exposing a surface, lawn, or building block to a more hydrophobic solvent (e.g., organic solvent or surfactant) by raising the temperature of the building block reversibly immobilized by hydrophobic interactions. Or by reducing the ionic strength around the building block. In one embodiment, the organic solvent includes acetonitrile, acetic acid, alcohol, tetrahydrofuran (THF), dimethylformamide (DMF), hydrocarbons such as hexane or octane, acetone, chloroform, methylene chloride, and the like, or mixtures thereof. In one embodiment, the surfactant comprises a nonionic surfactant such as nonylphenol ethoxylate or the like. Building blocks that move onto the support can interact with hydrophobic interactions or increased ionic strength, for example, by lowering the temperature, exposing the surface, lawn, or building blocks to more hydrophilic solvents (e.g., aqueous solvents). Can be reversibly immobilized.

  A building block reversibly immobilized by hydrogen bonding can be moved by increasing the ionic strength, the concentration of the hydrophilic solvent, or the concentration of competing hydrogen bonds around the building block. The building blocks that move on the support can be reversibly immobilized via electrostatic interactions, such as by reducing the ionic strength of the hydrophilic solvent.

  A building block reversibly immobilized by electrostatic interaction can be moved by increasing the ionic strength around the building block. The ionic strength can be increased to disrupt the electrostatic interaction. The building blocks that move on the support can be reversibly immobilized via electrostatic interactions by reducing the ionic strength.

  Building blocks reversibly immobilized by imine, acetal, ketal linkages can be moved by decreasing the pH or increasing the concentration of nucleophilic catalyst around the building block. In one embodiment, the pH is from about 1 to about 4. Imines, acetals, and ketals undergo acid-catalyzed hydrolysis. The building blocks that move on the support can be reversibly immobilized by reversible covalent interactions such as by increasing the pH by forming imine, acetal, or ketal bonds.

  In one embodiment, the building block can be moved away from the support and placed in a fluid (eg, liquid) phase separate from the support or lawn (exchange). For example, the building blocks can be exchanged on and / or away from the support. Exchange can be used, for example, to remove building blocks on the support that are not bound to the test ligand from the support. Exchange can be used, for example, to add additional building blocks to the support. The added building block may have a structure that is selected based on knowledge of the structure of the building block in the artificial receptor that binds the test ligand. The added building block can have a structure selected to provide additional structural diversity. The added building blocks can include all of the building blocks.

  Building blocks reversibly immobilized by hydrophobic interactions can be released from the support, for example, by raising the temperature of the support and / or artificial receptor. For example, hydrophobic interactions (e.g., hydrophobic groups on a support or lawn and on a building block) can be selected to provide an immobilized building block at or near room temperature, Release can be achieved at temperatures above room temperature. For example, hydrophobic interactions can be selected to provide an immobilized building block at approximately refrigerator temperature (e.g., 4 ° C) or lower, and release can be performed at, for example, room temperature or higher Can be achieved. To further illustrate, a building block can be reversibly immobilized by hydrophobic interactions, for example, by contacting a surface or artificial receptor with a fluid containing the building block and below room temperature. .

  The building blocks reversibly immobilized by hydrophobic interactions can be released from the support, for example, by contacting the artificial receptor with a sufficiently hydrophobic fluid (eg, an organic solvent or surfactant). Can do. In one embodiment, the organic solvent includes acetonitrile, acetic acid, alcohol, tetrahydrofuran (THF), dimethylformamide (DMF), hydrocarbons such as hexane or octane, acetone, chloroform, methylene chloride, and the like or mixtures thereof. In one embodiment, the surfactant comprises a nonionic surfactant such as nonylphenol ethoxylate or the like. Such reversible immobilization can also be achieved by contacting the surface or artificial receptor with a hydrophilic solvent and partitioning the somewhat lipophilic building block onto the hydrophobic surface or lawn. .

  The building blocks reversibly immobilized by imine, acetal, or ketal linkages can be released from the support, for example, by contacting the artificial receptor with a fluid having an acid pH or containing a nucleophilic catalyst. it can. In one embodiment, the pH is from about 1 to about 4. The building block is reversible by reversible covalent interactions, such as by forming an imine, acetal, or ketal bond by contacting a surface or artificial receptor with a fluid having a neutral or basic pH. Can be immobilized.

  A building block reversibly immobilized by electrostatic interaction can be released, for example, by perturbing electrostatic interaction by contacting the artificial receptor with a fluid having sufficiently high ionic strength. . The building block is reversible via electrostatic interaction by contacting the surface or artificial receptor with a fluid having an ionic strength that promotes electrostatic interaction between the building block and the support and / or the lawn. Can be fixed.

  The invention will be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the invention and are not intended to limit the scope of the invention.

Examples Example 1-Synthesis of building blocks Selected building blocks representing alkyl-aromatic-polar spans of building block embodiments are synthesized and the effectiveness of these building blocks to create candidate artificial receptors. Showed sex. These building blocks were created on a framework that can be represented by tyrosine and contains numerous pairs of recognition elements. These recognition element pairs represent a complete set of interactions of 81 such building blocks and a significant degree of functionality, including a sufficient range from alkyl to aromatic to polar.

Synthesis Building block synthesis used the general procedure outlined in Scheme 7, which specifically illustrates the synthesis of the building block recognition element pair A4B4 on the tyrosine framework. The general procedure is TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA6B2, TyrA6B4, TyrA6B6, TyrA6B6, TyrA2B6, TyrA2B6, TyrA2B6 Used for the synthesis of building blocks containing

Results The synthesis of the desired building blocks has generally proved simple. These syntheses consist of two recognitions having different structural features or structures once building blocks with corresponding recognition elements (eg A2B2, A4B4, etc.) are prepared via their X BOC intermediates. Figure 2 shows the relative simplicity of preparing building blocks with elements (eg, A4B2, A6B3, etc.).

  Conversion of one of these building blocks into a building block having a lipophilic linker can be accomplished, for example, by reacting the activated building block with dodecylamine.

Example 2 Preparation and Evaluation of Candidate Artificial Receptor Microarrays Candidate artificial receptor microarrays were created and evaluated for the binding of several protein ligands. The results obtained are: 1) ease of preparing candidate artificial receptor microarrays, 2) reproducibility of binding affinity and binding patterns, 3) against the building block heteroreceptor environment when compared to their respective homologous controls It showed significantly improved binding, and 4) a ligand-specific binding pattern (eg, acting receptor complex).

Materials and Methods Building blocks were synthesized and activated as described in Example 1. The building blocks used in this example were TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B2, TyrA6B4, and TyrA6B6. The abbreviation for the building block containing the linker, tyrosine framework, and recognition element AxBy is TyrAxBy.

For a microarray for evaluation of 130 n = 2 and n = 3 and for evaluation of 273 n = 2, n = 3, and n = 4, the candidate receptor environment is determined by modification of known methods: It was prepared as follows. As used herein, “n” is the number of different building blocks used in the receptor environment. Briefly: Modified amine (amine “lawn”; superamine microarray plate) Microarray plates were purchased from Telechem Inc., Sunnyvale, CA (www.arrayit.com). These plates were made specifically for microarray preparation and, according to the manufacturer, had a nominal amine loading of 2-4 amines per nm square. CAM microarrays are typically produced from Telechem Inc. (SpotBot ^™ Arrayer) with 200 um diameter spotting pins from Telechem Inc. (Stealth Micro Spotting Pins, SMP6) and 400-420 um spot spacing. Prepared using a potter instrument.

