EP1210607A1 - Dispositifs a reseaux de descripteurs moleculaires microelectroniques, methodes, procedures et formats de selection combinatoire de structures de fixation de ligands intermoleculaires et de criblage de medicaments - Google Patents

Dispositifs a reseaux de descripteurs moleculaires microelectroniques, methodes, procedures et formats de selection combinatoire de structures de fixation de ligands intermoleculaires et de criblage de medicaments

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Publication number
EP1210607A1
EP1210607A1 EP00955293A EP00955293A EP1210607A1 EP 1210607 A1 EP1210607 A1 EP 1210607A1 EP 00955293 A EP00955293 A EP 00955293A EP 00955293 A EP00955293 A EP 00955293A EP 1210607 A1 EP1210607 A1 EP 1210607A1
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Prior art keywords
ligand binding
rna
formation
detection
supramolecular
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00955293A
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German (de)
English (en)
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EP1210607A4 (fr
Inventor
Michael J. Heller
Norbert Windhab
Richard R. Anderson
Donald E. Ackley
Tina S. Nova
Hans-Ullrich Hoppe
Christian J. Hamon
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Aventis Research and Technologies GmbH and Co KG
Nanogen Inc
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Aventis Research and Technologies GmbH and Co KG
Nanogen Inc
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Publication of EP1210607A1 publication Critical patent/EP1210607A1/fr
Publication of EP1210607A4 publication Critical patent/EP1210607A4/fr
Withdrawn legal-status Critical Current

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    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
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    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/171—Systems in which incident light is modified in accordance with the properties of the material investigated with calorimetric detection, e.g. with thermal lens detection
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    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00729—Peptide nucleic acids [PNA]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the field of this invention relates to devices and methods for carrying out multi- step and multiplex affinity binding reactions in microscopic formats.
  • those devices and methods rapidly carry out higher order selectivity of combinatorially produced intermolecular ligand binding components, supramolecular structures and supramolecular complexes by application of unique stringency parameters.
  • this invention relates to microelectronic array devices, procedures, methods and formats for molecular recognition processes, new drug discovery, generation of new affinity reagents, generation of synthetic antibodies and for immunoassays.
  • ligand binding component structures can be designed such that they can assemble into larger structures, "supramolecular structures", which have molecular recognition properties and the potential for forming selective and stable complexes with important biologically active molecules and structures. Methods for rapidly carrying out combinatorial selection of large numbers of these ligand binding structures could lead to considerable information concerning the nature of the ligand binding process, particularly for known drugs and biologically active compounds.
  • Supramolecular binding structures have been described by a number of workers in the field. Classes of supramolecular polymers with repeating units have been described by Lehn J.M., in J. Chem. Soc. Chem. Commun. 479, 1990. Polymeric tubular type supramolecular structures formed, form cyclic peptides has been described by Ghadiri, M. R., et al., in Nature 366, pp. 324-327, 1993. Two and three dimensional DNA structures have been designed and synthesized enzymatically by Zhang, Y., and Seeman, N.C., in J. Am. Chem. Soc, 116, pp. 1661-1669, 1994.
  • nucleic acid hybridization analysis generally involves the detection of very small numbers of specific target nucleic acids (DNA or RNA) with probes among a large amount of non- target nucleic acids.
  • hybridization is normally carried out under the most stringent condition, achieved through a combination of temperature, salts, detergents, solvents, chaotropic agents, and denaturants.
  • Multiple sample nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats (see G. A.
  • Another problem relates to the high complexity of DNA in most samples, particularly in human genomic DNA samples.
  • a sample is composed of an enormous number of sequences that are closely related to the specific target sequence, even the most unique probe sequence has a large number of partial hybridizations with non-target sequences.
  • a third problem relates to the unfavorable hybridization dynamics between a probe and its specific target. Even under the best conditions, most hybridization reactions are conducted with relatively low concentrations of probes and target molecules. In addition, a probe often has to compete with the complementary (target) strand for the actual target sequence.
  • a fourth problem for most present hybridization formats is the high level of non-specific background signal. This is caused by the affinity of DNA probes to almost any material.
  • Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH.
  • Southern did not address how to achieve optimal stringency condition for each oligonucleotide on an array. See, e.g., PCT WO89/10977, entitled “Analysing Polynucleotide Sequences", priority claim May 3, 1988.
  • Other workers using the reverse dot blot microarray format required very long hybridization and stringency washing time in order to achieve minimal single base mismatch discrimination (Z. Guo et al., Nucleic Acids Res., Vol. 22, #24, pp. 5456-5465, 1994).
  • the target DNA was a fluorescently labeled single-stranded 12-mer oligonucleotide containing only nucleotides A and C.
  • Drmanac et al. 260 Science 1649-1652, 1993, used the above discussed second format to sequence several short (116 bp) DNA sequences.
  • Target DNAs were attached to membrane supports ("dot blot" format).
  • Each filter was sequentially hybridized with 272 labeled 10-mer and 11-mer oligonucleotides.
  • a wide range of stringency conditions were used to achieve specific hybridization for each n-mer probe; washing times varied from 5 minutes to overnight, and temperatures from 0° C to 16° C. Most probes required 3 hours of washing at 16°C.
  • the filters had to be exposed for 2 to 18 hours in order to detect hybridization signals.
  • this invention relates to apparatus and methods for the use of active microelectronic array devices to carry out combinatorial processes where component structures form intermolecular or supramolecular ligand binding structures and subsequent supramolecular complexes (or aggregates) with improved speed and higher order specificity.
  • the intermolecular or supramolecular ligand binding structures are composed of programmable self-assembling/pairing components that contain one or more ligand binding components.
  • the supramolecular complexes or aggregates are formed when supramolecular ligand binding structures with the appropriate or selective molecular recognition and binding properties, bind or aggregate with a specific ligand molecule or structure which include, drug molecules, or other biologically active molecules or structures.
  • the array can then serve as a "molecular descriptor" device.
  • This "molecular descriptor” device can be used for a variety of applications, some of which include: new drug discovery and screening processes, generation of novel affinity reagents, or generation of high affinity/high specificity synthetic antibodies. Additionally, other versions of these arrays can be used for multiplex proteomic analyses and for multiple immunoassay applications.
  • the invention may be considered as a device for the formation and detection of supramolecular aggregates comprising an attachment surface, the attachment surface being located in a variable electronic environment, a molecular recognition system including at least a first and a second molecular recognition component, at least the first component being attached to the surface, a separate molecular species bound to the molecular recognition system, and a third structure for formation of a supramolecular aggregate with the separate molecular species.
  • the molecular recognition system comprises a pairing system.
  • the pairing system is a complementary and coded pairing system, such as p-RNA.
  • the separate molecular species comprises a peptide sequence.
  • microelectronic arrays are used to affect the affinity binding of small molecules (drugs, metabolites, metal ions, etc), large molecules (proteins, enzymes, antibodies, etc.), and larger structures (organelles, cells, etc.) to the supramolecular ligand binding structures. These processes enable the identification of those intermolecular ligand binding structures that form the most selective and/or stable supramolecular complexes in the presence of a drug or other biologically active molecule or structure.
  • the use of microelectronic arrays allows the combinatorial selection process to be carried out in what might be considered a near realtime evolutionary or learning mode.
  • This process not only identifies the more stable supramolecular complexes, but also provides information on the structure's important molecular recognition properties.
  • These molecular recognition properties include: conformational, chemical, physical and mechanistic reasons as to why certain supramolecular structures and their component intermolecular ligand binding structures have more optimal binding characteristics.
  • the molecular recognition properties of the array can provide more useful information about the structure/function relationships of the bound drug or molecule, the ligand binding components and process, and even the actual biological receptor site. Feedback of information on the supramolecular complex formation allows iterative processes to be designed either for more focused selection of specific classes of supramolecular ligand binding structures, or very wide screening for newer classes of supramolecular ligand binding structures.
  • Microelectronic arrays are able to achieve this higher order specificity because they provide the added parameter of
  • the application of electric field stringency to supramolecular complex formation provides a powerful parameter for selecting the improved and/or more specific supramolecular ligand binding structures.
  • the electric field can also be used to, in effect, perturb the intermolecular suprastructures themselves producing lower energy configurations which can lead to the formation of better ligand binding environments and complexes.
  • supramolecular complex binding patterns are first determined for known drugs, agonists, antagonists, inhibitors, toxins and other biological agents that are known to interact at a targeted biological receptor site.
  • the binding patterns of these known drugs or biological agents on the combinatorial microelectronic array can be correlated with both good or desirable characteristics (for example, drug effectiveness) and bad or undesirable characteristics (for example, drug toxicity).
  • good or desirable characteristics for example, drug effectiveness
  • bad or undesirable characteristics for example, drug toxicity
  • an intermolecular ligand binding structure relevant to this invention utilizes pyranosyl-RNA or p-RNA as the programmable self-assembling pairing component and peptides as the ligand binding component.
  • p-RNA is a nucleic acid-like molecule in which the sugar group is a pentopyranose (see Figure 1).
  • the peptide sequences containing various combinatorial arrangements of amino acids, form the actual ligand binding structures.
  • Other classes of self-assembling intermolecular ligand binding supramolecular structures can be produced using nucleic acids (DNA or RNA) and peptides. In these suprastructures, the nucleic acid moiety provides the programmable self-assembling pairing component, and the peptide moieties serve to form the ligand binding component.
  • the p-RNA and the DNA (or RNA) based classes represent just some of the possible intermolecular ligand binding suprastructures that are relevant to this invention. (It should be pointed out that "supramolecular structure" and “suprastructure” are used interchangably in describing this invention).
  • intermolecular ligand binding components leads to supramolecular structures that produce a "triad" type ligand binding configuration.
  • These "triad" binding structures are produced by designing two short p-RNA sequences (A) and (B), which are complementary to a third longer capture p-RNA sequence (C).
  • the two p- RNA sequences (A) and (B) are preferably designed so as to be contiguous, when paired (hybridized) with the complementary capture p-RNA (C) sequence (see Figure 3).
  • the combinatorial binding formats and assays that utilize the "triad"-type ligand binding suprastructure involve producing a microelectronic array in which the capture p-RNA- peptide (C) ligand binding component structures are selectively addressed and immobilized to specific test sites or microlocations on the array.
  • the p-RNA component is one common or generic sequence that is complementary for the two p-RNA (A) and (B) sequences.
  • each test site (microlocation) on the array is addressed with a p-RNA-peptide set containing a "different peptide sequence" from the known combinatorial peptide library.
  • the peptides sequences on the p-RNA's (A) and (B) components preferably contain the same sets of known peptide sequences as does the immobilized capture p-RNA-peptides on the array.
  • the p-RNA-peptides (A), (B) and (C) form peptide sublibraries.
  • the two complementary sequences of the p- RNA-peptide (A) and (B) components hybridize to the complementary sequence of the capture p-RNA-peptide (C) sequence, they produce the intermolecular ligand binding suprastructure in which the three peptide strands form the triad binding structure or pocket.