  Nine building blocks were activated in aqueous dimethylformamide (DMF) solution as described above. To prepare a 384 well feed plate, the activated building block solution was added to DMF / H2O / PEG400 (90/10/10, v / v / v; PEG400 is polyethylene glycol. Nominal 400 FW, Aldrich (Chemical Co., Milwaukee, Wis.). These stock solutions were aliquoted (10 μl / aliquot) into wells of a 384 well microwell plate (Telechem Inc.). Separate series of controls were prepared by aliquoting 10 μl of building blocks with either 10 μl or 20 μl of activated [1-1] solution. The plate was covered with aluminum foil and placed on a rotary stirrer bed at 1,000 RPM for 15 minutes. The master plate was stored covered with aluminum foil at −20 ° C. when not in use.

To prepare a 384-well SpotBot ^™ plate, a well-to-well transfer from a feed plate to a second 384 well plate (eg, A-1 to A-1, A-2 to A-2, etc.) This was done using a 4 μl transfer pipette. The plate was stored sealed with aluminum foil at −20 ° C. when not in use. SpotBot ^™ was used to prepare up to 13 microarray plates / column using 4 μl microwell plates. SpotBot ^™ was programmed to spot each microwell in four ways. The cleaning station on SpotBot ^{™ used} EtOH / H 2 O (20/80, v / v) cleaning solution. This washing solution was also used to rinse the microarray upon completion of the SpotBot ^™ print section. The plate was finally rinsed with deionized (DI) water, dried using a stream of compressed air, and then stored at room temperature.

  In addition, certain microarrays were modified by reacting the remaining amine with succinic anhydride to form a carboxylic acid lawn instead of an amine lawn.

The following test ligands and labels were used in these experiments:
1) r-phycoerythrin, a commercially available and intrinsically fluorescent protein with 2,000,000 FW
2) Ovalbumin labeled with Alexa ^TM fluorophore (Molecular Probes Inc., Eugene, OR)
3) BSA, bovine serum albumin, labeled with activated rhodamine (Pierce Chemical, Rockford, IL) using a known activated carboxyl protocol. BSA had a FW of 68,000; the material used in this study had approximately 1.0 rhodamine / BSA.
4) Horseradish peroxidase (HRP) modified with excess amine and labeled as acetamide derivative or with 2,3,7,8-tetrachlorodibenzodixoin derivative was available through known methods . Fluorescence detection of these HRP conjugates was based on the Alexa 647-tyramide kit available from Molecular Probes, Eugene, OR.
5) Cholera toxin labeled with Alexa ^TM fluorophore (Molecular Probes Inc., Eugene, OR)

  Microarray incubation and analysis was performed as follows: in test-ligand incubation with microarray in typical concentrations of PBS-T (PBS with 20 μl / L Tween-20) at 10, 1.0 and 0.1 μg / ml A target protein solution (for example, 500 μl) was placed on the surface of the microarray and allowed to react, for example, for 30 minutes. The microarray was rinsed with PBS-T and DI water and dried using a stream of compressed air.

  Incubated microarrays were scanned using an Axon Model 4200A fluorescence microarray scanner (Axon Instruments, Union City, Calif.). The Axon scanner and its associated software produce a pseudo-color 16-bit image of the fluorescence intensity of the plate. This 16-bit data gives fluorescence unit s values (range 0-65,536) for each spot on the microarray using Axon software. This data is then exported to an Excel file (Microsoft) for further analysis including daytime, standard deviation and variation calculation counts.

result
CARATM: combinatorial receptor array (Combinatorial Artificial Receptor Array ^TM) concept was performed using a microarray format. CARA microarrays were prepared based on N = 9 building blocks and evaluated for binding to several proteins and substituted protein ligands. The microarray contained 144 candidate receptors (18 n = 1 control plus 6 blanks; 36 n = 2 candidate receptors; 84 n = 3 candidate receptors). The microarrays are: 1) ease of CARA microarray preparation, 2) binding affinity and binding pattern reproducibility, 3) significantly improved binding to the building block heteroreceptor environment when compared to their respective homologous controls, And 4) showed a unique binding pattern of the ligand.

Array reading
A typical false / grayscale image of a microarray incubated with 2.0 μg / ml r-phycoerythrin is shown in FIG. This image shows the predicted range of binding so that both processes of preparing the microarray and probing it with a protein test ligand are seen in the visual range of relative fluorescence from dark spots to bright spots. Indicates that it has been generated.

  The starting point for the analysis of the data was to collect integrated fluorescence units for the array of spots and normalize to the observed values for the [1-1] building block control, followed by Standard deviation and variation calculation coefficients were included. Furthermore, control values for homogenous building blocks were obtained from building block plus [1-1] data.

First set of experiments The following protein ligands were evaluated for binding to candidate artificial receptors in a microarray. The resulting fluorescence unit versus candidate receptor environment data includes a 2D format where the candidate receptor is along the X axis, the fluorescence unit is shown on the Y axis, and the candidate receptor is placed in the XY format, The fluorescent unit is shown in both 3D formats shown on the Z axis. A key to the composition of each spot was developed (not shown). Keys for building blocks in each of the resulting 2D and 3D representations were also developed (not shown). Data shown are for 1-2 μg / ml protein concentration.

  Figures 15 and 16 show binding data for r-phycoerythrin (endogenous fluorescence). Figures 17 and 18 show binding data for ovalbumin (commercially available via fluorescent labeling). Figures 19 and 20 show bovine serum albumin (labeled with rhodamine). Figures 21 and 22 show binding data for HRP-NH-Ac (fluorescent tyramide readout). Figures 23 and 24 show binding data for HRP-NH-TCDD (fluorescent tyramide readout).

  These results not only apply the CARA microarray to candidate artificial receptor evaluation, but also a number of readout methods that can be used for high-throughput candidate receptor evaluation (eg, intrinsic fluorescence, fluorescent labeling, in situ fluorescent labeling) A few of are also shown.

  Evaluation of candidate receptors benefits from reproducibility. The following results show that the microarray of the present invention provided reproducible ligand binding.

  The microarray was printed with each combination of building blocks spotted in four ways. An inspection of a direct plot (FIG. 25) of raw fluorescence data (from the run shown in FIG. 14) for one block of binding data obtained for r-phycoerythrin revealed that the candidate receptor environment “ “Spot” indicates reproducible binding to the test ligand. Further analysis of the r-phycoerythrin data (Figure 14) yields only 9 (1.2%) of 768 spots that are deleted as outliers. Analysis of the four data for r-phycoerythrin for the entire array yields the mean standard deviation for each experimental quadruplicate set of 938 fluorescent units, with an average coefficient of variation of 19.8%.

  Although these values are acceptable, more realistic comparisons used standard deviations and coefficients of variation for more fluorescent receptors that bound more strongly. The overall mean standard deviation unrealistically raises the coefficient of variation for weakly bound, weaker fluorescent receptors. The coefficient of variation for 19 receptors with 10,000 or more fluorescent units of binding target is 11.1%, which is well within the range necessary to generate meaningful binding data.

  One goal of the CARA approach is the convenient preparation of a significant number of candidate receptors through a combination of structurally simple building blocks. The following results establish that both individual building blocks and building block combinations have a significant and favorable effect on test ligand binding.

  The binding data shown in FIGS. 23-24 show that the heterogeneous combination of building blocks (n = 2, n = 3) is a significantly better candidate receptor made from a single building block (n = 1). Indicates. For example, FIG. 16 shows both the binding diversity observed for n = 2, n = 3 candidate receptors with fluorescent units ranging from 0 to about 40,000. These data also indicate that an approximately 10-fold improvement in binding affinity was obtained by changing from a homogeneous (n = 1) to a heterogeneous (n = 2, n = 3) receptor environment.

  The effect of heterologous building blocks is by comparing selected n = 3 receptor environment candidate receptors, including 1 or 2 of their building blocks (n = 2 and n = 1 subsets thereof). The most easily observed. Figures 26 and 27 show this comparison for two different n = 3 receptor environments using r-phycoerythrin data. In these examples, it is clear that the progression from the homogeneous system (n = 1) to the heterogeneous system (n = 2, n = 3) significantly promoted the binding.

  Van der Waals interactions are an important part of molecular recognition, but it is important to establish that the observed binding is not a simple case of hydrophobic / hydrophilic partitioning. That is, the observed binding is the result of specific interactions between individual building blocks and targets. The simplest way to assess the effects of hydrophobicity and hydrophilicity is to compare the building block logP value to the observed binding. LogP is a known acceptable measurement of lipophilicity, which can be measured or calculated by known methods for each of the building blocks. Figures 28 and 29 establish that the observed target binding is not directly proportional to the building block logP, as measured by the fluorescence unit. The plots in FIGS. 28 and 29 show a non-linear relationship between binding (fluorescence unit) and building block logP.

  One advantage of the methods and arrays of the present invention is that the ability to screen the majority of candidate receptor environments provides useful target affinity combinations and significant target binding diversity. High target affinity is useful for specific target binding, isolation, etc., but binding diversity can provide a multiple target detection system. This example generated about 120 bonding environments using a relatively small number of building blocks. The following analysis of the data of the present invention clearly shows that even a relatively large number of binding environments can produce diverse and useful artificial receptors.

  Target binding experiments performed for this study used protein concentrations including 0.1 to 10 μg / ml. Considering BSA data as a representative, it is clear that some receptor environments immediately bind 1.0 ug / ml BSA concentration near the saturation value for the fluorescence unit (see, eg, FIG. 20). Based on these data and a formula weight of 68,000 for BSA, some receptor environments readily bind BSA at a concentration of about 15 pmol / ml or 15 nanomolar. Further experiments using lower concentrations of protein (data not shown) show that femtomole / ml or picomolar detection limits were obtained even with a small selection of candidate receptor environments.

  One goal of artificial receptor development is the specific recognition of specific targets. FIG. 30 compares the observed binding to r-phycoerythrin and BSA. Comparison of overall binding patterns shows some general similarities. However, a comparison of the specific characteristics of binding to each receptor environment shows that the two targets have characteristic recognition features, as indicated by (*) in FIG.

  One goal of artificial receptor development is to develop receptors that can be used for multiplex detection of specific targets. Comparison of r-phycoerythrin, BSA and ovalbumin data from this study (FIGS. 16, 18, and 20) was used to select representative artificial receptors for each target. FIGS. 31, 32 and 33 show the identification of each of these three targets by their characteristic binding pattern using the data obtained in the examples of the present invention.

Conclusion The optimal receptor for a particular target requires molecular recognition that is greater than the predicted sum of individual hydrophilic, hydrophobic, ionic, etc. interactions. Therefore, the identification of optimal (specific, sensitive) artificial receptors from the restricted pool of candidate receptors investigated in this prototype study was unexpected and unlikely. Rather, the aim was to show that all of the key elements of the CARA: Combinatorial Artificial Receptor Array concept can be collected to form a functional receptor microarray. This purpose has been successfully demonstrated.

  This study finally establishes that CARA microarrays can be easily prepared and target receptors and candidate ligands can be used to identify artificial receptors and test ligands. In addition, these results show that there is significant binding enhancement for building block heterogeneous (n = 2, n = 3, or n = 4) candidate receptors when compared to their homologous (n = 1) counterparts It shows that. Combining binding pattern recognition results and the demonstrated importance of both heterologous receptor elements and heterologous building blocks, these results clearly show that CARA candidate artificial receptors-> leading artificial receptors-> acting artificial Shows the significance of receptor strategy.

Example 3 Preparation and Evaluation of Candidate Artificial Receptor Microarrays Containing Reversibly Immobilized Building Blocks Candidate Artificial Receptor Microarrays Containing Immobilized Building Blocks via Van der Waals Interactions were Created The protein ligand binding was evaluated. The evaluation was carried out at several temperatures around the phase transition temperature for the vide infra.

Materials and Methods Building blocks 2-2, 2-4, 2-6, 4-2, 4-4, 4-6, 6-2, 6-4, 6-6 were prepared as described in Example 1. did. C12 amide was prepared using the aforementioned carbodiimide activation of carboxyl following the addition of dodecylamine. This produced a building block with a 12 carbon alkyl chain linker for reversible immobilization in the C18 lawn.

  Aminolone microarray plates (Telechem) were modified using the lawn modification procedure generally described in Example 2, A) dimethylformamide / PEG 400 solution (90:10, v / v, PEG 400 is polyethylene C18 lawn was generated by reaction of stearoyl chloride (Aldrich Chemical Co.) in a methylene chloride / TEA solution (100 ml methylene chloride, 200 μl triethylamine) with glycol average MW 400 (Aldrich Chemical Co.) or B).

  C18 loan plates were printed using the SpotBot standard procedure as described in Example 2. The building blocks were in a printing fluid prepared with approximately 10 mg solution of each building block in 300 μl methylene chloride and 100 μl methanol. To this stock was added 900 μl dimethylformamide and 100 μl PEG 400. Thirty-six combinations of 9 building blocks taken two at a time (N9: n2, 36 combinations) were prepared in 384-well microwell plates, which were then used with SpotBot to print the microarray in quadruplicate. A random selection of printing positions included only the printing fluid.

Selected microarrays were as follows: 1) The microarray was washed with methylene chloride, ethanol and water to make a control plate; then 2) The microarray was incubated at 4 ° C, 23 ° C, or 44 ° C Were used to incubate with a 1.0 μg / ml solution of cholera toxin subunit B, a test ligand labeled with an Alexa ^™ fluorophore (Molecular Probes Inc., Eugene, OR). After incubation, the plate (s) were rinsed with water, dried and scanned (AXON 4100A). Data analysis was as described in Example 2.

Results A control array from which building blocks had been removed by washing with organic solvent did not bind to cholera toxin (FIG. 34). Figures 35-37 show fluorescent signals from arrays printed identically but incubated with cholera toxin at 4 ° C, 23 ° C, or 44 ° C, respectively. A fluorescent spot is seen in each array with a highly emphasized spot generated by a 44 ° C. incubation. The fluorescence values for the spots in each of these three arrays are shown in FIGS. 38-40. The fluorescent signal generally increases with temperature, and many nearly equally large signals are observed after incubation at 44 ° C. A linear increase over temperature may reflect the predicted improvement in coupling with temperature. The non-linear increase reflects the rearrangement of building blocks on the surface to achieve improved binding that occurred on the phase transition to the lipid surface (vide infra).