  • This triad binding structure or pocket has the potential to be an affinity binding site for a specific drug, or other biologically active molecule or structure (see Figure 5).
  • One basic ELIAS combinatorial selection process involves using a microelectronic array selectively addressed with capture p-RNA-peptide (C) ligand binding components, which is subsequently contacted with a solution containing the drug molecule and the p-RNA-peptide (A) and (B) ligand binding components.
  • the combinatorial selection process is carried out under appropriate conventional and/or electronic stringency conditions for forming supramolecular ligand binding structures and supramolecular complexes.
  • the supramolecular complex patterns on the array are detected, and the specific ligand binding components (peptides) are subsequently identified. This process is called "ELIAS on a chip" or the ELOC process (see Figure 6).
  • This combinatorial selection or ELOC process can be carried out using either passive or active microelectronic arrays.
  • active microelectronic arrays for the ELOC process has significant advantages in certain circumstances.
  • Active microelectronic chip/array technologies have been demonstrated which provide capability for selectively addressing arrays with DNA sequences, carrying out rapid multiplex hybridization and then providing electronic stringency for improving DNA hybridization selectivity.
  • These same basic microelectronic arrays can be used for the combinatorial selection processes that are the subject of this invention.
  • the basic designs and procedures for fabricating microelectronic DNA chips and arrays, particularly higher density devices (10,000 active sites) are applicable to the combinatorial and ELOC processes of this invention.
  • the various electronic methods, procedures and formats for carrying out electronic addressing of the arrays, active electronic hybridization and electronic stringency all serve as a basis for developing the combinatorial and ELOC processes of this invention.
  • One important aspect of this invention involves microelectronic array based ELOC formats that provide speed and higher order selectivity for carrying out combinatorial selection processes.
  • One of several ELOC formats relevant to this invention involves a transient dynamic equilibrium process (Format 1).
  • Form 1 the general stringency condition on the microelectronic array is set at or near the Tm (thermal melting mid-point) for the hybridization of the two p-RNA (A) and (B) sequences to the complementary capture p-RNA (C) sequence.
  • ELOC formats include a homogeneous combinatorial selection process that is designed to allow a specific drug molecule to first complex with specific p-RNA-peptide (A) and (B) structures in the solution phase, and then bind to the array (ELOC Format 2); and a heterogeneous combinatorial selection process designed to initially form very large numbers of triad ligand binding superstructures on the microarray, and then select and bind the drug molecule (ELOC Format 3).
  • a first level can involve low electric field stringency to remove non-specific or partially bound structures.
  • the second level can involve application of a more precise intermediate level of electric field strength to select the more specific and/or stable supramolecular complexes involving p-RNA-peptide (A) and (B) sets.
  • the third level of electronic stringency can involve application of the most precise and the highest level of electric field strength to select the most stable supramolecular complexes involving both p-RNA-peptide sets (A) and (B), and the capture p-RNA- peptide sets (C); i.e., the true "triad" drug/peptide complexes.
  • the electric field can be used to effectively perturb the intermolecular superstructures producing lower energy configurations that can lead to better drag or target molecule binding environments and more stable supramolecular complexes.
  • the test site positions on the array at which the supramolecular complexes form identifies all the basic sets of intermolecular p-RNA-peptide structures which are responsible for the drag binding event.
  • This electronic ELOC process allows the combinatorial selection for a large number intermolecular ligand binding structures to be made very rapidly.
  • a 100 test site array with 100 different peptide sequences would select through 10 6 possible triad ligand binding structures for the most unique combinations.
  • a 1,000 test site array with 1000 different peptide sequences would select through 10 9 possible triad ligand binding structures for the most unique combinations.
  • a 10,000 test site array with 10,000 different peptide sequences would select through 10 12 possible triad ligand binding structures for the unique combinations. Once supramolecular ligand binding complex patterns are determined, and the unique p-RNA-peptide intermolecular ligand binding structures are identified for known drags or biologically active compounds, the microelectronic array can be used as a molecular descriptor for screening new drugs or unknown compounds.
  • Figure 1 shows the basic p-RNA unit structure and the phosphodiester backbone structure of the p-RNA molecule.
  • Figure 2 shows the basic double-stranded helical stracture of DNA compared to the p-RNA planar or ladder-like structure.
  • Figure 3 shows the basic structure of the three p-RNA sequences (A, B, and C) used to form the hybridizing or pairing system for the combinatorial ELIAS process.
  • Figure 4 shows the basic form of the three p-RNA peptide derivatized (A, B, and
  • Figure 5 shows formation of a basic supramolecular complex between a ligand molecule and the p-RNA-peptide "triad" structures.
  • the intermolecular stracture can form both with and without the ligand binding molecule. However, the intermolecular stracture with the bound ligand molecule ordinarily is a more stable form.
  • Figure 6 shows the basic features of the ELOC process in which supramolecular complexes form on specific test sites on the microelectronic array.
  • Figure 7 shows the complete structure for the fully hybridized 7-mer p-RNA (A), the 7-mer p-RNA (B), and the complementary 14-mer p-RNA (C).
  • Figure 8 shows the dimensional geometry and stereostracture of the hexamer peptide "triad” stracture. The figure also gives the relative distance of the amino acid R- groups from the peptide "triad" convergence point.
  • Figure 9 shows a molecular model of a full p-RNA-peptide triad suprastructure binding a biotin ligand molecule.
  • the biotin portion is constracted using a CPK space- filing model.
  • Figure 10a shows the basic stracture of the acetylcholine molecule.
  • Figure 10b shows the acetylcholine molecule in a favorable peptide triad binding structure within a negative binding pocket.
  • Figure 10c shows an unfavorable peptide triad binding structure with positive binding pocket.
  • Figure 11a shows an intermolecular ligand binding supramolecular structure with a duplex peptide ligand binding stracture
  • Figure 1 lb shows shows an intermolecular ligand binding supramolecular structure with a quadraplex peptide ligand binding stracture
  • Figure l ie shows an intermolecular ligand binding supramolecular structure with multiplex triad peptide ligand binding structures.
  • Figure 12 shows an example of a molecular descriptor array surface in which certain test site areas indicate supramolecular complex formation relating to drag effectiveness and other areas relate to toxicity or ineffectiveness.
  • Figures 13a and 13b show an intermolecular ligand binding supramolecular stracture where electronic perturbation is used to flex an unfavorable p-RNA-peptide stracture (13 a) into a more favorable conformation (13b) that can now bind a specific drag molecule.
  • Figure 14 shows the addressing of a portion of a 10,000 test site microelectronic array having 30 micron test sites with p-RNA complementary and non-complementary sequences and subsequent hybridization with the complementary fluorescent labeled p- RNA sequence.
  • Figure 15 shows the ELOC Format 1, also known as the Transient Dynamic
  • Figures 16a, 16b, and 16c show the ELOC Format 2 or Homogeneous Triad Formation Process.
  • Figure 17 shows the ELOC Format 3 or Heterogeneous Triad Formation Process.
  • Figure 18 shows multiplexing using p-RNA-peptide triads as synthetic antibodies.
  • Figure 19 shows modular immunoassays using p-RNA-antibody conjugates and a microelectronic array.
  • Figure 20b also shows the mass spectrum for the molecule.
  • Figure 21 shows the stracture of the Texas Red labeled TR90 sequence (4'-I-G-A- A-G-G-G-TR-2') hybridized with a complementary biotinylated B92 sequence (4'-Biotin- C-C-C-T-T-C-T-I-C-C-C-C-C-G-2').
  • Figure 21 also shows the UV hypochromicity curves for the thermal denaturation of the hybridized pairs.
  • Figure 22 shows the structure of the Texas Red labeled TR90 sequence (4'-I-G-A-
  • FIG. 22 shows the UV hypochromicity date for the thermal denaturation of the hybridized pairs.
  • Figure 23 shows an example of a p-RNA sequence with multiple-tryptamine linkers (4'-C-C-I-I-I-G-G-2') that has been synthesized and characterized. Figure 23 also shows the UV hypochromicity and melting characteristics for the hybridized pair.
  • Figure 24a shows a double tryptamine p-RNA structure with two peptide chains attached via the tryptamine linker.
  • Figure 24b shows the mass spectrum for the molecule.
  • Figure 25 shows four peptide residues inco ⁇ orated into one supramolecular stracture.
  • Figure 24b also shows the UV hypochromicity curves which demonstrate the melting of the double-stranded p-RNA-peptide stracture occurs around 50 °C.
  • Figure 26 shows a simulation of a typical measurement starting from a normalized intensity at three positions of a set of 30 points (due to 30 conditions, time, temperature, stringency etc. here normalized to a total of 1) superimposed with typical noise.
  • Figure 27 shows a graph of the underlying idealized signal development.
  • Figure 28 shows a plot of factory analysis clustering of signal development on a 10,000 site array with a simulation of noise of absolute +/- 25% (50%), showing factor 1 (horizontal axis) versus factor 2 (vertical axis).
  • this invention relates to the design and fabrication of addressable active microelectronic array devices, and the processes, procedures, techniques, formats, methods and uses of these devices to carry out multi-step and multiplex affinity binding reactions in microscopic formats.
  • this invention relates to the use of these microelectronic array devices to rapidly carry out combinatorial selection and molecular recognition processes involving large numbers of potential intermolecular binding components and supramolecular complexes.
  • this invention relates to using microelectronic arrays and the electronic stringency parameter to affect higher order selectivity of intermolecular and supramolecular ligand binding structures and the formation of supramolecular complexes.
  • this invention relates to using microelectronic arrays and the electronic stringency parameter to effectively perturb supramolecular structures to their lowest energy configurations, to dynamically flex the supramolecular structures, and to generally enhance the formation of selective and stable ligand binding complexes.
  • combinatorial array devices of this invention can be used for a variety of applications, some of which include:
  • this invention relates to the use of active microelectronic array devices to carry out combinatorial processes for the selection of supramolecular complexes with improved speed and higher order specificity.
  • the supramolecular complexes are formed by the affinity binding of various specific ligand molecules and structures to intermolecular/supramolecular ligand binding structures.
  • a specific ligand molecule or stracture is defined as a targeted molecule or structure that has specific affinity for another molecule, synthetic ligand binding structure, or a biological receptor.
  • a given specific ligand molecule or stracture is a biologically active molecule (drug, metabolite, hormone, peptide) or stracture (protein, antibody) that has a specific shape and fits into a specific biological or physiological receptor molecule, site or stracture, where one or more non-covalent binding interactions stabilize the complex.
  • Receptor sites can include, but are not limited to: chelates, peptides, proteins, antibodies, enzymes, nucleic acid/protein complexes, membranes, and cells.
  • the specific or selective fit of a given specific ligand molecule or stracture into its specific receptor site is sometimes referred to as a "lock and key" fit.
  • the non-covalent interactions which bind the specific ligand molecule to the receptor site can include: hydrogen bonding, hydrophobic bonding, aromatic ring stacking, electrostatic interactions, chelation (with metal ion ligands) and van der Waals interactions.
  • the ligands and receptor sites can also have stereoselective properties. For enantiomeric ligands or molecules where chirality is based on optical asymmetry at a single atom, there are no differences in physical/chemical properties in the absence of a dissymmetric receptor site or binding surface. However, diastereomer ligands with more than one asymmetric center can have different physical/chemical properties.
  • some other important classical biological ligand/receptor interactions include: substrate molecules with enzymes, haptens/antigens with antibodies, toxins and carcinogens with biological, receptors, biotin with avidin or streptavidin, hormones with their receptor sites and neurotransmitters with nerve cell receptors.
  • the same molecule or structure can serve both as a ligand and a receptor; for example, an antibody molecule may serve as a receptor for a specific hapten molecule and also as a ligand for another antibody.
  • Some of the specific ligand target molecules and stractures relevant to this invention include, but are not limited to: (1) Small Ligand Molecules - drags, therapeutic agents, agonists, antagonists, inhibitors, metabolites, amino acids, peptides, hormones, ACTH, angiotensins, brady- kinins, cytokines, endomo ⁇ hins, endo ⁇ hins, enkephalins, exo ⁇ hins, lymphokines, neurotransmitters, vitamins, nucleotides, oligonucleotides, synthetic anti-sense oilgonucleotide agents, lectins, haptens, sugars, lipids, fatty acids, biological co-factors (NADPH, FAD, thiamine pyrophosphate, etc.), metal ions, metal chelates, dyes, po ⁇ hyrins, toxins, carcinogens, mutagens and other natural or synthetic small molecules with biological activity or binding properties.
  • intermolecular ligand binding structures are defined as molecular stractures which have one component with programmable pairing properties, and a second component with ligand binding properties. These structures are designed such that they can self-assemble under appropriate conditions into larger intermolecular structures "supramolecular structures" via their programmable pairing properties.
  • the stractures are further designed such that in the presence of an appropriate specific ligand target molecule or stracture, the binding of the ligand molecule by two or more of the ligand binding components can also lead to the formation and stabilization of the larger supramolecular structures.
  • the binding of the specific ligand molecule by the ligand binding components effectively produces intermolecular stabilization of the self-assembled supramolecular stracture through the pairing components.
  • the formation of the complete specific ligand molecule/intermolecular ligand binding structure is called the "supramolecular complex".
  • a programmable pairing stracture or component, of particular importance to this invention, which can be used to form self-assembling intermolecular ligand binding stractures is pyranosyl-RNA or p-RNA.
  • p-RNA is a nucleic acid like molecule in which the sugar group is a pentopyranose.
  • Figure 1 shows the basic p-RNA stracture.
  • p-RNA characteristics which are different than DNA include: higher duplex stability and selectivity than DNA or RNA, p-RNA does not base pair with DNA or RNA, and p-RNA duplexes form quasi-ladder structures, not the classical helix.
  • p-RNA has some characteristics similar to DNA/RNA, the fact that it does not hybridize to DNA means that it is uniquely different from other nucleic acid variants or derivatives i.e., p-RNA represents a unique pairing system. Additionally, the replacement of the normal deoxyribose (or ribose) with the pentopyranose sugar leads to planar or ladder-like form for the hybridized double-stranded p-RNA stracture. The planar form of double-stranded p-RNA stracture provides unique attributes for forming stable self-assembling intermolecular stractures, suprastructures, and supramolecular complexes when ligand molecules are bound.
  • p-RNA molecules can be derivatized with many of the same components and by many of the same procedures that have been developed for DNA and RNA modification.
  • p-RNA can be derivatized (functionalized or modified) with biotin moieties, aromatic and aliphatic amine groups, aromatic and aliphatic thiol groups, aromatic and aliphatic aldehyde groups.
  • p-RNA can also be functionalized by inco ⁇ oration of a tryptamine ribopyranosyl (Tr) phosphoramidite at the terminal position or anywhere within the sequence.
  • p-RNAs functionalized with amines, thiols, aldehydes, and/or tryptamine (I) nucleosides can also be subsequently attached to solid supports and surfaces, these include, but are not limited to: glass, silicon, plastics, nylon, nitrocellulose, ceramics, metals, metal oxides, agarose, polyacrylamide and other polysaccharides.
  • p-RNAs can be functionalized at their 2' or 4' terminal positions or at any position within the sequence (see Example 2). Derivatization of p-RNA can be carried out via modification of the base moieties, sugar, or the phosphate groups.
  • CNA-peptide pairing systems are disclosed in WO 99/15509, entitled “Cyclohexyl and Heterocyclyl Nucleoside Derivatives, Method for Producing These Derivatives, and the Use of the Derivatives and Their Oligomers or Conjugates in Pairing and/or Testing Systems, filed September 22, 1997).
  • CNA's have an uncharged backbone stracture, which means that they could have advantages for forming pairing stractures under low ionic strength conditions.
  • another group of programmable pairing components which can be used to form self-assembling intermolecular ligand binding stractures includes, but is not limited to: deoxyribonucleic acids (DNA), ribonucleic acids (RNA), synthetic polynucleotides, synthetic oligonucleotides, methylphosphonate nucleic acid analogues, phosphorothioate nucleic acid analogues, phosphorodithioate nucleic acid analogues, peptide nucleic acids (PNA) and other modified nucleic acids.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • synthetic polynucleotides synthetic polynucleotides
  • synthetic oligonucleotides synthetic oligonucleotides
  • nucleic acid moieties serve to form the intermolecular pairing system via classical hybridization. (It is important to point out again that the pairing system for p-RNA is distinct from DNA RNA and all other nucleic acid derivatives).
  • intermolecular pairing systems may be based on peptides, proteins, modified polysaccharides, lectins, electrostatic (cation/anion) type polymers, and metal/chelate systems, and a variety of synthetic stractures which can be designed with programmable self-assembling pairing properties.
  • peptides proteins, modified polysaccharides, lectins, electrostatic (cation/anion) type polymers, and metal/chelate systems, and a variety of synthetic stractures which can be designed with programmable self-assembling pairing properties.
  • the second important component of the intermolecular ligand binding structure is the actual ligand binding stracture itself.
  • the ligand binding stractures of this invention are defined as those stractures which, in the presence of an appropriate specific ligand target molecule or structure, can provide one or more of the following;
  • a first class of ligand binding stractures of this invention includes, but is not limited to: amino acids, peptides, cyclic peptides, antibodies, proteins, avidin, streptavidin, lectins, carbohydrates, polysaccharides, chelates, metal chelates, membranes, micelles, fluorophores, chromophores, crown ethers, cyclodextrins, cells and composites of two or more of any of the above.
  • a second class of ligand binding structures includes, but is not limited to: p-RNA, CNA, DNA, RNA, aptamers, oligonucleotides, PNA, and composites of two or more of the above.
  • the p-RNA and/or nucleic acid components are used for both pairing and ligand binding.
  • Ligand binding stractures can be composed of components from both group one and group two.
  • Programmable pairing components and ligand binding components can be combined in an almost unlimited number of configurations.
  • the programmable pairing component and the ligand-binding component are covalently coupled.
  • a peptide ligand-binding component could be covalently coupled to a p-RNA pairing component to form an intermolecular ligand binding structure.
  • Intermolecular ligand binding stractures of this invention include, but are not limited to, the following:
  • p-RNA molecular derivatives, structures and/or compositions include: p-RNA-amino acid(s), L, D, natural, un-natural forms, p-RNA-peptide, p-RNA-DNA-peptide (double recognition/pairing with ligand binding), p-RNA-antibody (protein, enzyme), • p-RNA-hapten derivative, p-RNA-drag derivative, p-RNA-neurotransmitter derivative, p-RNA-hormone derivative, p-RNA-toxin derivative, • p-RNA-radioisotope derivative, p-RNA-lectin (carbohydrate, disaccharide, polysaccharide derivatives), p-RNA-DNA (RNA, PNA, methylphosphonate), p-RNA-DNA (RNA, PNA, methylphosphonate)-peptide, p-RNA-fluorophore(s), • p-RNA-donor/acceptor fluorophores (arrangements for photonic energy transfer), p-
  • p-RNA nanoscale, meso-scale and/or solid support stractures include: • p-RNA-nanoparticles/nanostractures/nanodevices (metal nanoparticles, gold nanoparticles, metal oxide nanoparticles, quantum dots, carbon nanotubes, polysaccharide nanobeads, and organic polymer nanobeads),
  • p-RNA-meso-scale structures/devices metal particles/structures/devices, metal oxide particles/stractures/devices, silicon particles/structures/devices, semi- conductor particles/stractures/devices, gallium arsenide particles/structures/- devices, lift-off microelectronic devices, lift-off photonic devices, lift-off mechanical devices, and micro sensor devices),
  • p-RNA-macroscopic surface/support materials glass, quartz, mica, metal, metal oxides, silicon, silicon dioxide, GaAs, plastics, organic polymers, natural polymers, cell surfaces, agarose gels, polyacrylamide gels, hydrogels, silica gels, nylon, nitrocellulose, and ceramics
  • p-RNA and peptides nanoscales, mesoscales and solid support stractures include: • p-RNA-peptide-nanoparticles/nanostractures/nanodevices (metal nanoparticles, gold nanoparticles, metal oxide nanoparticles, quantum dots, carbon nanotubes, polysaccharide nanobeads, and organic polymer nanobeads),
  • p-RNA-peptide-meso-scale structures/devices metal particles/structures/devices, metal oxide particles/stractures/devices; silicon particles/structures/devices, semiconductor particles/stractures/devices, gallium arsenide particles/structures/devices, lift-off microelectronic devices, lift-off photonic devices, lift-off mechanical devices, and micro sensor devices
  • metal particles/structures/devices metal oxide particles/stractures/devices
  • silicon particles/structures/devices silicon particles/structures/devices, semiconductor particles/stractures/devices, gallium arsenide particles/structures/devices
  • lift-off microelectronic devices lift-off photonic devices, lift-off mechanical devices, and micro sensor devices
  • p-RNA-peptide-macroscopic surface/support materials glass, quartz, mica, metal, metal oxides, silicon, silicon dioxide, GaAs, plastics, organic polymers, natural polymers, cell surfaces, agarose gels, polyacrylamide gels, hydrogels, silica gels, nylon, nitrocellulose, and ceramics).
  • p-RNA and peptide-flurophore nanoscale, mesoscale, and solid support stractures include: • p-RNA-peptide-fluorophore-nanoparticles/nanostractures/nanodevices (metal nanoparticles, gold nanoparticles, metal oxide nanoparticles, quantum dots, carbon nanotubes, polysaccharide nanobeads, and organic polymer nanobeads),
  • p-RNA-peptide-fluorophore-meso-scale structures/devices metal particles/stractures/devices, metal oxide particles/stractures/devices; silicon particles/stractures/devices, semiconductor particles/stractures/devices, gallium arsenide particles/stractures/devices, lift-off microelectronic devices, lift-off photonic devices, lift-off mechanical devices, and microsensor devices), and
  • p-RNA-peptide-fluorophore-macroscopic surface/support materials glass, quartz, mica, metal, metal oxides/silicon, silicon dioxide, GaAs, plastics, organic polymers, natural polymers, cell surfaces, agarose gels, polyacrylamide gels, hydrogels, silica gels, nylon, nitrocellulose, and ceramics).