  FIG. 41 can be compared with FIG. The fluorescent signal plotted in FIG. 39 resulted from binding to the reversibly immobilized building block on the 23 ° C. support. The fluorescent signal plotted in FIG. 41 resulted from binding to the reversibly immobilized building block on the 23 ° C. support. These figures compare the same combination of building blocks in the same relative position but immobilized in two different ways.

  In addition, binding to a building block immobilized by covalent bonding was evaluated at 4 ° C, 23 ° C, or 44 ° C. FIG. 42 shows the change in fluorescence signal from individual combinations of building blocks covalently immobilized at 4 ° C., 23 ° C., or 44 ° C. Bonding increased slightly with temperature. The average increase in binding was 1.3 times. A plot of the fluorescent signal for each of the artificial receptors immobilized covalently at 23 ° C. versus the signal at 44 ° C. (not shown) gave a linear correlation with a correlation coefficient of 0.75. This linear correlation shows that an average 1.3-fold increase in binding is a thermodynamic effect, not binding optimization.

  FIG. 43 shows the change in fluorescence signal from individual combinations of reversibly immobilized building blocks at 4 ° C., 23 ° C., or 44 ° C. This graph shows that at least one combination of building blocks exhibited a signal that remained constant as the temperature increased. At least one candidate artificial receptor exhibited an approximately linear increase in signal with increasing temperature. Such a linear increase indicates a normal temperature effect on the coupling. The candidate artificial receptor with the lowest binding signal at 4 ° C. became one of the best binding agents at 44 ° C. This indicates that the rearrangement of this receptor building block on the phase transition for a loan that increases the building block mobility increased binding. Other receptors characterized by a greater change in binding between 23 ° C. and 44 ° C. (compared to between 4 ° C. and 23 ° C.) also experienced mechanical affinity optimization.

  FIG. 44 shows the data shown in FIG. 42 (line marked A) and the data shown in FIG. 43 (line marked B). The increase in binding observed with reversibly immobilized building blocks is significantly greater than the increase observed with covalently bonded building blocks. Binding to the reversibly immobilized building block increased from 23 ° C. to 44 ° C. with a median value of 6.1 and a mean value of 24 times.

  This confirms that reversibly immobilized building block movement within the receptor increased binding (ie, the receptor experienced dynamic affinity optimization).

  A plot of the fluorescence signal for each of the reversibly immobilized artificial receptors at 23 ° C. versus the signal at 44 ° C. (not shown) gives no correlation (correlation coefficient of 0.004). Also, a plot of the fluorescence signal for each of the reversibly immobilized artificial receptors at 44 ° C. versus the signal for the corresponding covalently immobilized receptor (not shown) produces no correlation (0.004 Correlation coefficient). This lack of correlation provides further evidence that reversibly immobilized building block movement within the receptor increases binding.

  FIG. 45 shows a graph of the 44 ° C. fluorescence signal divided by the 23 ° C. signal versus the fluorescence signal obtained from the 23 ° C. binding to the reversibly immobilized receptor for the artificial receptor. This comparison shows that binding enhancement is not related to the initial affinity of the receptor for the test ligand.

  Table 1 identifies the reversibly immobilized building blocks that make up each of the artificial receptors, the fluorescent signal (binding strength) at 44 ° C. and 23 ° C., and the ratio of observed binding at these two temperatures. To list. These data indicate that each artificial receptor reflects the unique properties for each combination of building blocks for each individual building block role.

CONCLUSION This experiment showed that arrays containing reversibly immobilized building blocks bind protein substrates such as arrays with covalently immobilized building blocks. With increasing temperature, the bond increased non-linearly, indicating that building block movement increased the bond. Many candidate artificial receptors have shown improved binding in moving building blocks.

Example 4-Oligosaccharide moiety of GM1 competes with artificial receptors for binding to cholera toxin A microarray of candidate artificial receptors was created and evaluated for binding of cholera toxin. The array was also evaluated for perturbation of the binding. For disruption of binding, a compound that binds to cholera toxin, an oligosaccharide site from GM1 (GM1 OS) was used. The results obtained indicate that the protein ligand specifically disrupted protein binding to the microarray.

Materials and Methods Building blocks were synthesized and activated as described in Example 1. The building blocks used in this example are TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3, TyrA3B5, TyrA3B7, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B6, TyrA4B6, TyrA4B6, TyrA4B6 TyrA6B6, TyrA6B8, TyrA7B3, TyrA7B5, TyrA7B7, TyrA8B2, TyrA8B4, TyrA8B6, and TyrA8B8. The abbreviation for the building block containing the linker, tyrosine framework, and recognition element AxBy is TyrAxBy.

A microarray for evaluation of the 171 n = 2 candidate receptor environment was prepared as follows by modification of known methods. The “n = 2” receptor environment includes two different building blocks. Briefly: Modified amine (amine “lawn”; SuperAmine microarray plate) Microarray plates were purchased from Telechem Inc., Sunnyvale, Calif. These plates were specifically manufactured for microarray preparation and, according to the manufacturer, had a nominal amine load of 2-4 amine / nm square. The microarray is typically a 200 μm diameter spotting pin (Stealth Micro Spotting Pins, SMP6) from Telechem Inc. and a pin microarray spotter instrument (SpotBot ^™ Arrayer from Telechem Inc. with 400-420 μm spot spacing. ).

  Nineteen building blocks were activated in aqueous dimethylformamide (DMF) solution as described above. To prepare a 384-well feed plate, the activated building block solution was added to DMF / H2O / PEG400 (90/10/10, v / v / v; PEG400 is a polyethylene glycol nominal 400 FW, Aldrich Chemical Co. , Milwaukee, WI). These stock solutions were aliquoted (10 μl / aliquot) into wells of a 384 well microwell plate (Telechem Inc.). The control spot contained the building block [1-1]. The plate was covered with aluminum foil and placed on a rotary stirrer bed at 1,000 RPM for 15 minutes. The master plate was stored covered with aluminum foil at -20 ° C. when not in use.

To prepare a 384 well SpotBot ^™ plate, a well-to-well transfer (eg, between A-1, A-2, etc.) from the feed plate to the second 384 well plate was performed using a 4 μl transfer pipette. . The plate was stored sealed with aluminum foil at −20 ° C. when not in use. SpotBot ^™ was used to prepare up to 13 microarray plates / run using 4 μl microwell plates. SpotBot ^™ was programmed to spot from each microwell in four ways. The cleaning station on SpotBot ^{™ used} EtOH / H 2 O (20/80, v / v) cleaning solution. The wash solution was adjusted to pH 4 with 1 M HCl and used to rinse the microarray upon completion of the SpotBot ^™ printing run. The plate was finally rinsed with deionized (DI) water, dried using a stream of compressed air, and stored at room temperature. In addition, the microarray was modified by reacting the remaining amine with acetic anhydride to form an acetamide lawn instead of an amine lawn.

The test ligand used in these experiments was an cholera toxin labeled with an Alexa ^™ fluorophore (Molecular Probes Inc., Eugene, OR). The candidate disruptor used in these experiments was GM1 OS (GM1 oligosaccharide), a known ligand for cholera toxin.