  • CNA BASED INTERMOLECULAR LIGAND BINDING STRUCTURES examples include:
  • p-RNA-peptide stractures represent the class of intermolecular ligand binding stractures that are of major importance to this invention.
  • the p-RNA moiety provides a programmable self-assembling intermolecular pairing system (via base sequence and hybridization), and the peptide moieties serve to form the actual ligand binding stractures.
  • One particular advantage of using p-RNA is that upon pairing (hybridization) the double-stranded p-RNA is a planar or ladder-like structure.
  • FIG. 2 shows the basic double-stranded p-RNA planar/ladder stracture.
  • the planar or ladder-form of paired double-stranded p-RNA provides more predicable supramolecular ligand binding stractures and complexes
  • p- RNA has further advantages of providing more stable and selective duplex stractures than does DNA or RNA.
  • the fact that p-RNAs are much more stable than DNA, means that significantly shorter sequences can be used to form the double-stranded planar structures.
  • p-RNA also does not pair or hybridize with DNA. This means that any extraneous DNA in a sample does not interfere with p-RNA pairing/hybridization.
  • DNA can be used to create a separate pairing system or composite system with two programmable pairing components (e.g., p-RNA-DNA or p- RNA-RNA).
  • p-RNA represents a unique programmable pairing system, involving the same basic purine and pyrimidine base pairing (adenine with thymidine, and guanine with cytosine) as DNA or RNA, but with no affinity to DNA or RNA.
  • the p-RNA stracture can be derivatized, functionalized and modified in the same fashion and by most of the same procedures that are used for DNA or RNA.
  • p-RNA molecules can be derivatized with peptide sequences at basically any position in the sequence.
  • the peptide sequences can contain any arrangement of amino acids, to form large combinatorial groups of potential ligand binding stractures.
  • One particular useful form of p-RNA-peptide intermolecular ligand binding structures are able to self-assemble into a "triad" type ligand binding superstructure.
  • p-RNA sequence (A) and p-RNA sequence (B) which are complementary to a third longer p-RNA sequence (p-RNA sequence (C)) produces these triad ligand binding suprastructures.
  • the longer p-RNA sequence (C) functions as a template for positioning the two shorter sequences (A and B).
  • the two shorter p-RNA sequences (A and B) are designed so as to be contiguous, or nearly contiguous, when hybridized to the complementary "template” p-RNA sequence (C).
  • Figure 3 shows the generic design for the three complementary p-RNA sequences (A, B, & C), and the pairing or hybridized double-stranded planar/ladder stracture.
  • the third "template” p-RNA sequence (C) can be further functionalized so it can be immobilized on beads, particles, or support material (glass, silicon, silicon dioxide, silicon nitride, metals, metal oxides, plastic, nylon, agarose, polyacrylamide, hydrogels, silica gels, sol-gels, etc.).
  • the appropriate functionalization allows the template p-RNA sequence (C) to be either covalently or noncovalently attached to a specific test site on the microelectronic array.
  • the p-RNA can be functionalized with a biotin moiety which allows it to be attached to the microelectronic array test sites via streptavidin inco ⁇ orated in the permeation layer of test site. It is also possible to covalently attach or immobilize p-RNA sequences that have been functionalized with amines, thiols, aldehydes, carboxyl groups, hydrazines, azido groups, and with phenylboronic acid.
  • the template p-RNA sequence (C) is functionalized at either its 4' or 2' terminal position when attachment to a solid support is the objective.
  • the peptide sequences that form the ligand binding component of the intermolecular ligand binding stractures are generally covalently coupled to the p-RNA sequence.
  • non-covalent attachments are possible (biotin avidin, etc.), and can sometimes have applicability.
  • Covalent coupling of the peptide can be achieved by either using a functional "R" group provided by one or more of the amino acids in the peptide itself, or by inco ⁇ orating additional functionalization into the peptide sequence.
  • Functional groups which can provide by one or more of the amino acids in the peptide sequence itself, include the following:
  • Amino Acid Functional "R” Groups for Coupling include: • Cysteine (thiol),
  • an amino acid functional (R) group for coupling to the p- RNA means that, the "R” group from amino acid used for coupling will play less of a role in the actual ligand binding process. Also, consideration must be given to the full synthetic route, including peptide synthesis, de-blocking procedures, and the final p- RNA peptide coupling procedure, so that amino acid "R" groups which are expected to be available for ligand binding and combinatorial selection remain viable after coupling to the p-RNA. Coupling of the peptide sequence to the p-RNA molecule can generally be carried out at any position within either the p-RNA sequence or the peptide sequence. This includes:
  • Functional groups for coupling reactions that can be inco ⁇ orated into p-RNA include: tryptamine nucleotides, amines, thiols, aldehydes, hydroxyl (ribose), carboxyl, phosphate, maleimides, and a number of others.
  • One particular method relevant to this invention for coupling a peptide via the cysteine thiol group involves using a peptide sequence with a cysteine in the terminal position, which is then reacted with one or more tryptamine nucleotides within the p-RNA sequence.
  • the procedure for linking cysteine peptides to tryptamine (I) containing p-RNA sequences is given in the Experimental Section (see Example 3).
  • spacer groups can include, but are not limited to: short run of amino acids (-gly-gly-gly-), aliphatic chains (-CH 2 -CH 2 -CH 2 - CH 2 -), polyglycols, and polysaccharide stractures.
  • Spacer groups can be designed to provide either ridge or flexible intervening stractures between the p-RNA and the peptide.
  • Another general class of intermolecular ligand binding stractures relevant to this invention can utilize nucleic acids as the pairing system component and peptides as ligand binder component. These structures can include DNA-peptides, RNA peptides, or various nucleic acid analogues with peptides attached.
  • DNA is used to form the pairing system, the double-stranded stractures that are formed are helical.
  • the helical nature of the DNA produces a relatively complex stracture; this can make it somewhat more difficult to determine the best positions for the peptide ligand binding structures.
  • the various combinatorial binding assays, formats, and procedures which are relevant to this invention first involve producing a microelectronic array in which a first group of capture p-RNA-peptide intermolecular ligand binding structures (each with a different peptide sequence) are selectively addressed and immobilized to specific test sites on the array. Addressing of p-RNA-peptides to the microelectronic array can be carried out by electronic addressing (as is described in the Experimental Section of this invention), or by other mechanical deposition (ink jetting, micropipeting, microcapillary application, etc.).
  • the capture p-RNA (C) component has a common or generic sequence that is complementary for the other two p-RNA sequences (A & B).
  • p-RNA sequences A and B are not complementary to each other, but only pair/hybridize with their own complementary section of capture p-RNA (C) sequence.
  • the capture p-RNA (C) sequence is the same for all test sites, each test site on the array contains a different peptide sequence from a known peptide library. The use of the term "each" is not meant to exclude the situation where repetitive or redundant test sites are included within the array.
  • Peptide libraries can be created with any given number of peptides containing permutations of any number of amino acids within the sequence.
  • One particularly useful peptide library that is relevant to the combinatorial aspects of this invention includes a group of 10,000 hexamer peptide (containing six amino acids).
  • the C-terminus amino acid is usually a glycine, which is generally used as the starting amino acid resin in the synthesis procedure
  • the N-terminus amino acid is usually a cysteine which provides a thiol group that is used for coupling to a tryptamine within the p-RNA molecule.
  • the four internal amino acids of the hexamer peptide are preferably chosen form a group often amino acids that include:
  • Phenylalanine aromatic ring R-group: hydrophobic & ring stacking interactions
  • Proline closed ring amino acid: stereochemistry, produces bend in peptide chain
  • hexamer peptide library or other peptide libraries can be constructed using reagents and automated solid phase peptide synthesis procedures that are well known to those who practice the art of peptide synthesis.
  • libraries which have longer peptides e.g., 7-mers, 8-mers, 9-mers, and up to 100-mers, and various sub library combinations
  • libraries with shorter peptides e.g., 4-mers, 3-mers, 2-mers, and single amino acids, and various sublibrary combinations
  • libraries which include all twenty one amino acids. Also, envisioned by this invention are libraries with combinations of peptides with other moieties; as well as combinations of non-peptide ligand binding moieties, examples of which include, but are not limited to:
  • a completed addressed microelectronic array there is a known peptide sequence from a peptide library at a known test site on the array. While the peptide sequence at each site is different, the capture p-RNA (C) sequence is generic or common and complementary to both p-RNA sequences (A & B). Generally, the p-RNA sequences A and B have been coupled with the same sets of peptide sequences (from the known peptide library) as were the immobilized capture p-RNA-peptides. The p-RNA peptide of groups A, B, and C now represent sublibraries of the original peptide library. When the three groups of p-RNA-peptides are mixed together under hybridization conditions, a large number of intermolecular ligand binding superstructures are formed (the cube of number of peptides in the library or on the array).
  • This process represents the formation of an "exponential library by aggregation of sublibraries” and is called “ELIAS”.
  • the basic ELIAS concept is shown in Figure 4.
  • the two complementary sequences of the p-RNA-peptide (A) and (B) components hybridize to complementary sequence of the capture p-RNA-peptide (C) sequence, they produce the intermolecular ligand binding structure in which the peptide strands form a "triad" binding structure or pocket.
  • This "triad" binding stracture or pocket has the potential affinity for a specific ligand molecule or structure (see Figure 5).
  • a 100 test site array addressed with 100 different sets of p- RNA-peptide (C), and contacted with 100 different sets of p-RNA-peptides (A) and 100 different sets p-RNA-peptides (B) under hybridization conditions would select through 10 6 possible supramolecular or triad ligand binding structures for the unique combinations.
  • ELIAS the process is called “ELIAS on a chip", or "ELOC”.
  • the basic ELOC concept is shown in Figure 6.
  • the microelectronic array based ELOC process and the various electronic formats represent an important aspect of this invention in certain embodiments. Depending on the objective of a particular combinatorial assay and the nature of the specific target molecules or stractures, different formats can be more effective in providing information about the structure-function relationships between the ligand and the binding site and lead to more useful "molecular descriptor" devices.
  • the basic ELOC combinatorial selection processes can be carried out in a number of different formats that involve the use of different conventional and electronic stringency parameters to affect the p-RNA pairing/hybridization process and to affect and/or perturb the peptide triad structures and the ligand binding process. Additionally, the formats can involve different order as to when and how ligands and p-RNA-peptide components A and B are added to the system.
  • the ELOC processes and formats, and the p-RNA-peptide stractures themselves are designed to achieve the following:
  • a particular group of p-RNA peptide intermolecular stractures relevant to this invention are those structures that can self-assemble, via the p-RNA pairing/hybridization, into arrangements that produce a "triad" peptide ligand binding stracture.