  Microarray incubation and analysis were performed as follows: a solution of cholera toxin in PBS-T (PBS with 20 μl / L Tween-20) at a concentration of 1.7 pmol / ml (0.1 μg / ml) (e.g. , 500 μl) was placed on the surface of the microarray and allowed to react for 30 minutes. For disruptor incubation with the microarray, the desired solution in cholera toxin (1.7 pmol / ml, 0.1 μg / ml) (e.g. 500 μl) and PBS-T (PBS with 20 μl / L Tween-20) A concentration of GM1 OS was placed on the surface of the microarray and allowed to react for 30 minutes. GM1 OS was added at 0.34 and 5.1 μM in separate experiments. After either of these incubations, the microarray was rinsed with PBS-T and DI water and dried using a stream of compressed air.

  Incubated microarrays were scanned using an Axon Model 4200A fluorescent microarray scanner (Axon Instruments, Union City, Calif.). The Axon scanner and related software produce a false 16-bit image of the fluorescence intensity of the plate. This 16-bit data was integrated using Axon software to obtain fluorescence unit values (range 0-65,536) for each spot on the microarray. This data is then exported to an Excel file (Microsoft) for further analysis including mean, standard deviation and coefficient of variation calculation.

  Table 2 identifies the building blocks in each of the first 150 receptor environments.

Result Low concentration of GM1 OS
FIG. 46 shows that binding of cholera toxin to a candidate artificial receptor microarray, followed by washing with buffer, produced a fluorescent signal. These fluorescent signals indicate that cholera toxin binds strongly to a particular receptor environment and is weak to others and undetectable to some. Comparison with experiments including that reported in Example 2 shows that cholera toxin binding was reproducible between arrays and months.

  Cholera toxin binding was also performed by competition from GM1 OS (0.34 μM). FIG. 47 shows the fluorescence signal due to cholera toxin binding detected after this competition. Of note, many of the signals shown in FIG. 47 are significantly smaller than the corresponding signals recorded in FIG. The small signal observed in FIG. 47 represents cholera toxin binding to fewer arrays. GM1 OS significantly disrupted cholera toxin binding to many receptor environments.

  The disruption of cholera toxin binding caused by GM1 OS can be visualized as the ratio of the amount bound in the absence of GM1 OS to the amount bound in competition with GM1 OS. This ratio is shown in FIG. The greater the percentage, the less cholera toxin remained bound to the artificial receptor after competition with GM1 OS. The ratio can be as large as about 30. The ratio is independent of the amount bound in the control.

High concentration of GM1 OS
Binding of cholera toxin to the candidate artificial receptor microarray, followed by washing with buffer, produced the fluorescent signal shown in FIG. As before, cholera toxin is reproducible and binds strongly to specific receptor environments, weakly others, and undetectably bound to some. FIG. 50 shows the fluorescent signal detected for cholera toxin binding detected upon competition with GM1 OS at 5.1 μM. Again, GM1 OS significantly disrupted cholera toxin binding to many receptor environments.

  This perturbation is represented in FIG. 51 as the amount of binding in the absence of GM1 OS versus the amount of binding after contact with GM1 OS. The percentage ranges up to about 18 and is independent of the amount bound in the control.

Conclusion This experiment showed that the binding of test ligands to the artificial receptors of the present invention can be reduced (eg, competing) by candidate disruptor molecules. In this case, the test ligand was a protein cholera toxin and the candidate disruptor was a compound known to bind to cholera toxin, GM1 OS. The extent to which the test ligand binding was broken was independent of the extent to which the test ligand bound to the artificial receptor.

Example 5-GM1 competes with artificial receptors for binding to cholera toxin A microarray of candidate artificial receptors was created and evaluated for cholera toxin binding. The array was also evaluated for perturbation of the binding. For binding disruption, a compound that binds to cholera toxin, liposaccharide GM1 was used. The results obtained indicate that protein ligands specifically disrupt protein binding to the microarray.

Materials and Methods Building blocks were synthesized and activated as described in Example 1. The building blocks used in this example were TyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B2, TyrA6B4, and TyrA6B6 in groups of 4 building blocks per artificial receptor. It was. The abbreviation for building block containing the linker, tyrosine framework, and recognition element AxBy is TyrAxBy.

A microarray for evaluation of 126 n = 4 candidate receptor environments was prepared as described above for Example 4. The test ligand used in these experiments was cholera toxin labeled with Alexa ^™ fluorophore (Molecular Probes Inc., Eugene, OR). Cholera toxin was used at 5.3 nM in both control and competition experiments. The candidate disruptor used in these experiments was GM1, a known ligand for cholera toxin that competed at concentrations of 0.042, 0.42, and 8.4 μM. Microarray incubation and analysis were performed as described for Example 4.

  Table 3 identifies the building blocks in each receptor environment.

Results FIG. 52 shows the fluorescent signal produced by binding of cholera toxin to the candidate artificial receptor microarray, competing alone and with each of the three concentrations of GM1. The magnitude of the fluorescent signal gradually decreases as the concentration of GM1 increases. The amount of reduction was not quantitatively the same for all receptors, but each receptor experienced a decrease in cholera toxin binding. These reductions indicate that GM1 competed with the artificial receptor for binding to cholera toxin.

  The decrease indicates a pattern of relative competition for binding sites on cholera toxin. This can be shown through a graph of the fluorescent signal (not shown) obtained with a specific concentration of GM1 versus the fluorescent signal in the absence of GM1. Certain receptors appear at similar relative positions on these plots as the concentration of GM1 increases.

  Disruption of cholera toxin binding caused by GM1 can be visualized as the amount of binding in the absence of GM1 OS versus the amount of binding in competition with GM1. This ratio is shown in FIG. The higher the ratio, the more cholera toxin remained bound to the artificial receptor when competing with GM1. The ratio can be as large as about 14. The percentage is independent of the amount bound in the control.

  Interestingly, in some instances, a slight change in structure to an artificial receptor resulted in a significant change in proportion. For example, an artificial receptor comprising building blocks 24, 26, 46, and 66 differs from that comprising 24, 42, 46, and 66 only by replacement of a single building block. (xy represents building block TyrAxBy.) Substitution of building block 42 to 26 increased binding in the presence of GM1 by approximately 14-fold.

  According to a further example, an artificial receptor comprising building blocks 22, 24, 46, and 64 differs from that comprising 22, 46, 62, and 64 only by replacement of a single building block. Substitution of building block 24 for 62 increased binding in the presence of GM1 approximately 3-fold.

  Even replacement of a single recognition element affected binding. Artificial receptors containing building blocks 22, 24, 42, and 44 differ from those containing 22, 24, 42, and 46 only by the replacement of a single recognition element. Substitution of building block 44 for 46 (recognition element B6 to B4 change) increased binding in the presence of GM1 by approximately 3-fold.

Conclusion This experiment showed that the binding of test ligands to the artificial receptors of the present invention can be reduced (eg, competing) by candidate disruptor molecules. In this case, the test ligand was protein cholera toxin and the candidate disruptor was a compound known to bind to cholera toxin, GM1. Slight changes in the structure of the building blocks that make up the artificial receptors caused significant changes in competition.

Example 6-GM1 used as a building block modifies the binding of cholera toxin to the artificial receptor of the present invention.
A microarray of candidate artificial receptors was created and GM1 bound to the array and they were evaluated for cholera toxin binding. The results obtained indicate that adding GM1 as a building block in an array of artificial receptors can increase binding to specific receptors.

Materials and Methods Building blocks were synthesized and activated as described in Example 1. The building blocks used in this example were those described in Example 4. A microarray for evaluation of 171 n = 2 candidate receptor environments was prepared as described above for Example 4. The test ligand used in these experiments was cholera toxin labeled with Alexa ^™ fluorophore (Molecular Probes Inc., Eugene, OR). Cholera toxin was used at 0.01 ug / ml (0.17 pM) or 0.1 ug / ml (1.7 pM) in both control and competitive environments. GM1 was used as a test ligand for the artificial receptor and became the building block for the receptor used to bind cholera toxin. The array was contacted with GM1 at either 100 μg / ml, 10 μg / ml, or 1 μg / ml as described above for cholera toxin and then rinsed with deionized water. The array was then contacted with cholera toxin under the conditions described above. Microarray analysis was performed as described for Example 4. Table 2 identifies the building blocks in each receptor environment.

Results FIG. 54 shows the fluorescent signal generated by binding of cholera toxin to a microarray of candidate artificial receptors without pretreatment with GM1. Binding of GM1 to the candidate artificial receptor microarray, followed by cholera toxin binding, is shown in FIGS. 55, 56, and 57 (100 μg / ml, 10 μg / ml, and 1 μg / ml GM1, respectively). A fluorescent signal was generated.

  The promotion of cholera toxin binding caused by pretreatment with GM1 can be visualized as a ratio of the amount of binding in the presence of GM1 to the amount of binding in the absence of GM1. This ratio is shown in FIG. 58 for 1 μg / ml GM1. The higher the percentage, the more cholera toxin bound to the artificial receptor after pretreatment with GM1. The ratio can be as large as about 16.

  In some instances, a slight change in structure to an artificial receptor caused a significant change in proportion. For example, an artificial receptor comprising building blocks 46 and 48 differs from that comprising 46 and 88 only by replacement of a single recognition element on a single building block. (xy represents building block TyrAxBy.) Substitution with 48 instead of building block 88 (change of recognition element A8 to A4) reduces the ratio representing increased binding to the presence of the GM1 building block by approximately Increased from 0.5 to about 16. Similarly, an artificial receptor comprising building blocks 42 and 77 differs from that comprising 24 and 77 only by replacement of a single building block. Substitution with 42 instead of building block 24 increased the ratio representing increased binding to the presence of the GM1 building block from about 2 to about 14.

  Interestingly, several building blocks that exhibited high levels of cholera toxin binding (45,000 to 65,000 fluorescent unit signals), including building block 33, were not significantly affected by the presence of GM1 as a building block.

Conclusion This experiment showed that binding of GM1 to the artificial receptor of the present invention can significantly increase binding by cholera toxin. A slight change in the structure of the building block of the artificial receptor caused a significant change to the extent that GM1 enhanced binding of cholera toxin.

Discussion of Examples 4-6 We have already shown that working artificial receptor arrays bind to protein targets in a manner complementary to the specific environment represented by each region of the protein surface topology. . Thus, the pattern of protein target binding to an array of working artificial receptors can include surface structures involved in interactions such as protein-small molecule, protein-peptide, protein-protein, protein-carbohydrate, protein-DNA, etc. Describes the protein surface topology involved. Thus, the binding of the selected protein to the working artificial receptor array can be used to characterize these protein-small molecule, protein-peptide, protein-protein, protein-carbohydrate, protein-DNA, etc. interactions. Is possible. In addition, proteins can be used for array interactions to define a “lead” for perturbation of these interactions.

  Cholera toxin B subunit binds to GM1 on the cell surface. Studies identifying competitors for this binding event have shown that competitors for cholera toxin: GM1 binding interactions (binding sites) can utilize both sugar and alkyl / aromatic functionality (Pickens, et al., Chemistry and Bio1ogy, vol. 9, pp 215-224 (2002)). We have already shown that fluorescently labeled cholera toxin B subunits bind to the array of artificial receptors of the present invention to give a defined binding pattern that reflects the surface topology of cholera toxin B. For this study we tried to show that the binding of cholera toxin to at least some members of the array can be perturbed with the natural ligand for cholera toxin, GM1.

  The results shown in the drawings clearly show that these objectives have been achieved. Specifically, a binding pattern different from the cholera binding pattern control is obtained by competition between GM1 OS pentasaccharide or GM1 and an artificial receptor array for cholera binding. Furthermore, these results showed complementarity between several working artificial receptors that contained a naphthyl moiety as compared to a working artificial receptor that only contained phenyl functionality. These results are consistent with the active site competition studies in Pickens, et al. And show that naphthyl and phenyl derivatives represent good mimics / probes against cholera for GM1 interactions. The specificity of these interactions was shown by the observation that a change in a single building block out of four in the four building block system combination changed the non-competitive environment to a fairly competitive environment. These results also showed that selected working artificial receptors can be used to develop high-throughput screens for further evaluation of cholera: GM1 interactions.

  In addition, we can use affinity supports / membrane mimics to select artificial receptors that work for high-throughput screening of lead compounds that destroy, for example, “cholera: membrane-GM1 mimic” We wanted to show that the binding pattern would then be prepared by pre-incubating an array of artificial receptors with GM1 that binds / captures cholera toxin. GMl preincubation is a poor cholera binder, some of the acting artificial receptors, probably between cholera toxin and the immobilized GMl pentasaccharide site and the working artificial receptor building block environment. It was clearly shown that their affinity for cholera binding was significantly increased via affinity interactions.

  As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” are used unless the content clearly dictates otherwise. It should be noted that it includes multiple indication objects. Thus, for example, reference to a composition containing “a compound (a)” includes a mixture of two or more compounds. It should be noted that the term “or” is generally used in its sense including “and / or” unless the content clearly dictates otherwise.

  Also, as used herein and in the appended claims, the term “adapted and configured” refers to a system constructed or configured to perform a specific task or to adapt a specific configuration; It should be noted that the device or other structure is described. The term “adapted and configured” can be used interchangeably with similar terms such as arranged and configured, constructed and arranged, adapted, constructed, manufactured and arranged, etc.

  All references and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains.