  • Ability to form the "triad" peptide ligand binding stracture is achieved by appropriate design of the p- RNA-peptide structures.
  • the capture p-RNA (C) sequence is functionalized in the 4 '-terminal position with a biotin moiety for subsequent immobilization to microelectronic array test sites, via streptavidin within the agarose permeation layer of the test site.
  • the immobilized capture p-RNA has a "test site surface-biotin-4'-p-RNA-2' terminal" orientation.
  • the lengths for capture p-RNA (C) sequences can most broadly range from about six nucleotides (bases) to over one hundred nucleotides (bases); more ideal lengths are from about seven nucleotides to sixty nucleotides; and the most ideal lengths are from about eight nucleotides to forty nucleotides.
  • the peptide sequence for the capture p-RNA (C) sequence is coupled at or near (within three bases), of the middle of the p-RNA (C) sequence.
  • the peptide is coupled through the modified tryptamine nucleoside (I) within the p-RNA sequence (see Example 3).
  • the p- RNA-peptide sequence (A) is designed to hybridize with the 2' end of the capture p-RNA (C) sequence.
  • the lengths for the p-RNA (A) sequences can most broadly range from about three nucleotides (bases) to over one hundred nucleotides (bases); more ideal lengths are from about four nucleotides to thirty nucleotides; and the most ideal lengths are from about five nucleotides to twenty nucleotides.
  • the peptide sequence for the p-RNA (A) sequence is coupled at or near (within three bases) of the 2'-terminus of p- RNA (A).
  • the coupling of the peptide to the p-RNA (A) is via the cysteine thiol (peptide) to the modified tryptamine nucleoside (I) at or near the 2' end of p-RNA (A), using the procedure described above for the p-RNA (C).
  • the p-RNA sequence (B) is designed to hybridize with the 4' end of the immobilized capture p-RNA (C) sequence.
  • the lengths for the p-RNA (B) sequences can most broadly range from about three nucleotides (bases) to over one hundred nucleotides (bases); more ideal lengths are from about four nucleotides to thirty nucleotides; and the most ideal lengths are from about five nucleotides to twenty nucleotides.
  • the peptide sequence for the p-RNA (B) sequence is coupled at or near (within three bases) of the 4'-terminus of p-RNA (B).
  • the coupling of the peptide to the p-RNA (B) sequence is via the cysteine thiol (peptide) to the modified tryptamine (I), at or near the 4' end of p-RNA (B), using the procedure described above for the p-RNA (C)
  • reporter group(s) fluorophores; however, also included in the invention are chromophores, biotin/avidin detection systems, chemiluminescent agents, metal chelates, radioisotopes, proteins, enzyme detection systems, antibodies and nanoparticles.
  • p-RNA-peptide (A) might be labeled with a Cyanine-3 fluorophore (Ex 530 nm, Em 570 nm) and p-RNA-peptide (B) might be labeled with a Texas Red fluorophore (Ex 590 nm, EM 620 nm). See Example 2.
  • two color fluorescent analysis can be used to detect the formation of intermolecular ligand binding complexes on the array surface.
  • Fluorescent analysis is not limited to these two fluorophores, as a variety of fluorescent labels may be used (fluorescein, Rhodamine, Bodipy Texas Red, Bodipy Far Red, Cyanine-5, etc.)
  • fluorescent labels fluorescein, Rhodamine, Bodipy Texas Red, Bodipy Far Red, Cyanine-5, etc.
  • Most detection formats and systems used for DNA hybridization and immunodiagnostic analysis which are well know to those practicing the art, can also be employed for many combinatorial applications. These general detection methodologies are hereby inco ⁇ orated into this invention.
  • each chain extends a distance of approximately 2.0 nanometers (nm) form the focal point (see Figure 8).
  • the first amino acid (cysteine) and its thiol R-l group (-SH) are involved in the covalent attachment to the p-RNA.
  • the second amino acid with the R-2 group is potentially available for ligand interaction and is approximately 0.6 nanometer (nm) form the attachment focal point.
  • the third amino acid with the R-3 group is approximately 0.95 nm from the focal point.
  • the fourth amino acid with the R-4 group is approximately 1.3 nm from the focal point.
  • the fifth amino acid with the R-5 group is approximately 1.7 nm from the focal point.
  • the sixth and last amino acid is approximately 2.0 nm from the focal point.
  • the R-6 group is a hydrogen atom (-H) which would not be expected to have significant interaction with ligand molecules.
  • the glycine's free C-terminal carboxyl (-COO ) could potentially be involved in ligand binding interactions.
  • Figure 9 shows a molecular model of a full p-RNA-peptide triad suprastructure binding a biotin ligand molecule. The biotin portion is constracted using a CPK space filling model system.
  • Acetylcholine is a relatively small molecule (MW 146) which is the acetyl ester of choline (2-hydroxy- N,N,N-trimethylethanaminium).
  • the acetylcholine molecule carries a formal positive charge which plays a role in binding and orienting the molecule in the enzyme active site and in receptor sites (see Figure 10a).
  • a potentially favorable ligand binding "triad" structure might be composed of the following hexamer peptide (A, B, and C) sequences:
  • Acetylcholine agonist Natural Muscarine, Phenyltrimethylammonium, etc.
  • antagonist d-Tubocurarine, Tr ⁇ methaphan, Hexamethomium, Decamethonium, Atropine, etc.
  • the first peptide triad stracture and unfavorably by the second peptide triad structure.
  • one can predict to some degree what might be a generically favorable or unfavorable binding ligand structures for certain ligands it would be very difficult to empirically determine which are the "optimal" stractures that would provide the basis for the molecular descriptor device.
  • An important aspect of this invention involves electronic combinatorial selection processes and formats where microelectronic arrays are used to affect the affinity binding of small molecules (drugs, agonist, antagonists, substrates, metabolites, metal ions, etc.), large molecules (proteins, enzymes, antibodies, etc.) and larger stractures (organelles, cells, etc.) to various supramolecular stractures.
  • small molecules drug, agonist, antagonists, substrates, metabolites, metal ions, etc.
  • large molecules proteins, enzymes, antibodies, etc.
  • larger stractures organelles, cells, etc.
  • a supramolecular stracture which is important to this invention is a p-RNA-peptide intermolecular triad ligand binding stracture.
  • the use of microelectronic arrays allows the combinatorial selection process to be carried out in what might be considered a near real time evolutionary or learning mode.
  • Microelectronic arrays have the potential to achieve higher order specificity for combinatorial selection processes, because they provide the added parameter of selective electric field stringency control at each binding (test) site on the array.
  • stringency parameters which include: temperature, pH, ionic strength, and chemical agents (detergents, denaturants, chaotropic agents)
  • the application of an electric field stringency to supramolecular complex formation provides a totally new and powerful parameter for selecting the improved and more specific intermolecular ligand binding stractures.
  • Active microelectronic chip/array technologies have been demonstrated which provide capability for rapid multiplex hybridization and electronic stringency for improving hybrid selectivity. This technology also includes procedures and methods for selective addressing DNA sequences, oligonucleotides, amplicons, and other moieties to the array device.
  • Example 5 shows further experiments which demonstrate supramolecular complex formation and "significant" increase in intermolecular complex stability (increase electronic Tm) for p-RNA histidine rich peptides in the presence of nickel ions. This is a particularly important experiment as it provides verification that certain intermolecular complexes can be significantly stabilized upon proper ligand molecule binding.
  • Microarrays can be selectively addressed with p-RNA peptides sets by either electronic or mechanical means.
  • Electronic addressing of p-RNA-peptides to the microelectronic array involves exposing the array to a solution of the specific p-RNA peptide (C), and biasing the selective site on the array positive relative to a set of negatively biased sites or counter electrodes on the array.
  • the addressing solution contains between 1 to 100 nM of the biotinylated p-RNA-peptide (C) sequence in a 50-100 mM hisitidine (zwiterionic) solution.
  • Electronic addressing is carried out by application of between 200nA to 600nA (to the positively biased site) for a period of 60 to 120 seconds.
  • Three basic electronic ELOC formats are described which provide advantages for carrying out combinatorial selection processes that involve different libraries (smaller and larger), different ligands, and/or require different combinatorial selection criteria such as speed, selectivity and sensitivity.
  • the formats are designed to take advantage of both the self-assembling properties of the p-RNA sequences (hybridization of p-RNA A and B sequences to an immobilized p-RNA C sequence) and the overall stabilization of intermolecular stractures that occurs upon ligand binding.
  • the formats are also designed to utilize conventional stringency (temperature, pH, ionic strength, etc.), electronic assisted hybridization, electronic stringency and electronic perturbation to overcome the combinatorial complexity issues which lead to non-specific binding and non-specific background problems.
  • the formats are designed to produce the maximum number of selective and stable supramolecular complexes and allow them to be detected as rapidly as possible.
  • This format is designed to take advantage of transient supramolecular structure formation produced by rapid forming and breaking p-RNA-peptides (A) and p-RNA peptides (B) hybrids with the immobilized p-RNA-peptides (C) site on the array. Additionally, the format takes advantage of the stabilization effect that occurs when a favorable ligand binding event stabilizes a specific triad stracture that is produced in the transient process.
  • This electronic ELOC format involves a first step in which the general stringency condition on the microelectronic array is, at or near (plus/minus 5° C), the thermal melting mid-point (Tm) for the hybridization of the two p-RNA (A) and (B) sequences to the immobilized complementary capture p-RNA (C) sequence.
  • the p-RNA- peptide (A) and (B) sublibraries can be added at concentrations from about InM to 100 ⁇ M of each sublibrary. For a 10,000 member combinatorial peptide library this is a concentration of about 100 fM to 10 nM for each peptide sequence.
  • These p-RNA peptide libraries can be in either low conductance solution or higher conductance solutions.
  • electronic assisted hybridization can be used to accelerate both the on and off rates for hybrid formation.
  • hybridization can proceed at its normal rate and electronic stringency or perturbation can be used to accelerate the hybrid off rates.
  • Higher conductance solutions can be composed of 1 mM to 1M of sodium chloride/sodium phosphate, sodium citrate, Tris, or any common buffer system used for normal hybridization or affinity binding reactions that are well known this art. These procedures are designed to achieve the condition where the supramolecular triad structure formation for most of the potential p-RNA-peptides (A, B, and C) exist in a transient or dynamic equilibrium state.
  • the ligand molecules can be added into the system at any time during this process, however depending on the nature of the ligand, addition after the dynamic equilibrium state is reached may sometime be preferred.
  • Ligand molecule concentrations can range from about 1 pM to 100 nM. Under these conditions, the binding of a ligand molecule to any favorable triad combination of peptide sequences will effectively stabilize that particular supramolecular structure/complex formation. This is an intermolecularly produced stabilization that effectively raises the thermal melting temperature (Tm) for the hybridization of those selected p-RNA-peptide (A) and/or (B) components to the selected capture p-RNA-peptide (C) site(s) on the array.
  • Tm thermal melting temperature
  • Both positive and negative electric field stringency in contiguous or pulsed DC and/or offset AC scenarios can be used to perturb peptide triad stractures and ligand/peptide triad complexes.
  • the multitude of combinatorial triad stractures that can potentially be produced would have numerous peptide combinations containing both positively and negatively charged amino acid R-groups. These charged peptide R-groups would be strongly influenced by the application of the electric field.
  • the electric field could be used to flex the peptide triad stractures, helping them to achieve lowest energy conformations that are more suitable to ligand binding.
  • the electric field can be used to perturb the ligand/supramolecular complexes allowing them to find more stable configurations.
  • any charged ligand molecules can themselves be influenced or perturbed by application of negative and/or positive electric field.
  • Figure 13a and 13b show an example of the electronic perturbation effect on charged p-RNA-peptide structures.
  • an acetylcholine ligand molecule that has a formal positive charge would be moved toward and concentrated at a microelectronic array test site that is biased positive, and moved away from a test site which is biased negative.
  • a positively charged ligand molecule could be removed from its ligand binding site by application of positive electronic stringency to that site.
  • a methotrexate ligand molecule which has a formal negative charge
  • application of a negative bias would move the molecule away from the test site
  • application of a positive bias would move the molecule toward and concentrate it at that test site.
  • a negatively charged ligand molecule could be removed from its ligand binding site by application of negative electronic stringency.
  • electric field stringency can be used at three other levels to achieve higher selectivity and sensitivity for detecting supramolecular complexes.
  • the first level involves low electric field stringency to remove nonspecific or partially bound stractures.
  • the second level involves application of a more precise intermediate level of electric field strength to select only the most selective and/or stable supramolecular complexes involving p-RNA peptide (A) and (B) sets.
  • the third level of electronic stringency involves application of the most precise and highest level of electric field strength to select the most stable supramolecular complexes involving both p-RNA-peptide sets (A) and (B), and the capture p-RNA peptide sets (C); i.e., the optimal "triad" peptide/ligand complexes.
  • the positions on the array at which the supramolecular complexes form, identifies all the basic sets of intermolecular p-RNA-peptide structures which are responsible for the ligand binding event.
  • This electronic ELOC format allows the combinatorial selection for a large number intermolecular ligand binding structures to be made very rapidly.
  • Figure 15a and 15b shows the ELOC Format 1 or the Transient Dynamic Equilibrium Triad Formation process.
  • a combinatorial selection process to produce a molecular descriptor array for screening new drags and therapeutics which affect the Nicotinic and Muscarinic Cholinergic receptors might be carried out in the following manner.
  • the combinatorial selection process involving the 10,000 member p-RNA-peptide library would be carried in electronic ELOC format 1 for a group of ligands which include the cholinergic substrate (acetylcholine) and related drugs, agonist, and antagonists (nicotine, muscarine, phenyltrimethylammonium, d-tubocurarine, hexamethonium, atropine,et ).
  • cholinergic substrate acetylcholine
  • related drugs, agonist, and antagonists nicotine, muscarine, phenyltrimethylammonium, d-tubocurarine, hexamethonium, atropine,et .
  • a 10,000 test site microelectronic array would be selectively addressed with all 10,000 p- RNA-peptide (C) sequences.
  • the ELOC format 1 would be carried out for each of the known cholinergic ligands (acetylcholine, nicotine, muscarine, etc.) with the p-RNA- peptide (A) and (B) sublibraries.
  • the positions of supramolecular complex formation on the microelectronic array would be detected and correlated for all the known chloinergic substrates, agonists, and antagonists.
  • the microelectronic array would now be a useful molecular descriptor for screening new drags which affect the cholinergic receptors.
  • Electronic array ELOC Format 2 Homogeneous Triad Formation Process
  • Electronic array ELOC format 2 is a combinatorial selection process that is designed to allow a specific ligand to complex with specific p-RNA-peptide (A) and (B) structures in the solution phase. Under one set of low stringency conditions (below the Tm for p-RNA hybridization) the homogeneous phase would be carried out separate from the microelectronic array. In this case, stringency conditions would be optimized for formation of ligand/p-RNA-peptide (A)/p-RNA-peptide (B) complexes.
  • the solution phase (homogeneous) complex formation can be placed on the microelectronic array that has been selectively addressed with the p-RNA-peptide (C) library.
  • the dimer complexes p-RNA-peptide A/ligand/p-RNA-peptide B
  • the dimer complexes are then allowed to form the triad supramolecular complexes by binding to specific positions (p- RNA-peptide C) sites on the array.
  • ELOC Format 1 can now be used for carrying out combinatorial selection of the optimal supramolecular ligand complexes.
  • a second variation of Format 2 involves carrying out the homogeneous phase formation of the ligand/p-RNA-peptide (A)/p-RNA-peptide (B) complexes on the microlelectronic array.
  • the initial procedure has to be carried out at high stringency or above the Tm for p-RNA-peptide hybridization.
  • the second phase would involve lowering the general stringency conditions to be at or near the Tm for p-RNA hybridization, so that triad complex formation could start occurring on the array.
  • Electronic assisted hybridization, stringency and perturbation can be used in several modes both during and after the second phase.
  • the electric field can be used to effectively perturb the intermolecular superstructures producing lower energy configurations that can lead to better ligand binding environments and to more stable complexes.
  • Both positive and negative electric field stringency in contiguous or pulsed DC and/or offset AC scenario can be used to perturb peptide triad stractures and ligand/peptide triad complexes.
  • the multitude of combinatorial triad stractures that can potentially be produced would have numerous peptide combinations containing both positively and negatively charged amino acid R-groups. These charged peptide R-groups would be strongly influenced by the application of the electric field.
  • the electric field could be used to flex the peptide triad stractures, helping them to achieve lowest energy conformations that are more suitable to ligand binding.
  • the electric field can be used to perturb the ligand/supramolecular complexes allowing them to find more stable configurations. Any charged ligand molecules can themselves be influenced or perturbed by application of negative and/or positive electric field.
  • electric field stringency can be used at three other levels to achieve higher selectivity and sensitivity for detecting supramolecular complexes. The first level involves low electric field stringency to remove nonspecific or partially bound stractures.
  • the second level involves application of a more precise intermediate level of electric field strength to select only the most selective and/or stable supramolecular complexes involving p-RNA-peptide (A) and (B) sets.
  • the third level of electronic stringency involves application of the most precise and highest level of electric field strength to select the most stable supramolecular complexes involving both p-RNA peptide sets (A) and (B), and the capture p-RNA-peptide sets (C); i.e., the optimal "triad" peptide/ligand complexes.
  • the positions on the array at which the supramolecular complexes form, identifies all the basic sets of intermolecular p-RNA peptide structures which are responsible for the ligand binding event.
  • Figure 16a, 16b, and 16c show the ELOC Format 2 or Homogeneous Triad Formation process.
  • Electronic array ELOC format 3 is a combinatorial selection process designed to initially form a very large number of triad type ligand binding superstructures. This process involves a first step of carrying out a low stringency hybridization of the complete peptide sublibrary sets of the two p-RNA peptide (A & B) sequences to the capture p-
  • RNA sequence (C) immobilized to the test sites on the array The low stringency condition for hybridization on the array would be the equivalent of 5° C or more below the Tm (thermal melting mid-point) for the p-RNA hybrids. This low stringency condition allows a very large number p-RNA peptide triad superstructures to be rapidly formed on the array.
  • the ligand molecule drug or biologically active compound
  • Electronic stringency can be used in several modes both during and after the initial process. During the process, the electric field can be used to effectively perturb the intermolecular superstructures producing lower energy configurations that can lead to better ligand binding environments and to more stable complexes.
  • Both positive and negative electric field stringency in contiguous or pulsed DC and/or offset AC scenarios can be used to perturb peptide triad stractures and ligand/peptide triad complexes.
  • the multitude of combinatorial triad stractures that potentially can be produced would have numerous combinations of both positively and negatively charged amino acid R-groups. These would be strongly influenced by the application of the electric field.
  • the electric field could be used to flex the peptide triad structures, helping them to achieve lowest energy conformations that are more suitable to ligand binding.
  • the electric field can be used to perturb the ligand/supramolecular complexes allowing them to find more stable configurations.
  • Electric field stringency can be used further for three levels of selectivity.
  • the first level involves low electric field stringency to remove non-specific or partially bound structures.
  • the second level involves application of a more precise intermediate level of electric field strength to select only the most selective and/or stable supramolecular complexes involving p-RNA peptide (A) and (B) sets.
  • the third level of electronic stringency involves application of most precise and highest level of electric field strength to select the most stable supramolecular complexes involving both p-RNA peptide sets (A) and (B), and the capture p-RNA peptide sets (C); i.e., the true "triplex" peptide/ligand complexes.
  • FIG. 17a, 17b, and 17c shows the ELOC Format 3 or Heterogeneous Triad Formation process.
  • the identification of the ligand specific supramolecuar stractures can also lead to their direct use and applications for a number of other areas.
  • Some of these unique supramolecular structures include but are not limited to:
  • Specific p-RNA peptide supramolecular stractures with specific peptide sequences can be produced with common p-RNA sequences as was described for the combinatorial selection processes, or with specific p-RNA sequences.
  • Specific p-RNA- peptide supramolecular triads with common p-RNA sequences can be selectively addressed to the array using the same electronic procedures described for the electronic combinatorial processes.
  • Other formats can involve using specific p-RNA sequences A, B, and C, which would produce the "specific" p-RNA-peptide triad stractures when all the component stractures were simply mixed together.
  • the complexes can be selectively immobilized by hybridization to the complementary p-RNA sequences on the array.
  • the microelectronic arrays can be used for selective addressing of stractures, for electronic perturbation of stractures and complexes, and for applying electronic stringency to achieve better assay sensitivity and selectivity.
  • the above description represents just some of the many homogenous and heterogeneous synthetic antibody "immuno" type assay formats which are envisioned by this invention.
  • one important "immuno" type assay utilizing the synthetic antibodies of this invention would be designed to detect and quantify the levels of Cytokinins (TNF, IL-1, IL-6, IL-8, etc.) and other cellular agents (Bradykinins, tissue factors, adhesion molecules, etc.) which are the initial indicators of bacterial/endotoxin septic infections, systemic inflammatory response syndrome (SIRS), sepsis, and impending septic shock and multiple organ dysfunction syndrome (MODS).
  • Cytokinins TNF, IL-1, IL-6, IL-8, etc.
  • other cellular agents are the initial indicators of bacterial/endotoxin septic infections, systemic inflammatory response syndrome (SIRS), sepsis, and impending septic shock and multiple organ dysfunction syndrome (MODS).
  • Multiple or modular type immunodiagnostic assays can be developed by preparing specific p-RNA-antibody conjugates which are then selectively addressed to a microelectronic or other array type device or substrate (see Figure 19).