  The present invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

FIG. 1 schematically shows a two-dimensional representation of an embodiment of a receptor according to the present invention that uses four different building blocks to create a ligand binding site. FIG. 2 schematically shows a two- and three-dimensional representation of an example of the molecular composition of the four building blocks, where each building block has a recognition element, framework, and linker (fixed / fixed) coupled to the support. Anchor). FIG. 3 schematically illustrates an embodiment of the method and artificial receptor of the present invention using building block shuffling and replacement. FIG. 4 schematically illustrates an embodiment of a method for evaluating candidate artificial receptors for binding to a test ligand such as a molecule or cell. FIG. 5 schematically illustrates an embodiment of the invention using an array of candidate artificial receptors. FIG. 6 schematically shows a specific binding pattern for an array of working artificial receptors. FIG. 7 schematically shows an example of a method for developing a method and system for detecting a test ligand. FIG. 8 schematically shows a specific example of a method for detecting an agent that disrupts the binding interaction of a target molecule. FIG. 9 schematically shows a specific example of a method for detecting an agent that disrupts the binding interaction of a complex containing a target molecule. FIG. 10 schematically shows candidate disruptors that disrupt the protein: protein complex. FIG. 11 schematically illustrates a specific example of a method for producing or using an affinity support using the artificial receptors of the present invention. FIGS. 12A-B schematically illustrate evaluation of an array of candidate artificial receptors for test ligand binding and selection of one or more working artificial receptors for binding or manipulation to the test ligand. FIG. 13 schematically illustrates the identification of lead artificial receptors from among candidate artificial receptors. FIG. 14 schematically shows a pseudo-color fluorescence image of a labeled microarray according to an embodiment of the present invention. FIG. 15 schematically shows a two-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to phycoerythrin. FIG. 16 schematically shows a three-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to phycoerythrin. FIG. 17 schematically shows a two-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to a fluorescent derivative of ovalbumin. FIG. 18 schematically shows a three-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to a fluorescent derivative of ovalbumin. FIG. 19 schematically shows a two-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to a fluorescent derivative of bovine serum albumin. FIG. 20 schematically shows a three-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to a fluorescent derivative of bovine serum albumin. FIG. 21 schematically shows a two-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to acetylated horseradish peroxidase. FIG. 22 schematically shows a three-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to acetylated horseradish peroxidase. FIG. 23 schematically shows a two-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to a TCDD derivative of horseradish peroxidase. FIG. 24 schematically shows a three-dimensional plot of the data obtained for a candidate artificial receptor that contacts and / or binds to a TCDD derivative of horseradish peroxidase. FIG. 25 schematically shows a subset of the data shown in FIG. FIG. 26 schematically shows a subset of the data shown in FIG. FIG. 27 schematically shows a subset of the data shown in FIG. FIG. 28 schematically shows the correlation of phycoerythrin binding data to logP for building blocks that constitute artificial receptors. FIG. 29 schematically shows the correlation of phycoerythrin binding data to logP for building blocks that constitute artificial receptors. FIG. 30 shows the data obtained for a candidate artificial receptor that contacts and / or binds to phycoerythrin with a candidate artificial receptor that contacts and / or binds to a fluorescent derivative of bovine serum albumin. A two-dimensional plot comparing the data is shown schematically. FIGS. 31, 32, and 33 schematically show a subset of the data from FIGS. 16, 20, and 18, respectively, where an array of artificial receptors according to the present invention comprises three analytes, phycoerythrin, bovine serum albumin And obtaining a receptor discriminated between ovalbumin. FIG. 34 schematically shows a grayscale image of the fluorescent signal from a control plate scan prepared by washing the building blocks with organic solvent prior to incubation with the test ligand. FIG. 35 schematically shows a grayscale image of the fluorescent signal from a scan of an experimental plate incubated with 1.0 μg / ml cholera toxin B at 23 ° C. FIG. 36 schematically shows a grayscale image of the fluorescent signal from a scan of an experimental plate incubated with 1.0 μg / ml cholera toxin B at 3 ° C. FIG. 37 schematically shows a grayscale image of the fluorescent signal from a scan of an experimental plate incubated with 1.0 μg / ml cholera toxin B at 43 ° C. Figures 38-40 schematically show a plot of the fluorescent signal obtained from the candidate artificial receptors shown in Figures 35-37. FIG. 41 schematically shows a plot of the fluorescent signal obtained from the combination of building blocks used in the study of the present invention when the building blocks are covalently bound to the support. Binding was performed at 23 ° C. FIG. 42 schematically shows the change in fluorescence signal from individual combinations of building blocks covalently immobilized at 4 ° C., 23 ° C., or 44 ° C. FIG. 43 schematically shows a graph of the change in fluorescence signal from individual combinations of building blocks at 4 ° C., 23 ° C., or 44 ° C. 44 schematically illustrates the data shown in FIG. 42 (line marked A) and the data shown in FIG. 43 (line marked B). FIG. 45 is a graph of the fluorescence signal at 44 ° C. divided by the signal at 23 ° C. versus the fluorescence signal obtained from binding at 23 ° C. for an artificial receptor with a reversibly immobilized receptor. Is shown schematically. FIG. 46 shows the fluorescent signal generated by binding cholera toxin to a microarray of candidate artificial receptors of the present invention, followed by washing with buffer in the experiment reported in Example 4. FIG. 47 shows the fluorescence signal due to cholera toxin binding detected during competition with the experimental GM1 OS (0.34 μM) reported in Example 4. FIG. 48 shows the ratio of the amount bound in the absence of GM1 OS versus the amount bound in competition with GM1 OS (0.34 μM) in the experiment reported in Example 4. FIG. 49 shows a competition experiment using 5.1 μM GM1 OS generated by binding cholera toxin to a microarray of candidate artificial receptors of the present invention followed by washing with buffer in the experiment reported in Example 4. The fluorescent signal for comparison is shown. FIG. 50 shows the fluorescence signal due to cholera toxin binding detected in comparison with GM1 OS (5.1 μM) in the experiment reported in Example 4. FIG. 51 shows the ratio of the amount bound in the absence of GM1 OS versus the amount bound in competition with GM1 OS (5.1 μM) in the experiment reported in Example 4. FIG. 52 shows the fluorescent signal produced by binding of cholera toxin to a microarray of candidate artificial receptors alone and in competition with each of the three concentrations of GM1 in the experiment reported in Example 5. FIG. 53 shows the ratio of the amount bound in the absence of GM1 OS to the amount bound in competition with GM1 for the low concentration of GM1 used in Example 5. FIG. 54 shows the fluorescence signal generated by binding of cholera toxin to a candidate artificial receptor microarray without pretreatment with GM1 in the experiment reported in Example 6. FIGS. 55-57 show cholera on candidate artificial receptor microarrays by pretreatment with GM1 (100 μg / ml, 10 μg / ml, and 1 μg / ml GM1, respectively) in the experiment reported in Example 6. The fluorescent signal generated by the binding of the toxin is shown. FIG. 58 shows the ratio of the amount bound in the presence of 1 μg / ml GM1 to the amount bound in the absence of GM1 in the experiment reported in Example 6.

Claims (47)

A method of creating an artificial receptor for binding a test ligand comprising:
Contacting at least one candidate artificial receptor with a test ligand;
Wherein the candidate artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the candidate artificial receptor;
The building block is naive to the test ligand;
Detecting binding of the test ligand to at least one candidate artificial receptor; then selecting at least one candidate artificial receptor for which test ligand binding has been detected as an artificial receptor acting on the test ligand The method characterized by the above.   The method of claim 1, wherein the array of candidate artificial receptors is contacted with a test ligand.   The method of claim 2, wherein the array comprises at least about 100 candidate artificial receptors.   The method of claim 2, wherein the array comprises at least about 10,000 candidate artificial receptors.   The method of claim 2, wherein the array comprises at least about 1,000,000 candidate artificial receptors.   The method of claim 1, wherein the test ligand comprises at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism.   The method of claim 1, wherein the test ligand comprises at least one of an isomer, a protein conformation, and a proteome. And at least one candidate artificial receptor is contacted with the first protein to produce at least one protein-treated candidate artificial receptor;
Contacting said protein processing candidate artificial receptor with a proteome; wherein said proteome comprises a proteome protein;
Detecting the binding of at least one proteome protein to the at least one protein-treated candidate artificial receptor; 2. The method of claim 1, wherein the at least one proteome protein and the first protein in which binding is detected are selected for analysis. Contacting at least one candidate artificial receptor with a mixture comprising the test ligand and a candidate disruptor;
Detecting the binding of the test ligand to at least one candidate artificial receptor in the presence of the candidate disruptor; then test ligand binding occurred in the absence of the candidate disruptor, The method according to claim 1, wherein at least one candidate artificial receptor disturbed by the candidate disturbing substance is selected as an artificial receptor acting on the test ligand. Contacting at least one working artificial receptor with a sample suspected of containing a test ligand;
Wherein the working artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
The at least one working artificial receptor is known to bind the test ligand;
Monitoring binding to the at least one working artificial receptor; wherein the binding to the at least one working artificial receptor is indicative of the presence of a test ligand in the sample A method for detecting a test ligand.   The method of claim 10, wherein the at least one working artificial receptor comprises a plurality of artificial receptors.   The method of claim 11, wherein binding of the test ligand produces a detectable pattern in the plurality of artificial receptors.   11. The method of claim 10, wherein the test ligand comprises at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism.   11. The method of claim 10, wherein the test ligand comprises at least one of an isomer, a protein conformation, and a proteome. Contacting a plurality of candidate artificial receptors with a first test ligand;
Wherein each candidate artificial receptor independently comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
Detecting binding of the first test ligand to at least one candidate artificial receptor; and then rearranging the location or composition of the at least one artificial receptor bound to the first test ligand A method for detecting a test ligand. further:
Contacting one or more candidate artificial receptors with a second test ligand;
Detecting binding of the second test ligand to at least one candidate artificial receptor; and then rearranging the location or composition of the at least one artificial receptor bound to the second test ligand The method of claim 15 . further:
Comparing the position or composition of the at least one artificial receptor bound to the first test ligand with the position or composition of the at least one artificial receptor bound to the second test ligand; 17. The method of claim 16, further comprising organizing the compositional comparison.   The method of claim 17, further comprising distinguishing the first test ligand from the second test ligand based on the comparison. A method for detecting a compound that disrupts a test ligand interaction comprising:
Contacting at least one working artificial receptor with a mixture comprising the test ligand and a candidate disruptor;
Wherein the working artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
The at least one working artificial receptor is known to bind the test ligand:
Monitoring candidate disruptors that reduce binding of the test ligand to the at least one working artificial receptor; wherein reduced binding is that the candidate disruptor is a major disruptor The method characterized by showing.   20. The method of claim 19, wherein the test ligand comprises at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism.   20. The method of claim 19, wherein the test ligand comprises at least one of an isomer, a protein conformation, and a proteome.   21. The method of claim 19, wherein the candidate disruptor comprises a compound having a molecular weight of less than 500.   20. The method of claim 19, wherein the candidate disruptor comprises a polypeptide. A method for making an affinity support for a test ligand comprising:
Contacting at least one candidate artificial receptor with the test ligand;
Wherein the candidate artificial receptor comprises a plurality of building blocks independently coupled to the first support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
Detecting binding of the test ligand to at least one candidate artificial receptor; then selecting at least one candidate artificial receptor from which test ligand binding has been detected as a working artificial receptor for the test ligand;
The method, wherein the working artificial receptor is bound to a second support to form an affinity support.   25. The method of claim 24, wherein the test ligand comprises at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism.   26. The method of claim 25, wherein the test ligand comprises at least one of an isomer, a protein conformation, and a proteome. Contacting a sample containing a test ligand with a working artificial receptor;
Wherein the working artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
A method for isolating a test ligand, wherein the working artificial receptor is known to bind to the test ligand. A method of creating a reaction support for a reactant comprising:
Contacting at least one candidate artificial receptor with the reactant;
Wherein the candidate artificial receptor comprises a plurality of building blocks independently coupled to the first support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
Detecting binding of the test ligand to at least one candidate artificial receptor; then selecting at least one candidate artificial receptor for which test ligand binding has been detected as an acting artificial receptor for the test ligand;
Contacting at least one working artificial receptor with the reactant and monitoring the reaction of the reactant;
Selecting the acting artificial receptor for which the reaction was observed as the reactive artificial receptor;
The method wherein the reactive artificial receptor is bound to a second support to form a reactive support.   30. The method of claim 28, wherein the reactant comprises at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism.   30. The method of claim 28, wherein the reactant comprises at least one of an isomer, a protein conformation, and a proteome.   29. The method of claim 28, further comprising contacting at least one working artificial receptor with a second reactant. Contacting the sample containing the reactant with a working artificial receptor;
Wherein the working artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
A method of reacting a reactant characterized in that the acting artificial receptor is known to bind to the test ligand.   32. The method of claim 31, further comprising contacting the acting artificial receptor with a second reactant. A method of making a catalyst support for a reactant comprising:
Contacting at least one candidate artificial receptor with the reactant;
Wherein the candidate artificial receptor comprises a plurality of building blocks independently coupled to the first support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
Detecting the catalysis of the reaction of the reactants in the presence of at least one candidate artificial receptor; then selecting at least one candidate artificial receptor for which catalysis has been detected as an acting artificial receptor for the test ligand;
The method wherein the working receptor is bound to a second support to form a catalyst support.   35. The method of claim 34, wherein the reactant comprises at least one of an addictive drug, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, and a microorganism.   35. The method of claim 34, wherein the reactant comprises at least one of an isomer, a protein conformation, and a proteome.   35. The method of claim 34, further comprising contacting at least one working artificial receptor with a second reactant. Contacting the sample containing the reactant with a working artificial receptor;
Wherein the working artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
A method of catalyzing the reaction, wherein the acting artificial receptor is known to bind to the test ligand and catalyze the reaction.   40. The method of claim 38, further comprising contacting the acting artificial receptor with a second reactant. A method of creating a surface that does not bind a test ligand comprising:
Contacting at least one candidate artificial receptor with the test ligand;
Wherein the candidate artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
Detecting a lack of binding of the test ligand to at least one candidate artificial receptor;
The method comprising selecting at least one candidate artificial receptor in which a test deficiency of ligand binding has been detected as an unbound surface for the test ligand.   41. The method of claim 40, wherein the test ligand comprises a plurality of test ligands. A method for detecting a compound that disrupts the interaction of a test ligand with a second ligand comprising:
Binding the test ligand to at least one working artificial receptor;
Wherein the working artificial receptor comprises a plurality of building blocks independently coupled to the support in adjacent regions;
Two or more building block molecules together form the working artificial receptor;
The building block is naive to the test ligand;
The at least one working artificial receptor is known to bind to the test ligand;
Contacting said at least one working artificial receptor with a test ligand bound to a mixture comprising said second ligand and a candidate disruptor;
Monitoring for the candidate disruptor that reduces binding of the second ligand to the test ligand; wherein the decreased binding indicates that the candidate disruptor is a major disruptor The method characterized by the above.   43. The method of claim 42, wherein the test ligand comprises at least one of a peptide, polypeptide, oligonucleotide, polynucleotide, and microorganism.   43. The method of claim 42, wherein the candidate disruptor comprises a compound having a molecular weight of less than 500.   43. The method of claim 42, wherein the candidate disruptor comprises a polypeptide.   43. The method of claim 42, wherein the test ligand comprises a first protein and the second ligand comprises a second protein; wherein the first protein and the second protein form a complex.   43. The method of claim 42, wherein the test ligand comprises a protein and the second ligand comprises a polynucleotide; wherein the protein and the polynucleotide form a complex.

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