  • specific complementary p-RNA sequences capture sequences
  • specific p-RNA-antibody conjugates are reacted in solution with samples containing the target haptens or antigens.
  • These p-RNA-antibody conjugates have both a specific antibody and a specific p-RNA sequence which allows it to be subsequently captured (via hybridization) to its complementary p-RNA on the array.
  • the p-RNA-antibody/hapten/antigen complexes are then selectively immobilized by hybridization to the complementary p-RNA sequences on the array.
  • a number of procedures can be used to detect the antibody/hapten/antigen complex, one of which is a so-called sandwich format.
  • sandwich assay format the first specific p-RNA- antibody conjugate is used to selectively bind and immobilize the targeted hapten/antigen, and a second specific antibody labeled with a reporter group is used to detect the complex.
  • reporter groups which can be used to label the reporter antibody include but are not limited to fluorophores, chromophores, enzymes, chemiluminescent moities, biotin/avidin reporter complexes, gold particles, nanobeads, magnetic beads, and radioisotopes. Additionally, an important aspect of this invention involves the use of the electronic properties of the microelectronic array for:
  • Antibodies can easily be attached to p-RNA molecules by a number of covalent and non-covalent methods.
  • Example 7 of this invention describes a procedure where complementary p-RNA constructs were used as immobilization tethers for a protein conjugate consisting of Streptavidin and a goat anti-human F(ab') 2 antibody.
  • p-RNA No. 81 was used to provide a capture sequence for p-RNA No. 80 by binding the biotin of p- RNA No. 81 to Streptavidin which had been immobilized in the permeation layer covering a microelectronic array (APEX chip). The biotin of p-RNA No.
  • p-RNA #54 was immobilized to a permeation layer overlaying the microelectronic array by binding its 4' biotin to immobilized Streptavidin in the permeation layer.
  • p-RNA #79 was hybridized to its complementary strand #54 and the 4' biotin of #79 was used to immobilize the Streptavidin half of a solubilized conjugate of Streptavidin and goat anti-human F(ab') antibody.
  • the goat anti- human F(ab')2 antibody was then used as an immunosorbent to capture its target antigen which is human IgG F(ab') 2 antibody.
  • the immunological reagents from Example 3 Using the complementary pairs of p-RNA sequences from Example 3, the immunological reagents from Example 3, and including a second protein conjugate consisting of Streptavidin chemically coupled to a murine monoclonal anti-subunit of human Chorionic Gonadotropin a simultaneous immunological detection of two different antigens was accomplished.
  • the p-RNA capture strands successfully differentiated between their respective complementary strands such that the two antigen targets were differentially detected by the appropriate p-RNA capture strands.
  • Solid support derivatives derived from DMT-protected pyranosyl-nucleoside (A,T,G,C) precursers free at the 2'-position and benzoylated at the 3 '-position were used for the p- RNA synthesis.
  • CPG solid support materials were also used in carrying out the standard phosphoramidite DNA synthesis and for inco ⁇ orating the tryptamine ribopyranosyl (I) phosphoramidite monomer.
  • the synthesis protocol included the following steps: (1) DMT deblocking was carried out using 6% dichloroacetic acid (v/v) in dichloromethane (100 ml); (2) washing with dichloromethane (20ml), washing with acetonitrile (20ml), and flushing with argon; (3) coupling procedure involved first washing the CPG solid support material with the activator (0,5 M pyridinium hydrochloride in dichloromethane (0,2ml), then 30 minutes treatment with 1/1- activator (0,76ml of the phosphoramidites (8 eq; 0,1 M dissolved in acetonitrile); (4) washing with acetonitrile (20ml); (5) the capping procedure involved a 2 minute treatment with 50% Cap A (10,5ml) and 50% Cap B (10,5ml) regents from PerSeptive (Cap A: THF, lutidine, acetic-anhydride, Cap B: 1-methylimidazole, THF, pyridine); (6) washing with
  • the p-RNA-oligonucleotide is first allyl- deprotected at the phosphotriester linkages and at the guanine bases under the conditions described by Noyori and coworkers. (Y. Hayakawa, S. Wakabayashi, H. Kato, R. Noyori, J. Am. Chem. 1990, 112, 1691). This is carried out by suspending the support in a mixture of 272 mg of Pd(PPh 3 ) 4 , 272 mg of PPh 3 and 272 mg of Et 2 NH 2 HCO 3 in 15 ml of dichloromethane at room temperature. The suspension is vigorously shaken for 4 to 5 hours.
  • the support is then carefully washed with dichloromethane (30 ml), acetone (30 ml) and water (30 ml) suspended for 30 minutes in a 0,1M solution of sodium diethyldithiocarbamate in water, and washed again with water (15 ml), acetone (15ml) and dichloromethane (15 ml).
  • the cleavage from the solid support and the deacylation of the bases and sugars was effected by hydrazinolysis at 4°C within 25-40 hours (25% hydrazine hydrate in water, 6ml).
  • Hydrazine is removed from the crade oligonucleotide by desalting over a Sep-Pak-cartridge (elution with acetonitrile/triethyl-ammonium- hydrogencarbonate 0,1M). The oligonucleotide containing fractions were combined and evaporated to dryness.
  • the combined product fractions were evaporated to dryness and then dissolved in a 0,1 M triethyl-ammonium-hydrogencarbonate and desalted over a Sep-Pak-C18 (Waters) cartridge.
  • the eluted product was evaporated in vacuum, once dissolved with 2 ml of water and re-evaporated to dryness and then dissolved in 1 ml of water for the determination of the optical density.
  • the oligonucleotide was injected on an analytical RP18 column. (>95%).
  • the product was characterized and identified by ESI-MS.
  • Example 2 Procedure for Fluorescent Dye Labeling of p-RNA at 2 '-Terminus The following gives the basic procedure for inco ⁇ orating a fluorescent dye into the 2'-terminal position of a p-RNA sequence. This provides just one of the many options for inco ⁇ oration of reporter labels and other moieties into the p-RNA sequence.
  • 3 '-Amino-Modifier C3 CPG purchased by Glenn Research
  • 3 '-Amino-Modifier C3 CPG purchased by Glenn Research
  • the Fmoc protection group on the 3 '-amino moiety was removed by a 10-minute treatment with 3.5 ml of DMF/Piperidine (6/4).
  • the procedure was repeated a second time.
  • the deprotected 3'- amino-C3-CPG-support material was then washed with 10 ml of DMF and 10 ml Acetonitrile.
  • the 3'-amino-C3-CPG-support material was then dried under high vacuum for 10 minutes.
  • the Texas Red dye reacted 3'-amino-C3 CPG support material was then washed with 10 ml of DMF/Pyridine.
  • the reacted material had a blue color and red fluorescence.
  • the remaining free amino groups on the modified CPG were capped with 0.4 ml acetic anhydride (with 0.2 g DMAP) in 2 ml Pyridine for 30 minutes.
  • the dye modified CPG support was then washed with Pyridine, DMF, Methanol and Methyl-t-Butyl-Ether, and then dried under high vacuum.
  • the Texas Red labeled 3'- Amino-C3 CPG support was then ready for the synthesis of a p-RNA oligonucleotide sequence using standard phosphoramide synthesis procedure after deblocking the DMT protection group.
  • This 2'-labelling procedure can be used for those fluorescent dyes which are stable through the remaining p-RNA synthesis procedure.
  • Figure 20 The mass spectrum for the TR-90 molecule is also shown in Figure 20.
  • the structure of the TR90 sequence (4'-I-G-A-A-G-G-G-TR-2') hybridized with the complementary biotinylated B-92 sequence (4'-Biotin-C-C-C-T-T-C-T-I-C-C-C- C-C-G-2') is shown in Figure 21.
  • the UV hypochromicity curves for the thermal denaturation of the hybridized pairs are also shown in Figure 21. This hybridization was carried out using 5 ⁇ M concentrations of each of the p-RNA sequence in 0.01 M Tris/HCL pH 7/0.15 M NaCl 2 . The UV hypochromicity results shows the Tm for the hybridized pair was about 59 °C.
  • the following example is the procedure that would be used to attach a peptide moiety to a p-RNA oligomer containing a tryptamine group in place of a normal base moiety. This procedure first involves the inco ⁇ oration of a tryptamine nucleoside into the p-RNA sequence, which then provides an aliphatic amine group for the subsequent coupling of the peptide to the p-RNA.
  • the product was desalted and further purified by standard work-up procedures on a Sep PakTM cartridge.
  • the solution was poured over an activated Sep PakTM cartridge, washed with 20 ml 0.1 M TEAB buffer solution and eluted with pure acetonitrile.
  • the product yield was determined by UV abso ⁇ tion at 260 nm, and then the product was lyophilyzed to dryness using a vacuum centrifuge.
  • the iodacetylated p-RNA was dissolved in a buffer system containing Borate-HCL buffer and Na 2 EDTA (pH 7.6; 100 ⁇ l per 10 nmol oligo) and mixed with 30-60 equivalents of the peptide in 100 ⁇ l DMF.
  • Buffer system Borax/HCl-buffer; Riedel-de Haen, pH 8.0, mixed with an equal amount of a 10 mM solution of Na 2 EDTA in water, the pH was adjusted to 7.6 with HC1, the resulting solution contained ⁇ 5 mM Na 2 EDTA).
  • the reaction was carried out in the dark, at ambient temperature (25°C), for approximately 6- 12 hours. The completion of reaction was monitored by HPLC.
  • the HPLC buffer system was buffer A 0.1M triethylammonium acetate in water, and buffer B 0.1M triethylammonium acetate/CH 3 CN in water at a 1 to 4 ratio. Elution was started with 90% buffer A/10% buffer B, and proceeded to 50% buffer A/50% buffer B by 40 minutes; the elution was monitored at 260nm and 220nm, the HPLC column material used was Merck lO ⁇ M LiChrosphere TM100 RP-18 in a 250mm by 4mm column. After the iodacetylated oligomer had disappeared, the final product was isolated by the same HPLC procedure and desalted using the above Sep PakTM cartridge procedure.
  • p-RNA-peptide intermolecular architectures can be created by modification of p-RNA with multiple tryptamine linker sites within the same p-RNA oligonucleotide structure.
  • An example of one such p-RNA sequence with multiple-tryptamine linkers (4'- C-C-I-I-I-G-G-2') that has been synthesized and characterized is shown in Figure 23.
  • the UV hypochromicity and melting characteristics for the hybridized pair is also shown in Figure 23.
  • the melting temperatures (Tm) and thermodynamic properties for other hybridized pairs of p-RNA's with one, two and three tryptamine linkers are given in Table 1 below. TABLE 1
  • the solution hybridization properties for the sequences in Table 1 showed the desired cooperative behaviors, including Tm, melting curve, and thermodynamic properties. Additionally, the sequences were characterized by mass spectroscopy (see Table 2). Those examples demonstrate that two or three juxtaposed tryptamine linkers in a short p-RNA oligomer sequence still exhibit appropriate hybridization properties.
  • FIG. 24 An example of a multiple tryptamine p-RNA-peptide conjugate (4'-CCC-I*-I*- GGG-2' with *two-Cys-Phe-Pro-Tyr-T ⁇ -Gly peptides) is shown in Figure 24.
  • the NH 2 - Cys-Phe-Pro-Tyr-T ⁇ -Gly-CO H peptides were linked through the thiol group of the cysteine amino acid to the primary amino group of tryptamine via a heterobifunctional linker (succinimidyl ester of iodoacetic acid).
  • the mass spectrometric analysis of the p- RNA -peptide stracture is also shown in Figure 24, and in Table 3 below.
  • the double tryptamine p-RNA architecture allows four peptides residues to be inco ⁇ orated into one supramolecular structure when two p-RNA peptide sequences hybridize to the complementary capture p-RNA peptide sequence (this is shown in Figure 25).
  • the UV hypochromicity curves show the melting (T m ) of the double-stranded p- RNA-peptide stracture is around 50 °C. This is consistent with the predicated T m for these p-RNA sequences.
  • the fact that hybridization occurs in the presence of the large and bulky peptide stractures indicates that the tryptamine linker arrangement produces favorable stereochemistry for attachment of peptides. Additionally, measurement of the tryptophan fluorescence could be used to follow the formation of the p-RNA hybrids in solution.
  • This experiment involves the use of electronic stringency to determine the electronic T m for combinations of the p-RNA sequences #70:#72, #71:#72 and #71 :#72.
  • the sequences of the p-RNA's are shown below:
  • the #70 sequence is complementary to the 4' end of the #72 sequence
  • the #71 sequence is complementary to the 2' end of the #72 sequence.
  • the 4'-terminal (I) of the #70 sequence and the 2 '-terminal (I) of the #71 sequence also remain unpaired.
  • the Tryptamine (I) moieties are the points of attachment for adding peptide sequences or other ligand binding structures).
  • the thermal T m for the hybridized combination of the p-RNA sequences #70:#72 was 49° C; the #71 :#72 was 49° C; and fully paired structure #70/#71 :#72 was 49° C.
  • the electronic T m s for hybridized combination of the p-RNA sequences #70:#72 was 230 nanoamperes (nA); the #71:#72 was 215 nA; and #71:#72 was 250 nA.
  • the capture p-RNA structures used in this experiment were derived from p-RNA sequence #72 (see Example), and included a #72 p-RNA capture sequence with no peptide attached, the Cap-72-NP component, a # 72 p-RNA capture sequence with the hexapeptide N-Cys-His-His-His-His-Gly-C (-CHHHHG) attached, the Cap-72-CHHHHG component, and a # 72 p-RNA capture sequence with the hexapeptide N-Cys-Phe-Pro-Ser-Phe-Gly-C (-CFPSFG) attached, the Cap-72-CFPSFG component.
  • the #71 p-RNA sequence was derivatized with the hexapeptide N-Cys-His-His-His-His- Gly-C (-CHHHHG) and also with the fluorescent dye (Cy3) to form the Cy3-71- CHHHHG component.
  • the #70 p-RNA sequence was derivatized with the hexapeptide N- Cys-His-His-His-His-His-Gly-C (-CHHHHG) to form the 70-CHHHHG component.
  • An APEX chip with 10,000 sites using individual site current control was coated with an agarose:streptavidin permeation layer. 300 sites were simultaneously electronically biased with a positive potential and a current of 100 nanoamperes (nA). A solution containing 1 M of p-RNA #92 was overlayed over the biased sites and the p- RNA immobilized by binding between the permeation layer immobilized Streptavidin and the 4'-biotin.
  • the solution containing p-RNA #92 was removed and replaced with a solution containing 1 M of p-RNA #72 and an additional 100 sites were electronically biased with a positive potential at a current of 100 nanoamperes and the p-RNA immobilized by the binding of the 4 '-biotin to the immobilized Streptavidin.
  • the solution containing the p-RNA #72 was removed and replaced by a solution containing 1 M o f p- RNA #91 which had been labeled with the fluorescent dye Cyanine3.
  • the 400 sites represented by the previous two p-RNA binding events were electronically biased with a positive potential at a current of 100 nanoamperes and the mobile p-RNA oligmer, Cy3 labeled #91, was allowed to hybridize for 30 seconds with the immobilized capture strands, #92 and #72.
  • the fluid containing p-RNA #91 was removed and the chip washed to remove residual fluid and then imaged on a fluorimeter.
  • the resulting image demonstrated that the p-RNA oligmer #91 hybridized only to its matching complementary strand #92 and not to the non-complementary strand #72. This demonstrates moderate scale electronically mediated multi-site hybridization with hybridization mediated strand discrimination (see Figure 14).
  • Biotin- A-I-T-G-C-C-T-A-2' p-RNA #81 was used to provide a capture sequence for p-RNA #80 by binding the biotin of p-RNA #81 to Streptavidin which had been immobilized in the permeation layer covering an APEX chip. The biotin of p-RNA #80 was then used to bind to a mobile streptavidin which had been chemically conjugated to a goat anti-human F(ab') 2 antibody. The goat anti-human F(ab') 2 antibody was used to capture its specific antigen target which is human IgG F(ab') 2 antibody.
  • p-RNA #54 was immobilized to a permeation layer overlaying an APEX chip by binding its 4' biotin to Streptavidin which was immobilized in the permeation layer.
  • p-RNA #79 was hybridized to its complementary strand #54 and the 4' biotin of #79 was used to immobilize the Streptavidin half of a solubilized conjugate of Streptavidin and goat anti-human F(ab') 2 antibody.
  • the goat anti-human F(ab')2 antibody was then used as an immunosorbent to capture it's target antigen which is human IgG F(ab') 2 antibody.
  • Example 8 Demonstration of Novel Methods for Achieving A Simultaneous Multiple Homogeneous Assays Combined with Discrete Analyte Detection
  • Example 7 Using the complementary pairs of p-RNA sequences from Example 7, the immunological reagents from Example 3, and including a second protein conjugate consisting of Streptavidin chemically coupled to a murine monoclonal anti-subunit of human Chorionic Gonadotropin, a simultaneous immunological detection of two different antigens was accomplished.
  • the p-RNA capture strands successfully differentiated between their respective complementary strands such that the two antigen targets were differentially detected by the appropriate p-RNA capture strands.
  • a sandwich format detection may be utilized, such as where a fluorescently labeled antigen interacts with the antibody.
  • Detection may be by direct detection, e.g., electrical, optical or other direct detection, or by a sandwich format, such as through use of another fluorescently labeled antibody.
  • Example 9 Demonstration of an Analysis Algorithm for Deconvolution of Data from Targeting with a Sublibrary
  • the following is a description of the type of algorithms that will be used to deconvolute and analyze the large amount of data that would come off a ELOC combinatorial arrays after a drag/ligand binding experiment. This type of analysis will be important for determining the molecular descriptor properties of the ELOC arrays. Malinowski and Howery (E. R. Malinowski and D.G.
  • Any set of data that can be represented as a matrix can be treated by factor analysis (P. M. Fredericks, J. B. Lee, P. R. Osbom and D. A. J. Swinkels Materials Characterization using FT-IR Spectra. Part 2: Mathematical and Statistical Considerations, Applied Spectroscopy, 39, 2 (1989), p. 311).
  • Such data can be treated as a matrix where each column contains the value measured of one set (intensities against time, voltage concentration etc.).
  • a data matrix containing variables of the conditions (temperature, pH, ionic strength, stringency, etc.) coming from 30 moments or snapshots of the detected intensities at 10,000 test sites (on the combinatorial chip/array) throughout the experiment, would be a dynamic representation of signals evolving through 30 moments in time, temperature or any other changing parameter.
  • the intensity measurements may be regarded in first order as a representation of molecules found in the optical focus, the use of a linear correlation model proves very useful for cluster analysis of similar chip events per position as well as for a noise reduction and a reconstruction (recombination) of idealized signal evolution and signal correlation in reasonable time for the large data sets created.
  • the factor scores are the weighting factor attributed to each factors (1 to 30). Factors with contributions below the signal to noise ratio may be regarded as non- correlating noise without substantial physical or experimental meaning. Chip positions however, that show similar events (in shape or intensity) will show similar contribution in their major factors. A plot of the first two factor columns (variable loading matrix) often yields sufficient information to identify the position similarities with respect to the event.
  • Figure 26 shows a simulation of a typical measurement starting from a normalized intensity at three positions of a set of 30 points (due to 30 conditions, time, temperature, stringency etc. here normalized to a total of 1) superimposed with typical noise.
  • Figure 27 shows the underlying idealized signal development.
  • This procedure can be used with pretreated data to look for similarities in binding, clustering of consensus peptide sequences and the possibility to identify peptide sequences that correspond to the same set of subunits but distributed differently with respect to the immobilized species and the soluble subunits.

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Abstract

L'invention concerne des dispositifs à réseaux de descripteurs moléculaires microélectroniques, des méthodes, des procédures et des formats de sélection combinatoire de structures de fixation de ligands intermoléculaires et de criblage de médicaments. Plus particulièrement, ces dispositifs et ces méthodes mettent en application rapidement une sélectivité d'ordre supérieur de constituants de fixation de ligands intermoléculaires, de structures supramoléculaires et de complexes supramoléculaires produits de manière combinatoire par application de paramètres de stringence uniques. De préférence, l'invention consiste en la formation de banques exponentielles par agrégation de sous-banques par l'intermédiaire de l'effet de stringence électronique pour influer sur la formation ou la détection de structures ou de complexes supramoléculaires. De plus, cette invention concerne des dispositifs à réseaux microélectroniques, des procédures, des méthodes et des formats pour des processus de reconnaissance moléculaire, la découverte de nouveaux médicaments, la production de nouveaux réactifs à affinité, la production d'anticorps de synthèse et pour des immunodosages.
EP00955293A 1999-08-13 2000-07-28 Dispositifs a reseaux de descripteurs moleculaires microelectroniques, methodes, procedures et formats de selection combinatoire de structures de fixation de ligands intermoleculaires et de criblage de medicaments Withdrawn EP1210607A4 (fr)

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DE19950969A1 (de) * 1999-10-22 2001-05-10 Aventis Res & Tech Gmbh & Co Doppelstrang-Nukleinsäure-Sonden und deren Verwendung
DE10040289A1 (de) * 2000-08-17 2002-02-28 Aventis Res & Tech Gmbh & Co Verfahren zur Herstellung und Ermittlung geeigneter Effektoren von Zielmolekülen mit Substanzbibliotheken
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WO2002075312A1 (fr) * 2001-03-19 2002-09-26 Gyros Ab Caracterisation de variables de reaction
US20040161741A1 (en) 2001-06-30 2004-08-19 Elazar Rabani Novel compositions and processes for analyte detection, quantification and amplification
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WO2008016680A1 (fr) * 2006-08-02 2008-02-07 California Institute Of Technology Procédés et systèmes destinés à détecter et/ou à trier des cibles
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