WO2002083951A1 - Dosage de proteine/ligand de fixation multiplexes avec marques a proteine pna - Google Patents

Dosage de proteine/ligand de fixation multiplexes avec marques a proteine pna Download PDF

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WO2002083951A1
WO2002083951A1 PCT/US2002/011411 US0211411W WO02083951A1 WO 2002083951 A1 WO2002083951 A1 WO 2002083951A1 US 0211411 W US0211411 W US 0211411W WO 02083951 A1 WO02083951 A1 WO 02083951A1
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ligand
protein
nucleic acid
peptide nucleic
species
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Roger W. Giese
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Northeastern University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the field of the invention is detection of binding between proteins and ligands for the purpose of ligand or protein discovery and/or quantitation.
  • MS Mass spectrometry
  • the invention is directed to multiplexed methods for discovering and quantifying ligands, proteins and ligand-protein interactions and to novel peptide nucleic acid-tagged ligands for carrying out such methods .
  • the methods of the invention provide the advantages of ultra-multiplicity, such as testing an entire combinatorial synthesis library all at once against an entire proteome; practicality, since unlabeled proteins can be tested; and ultra-sensitivity, in that a proteome from a single cell can be studied.
  • the methods of the invention make possible advances in protein chips and in multiplexing competitive and noncompetitive ligand assays that use known proteins as binding reagents. They also enable ligand-based sandwich assays of protein analytes.
  • one or more ligand candidates preferably from a combinatorial library and each labeled with a unique peptide nucleic acid (PNA) , are combined simultaneously with one or more proteins, and complementary ligand/protein combinations are discovered.
  • PNAs are advantageous as labeling entities because of their small size, high coding properties, ease of detection by means of hybridization and opportunity for ultrasensitivity based on detection by a polymerase chain reaction (PCR) or related nucleic acid amplification reaction.
  • complementary (positive) PNA-ligand/protein complexes can be separated by ultrafiltration from nonbonding (negative) PNA-labeled ligands, followed by dissociation of the positive complexes, and then hybridization-based detection of the released positive PNA-ligand species on an array of PNA or DNA spots on a solid surface can be carried out.
  • one or more PNA molecules are used as labels in a multiplexed competitive or noncompetitive ligand assay in which known binding proteins ' are employed to quantify known analytes.
  • a second label such as biotin, a common PNA sequence, or a fluorescent tag is employed as well.
  • detection takes place via hybridization of the PNA labels onto an array of complimentary PNA or DNA spots on a solid surface.
  • the methods of the invention bring together, for the first time in the field of proteomics, the techniques of combinatorial synthesis, peptide nucleic acid coding, detection signaling, ultrafiltration separation, PNA (or DNA) detection assays, multiplexed ligand assays and detection of nonlabeled proteins. Furthermore, the invention gives rise to technology that can be used for simultaneous multiplexed ligand/multiplexed protein discovery and also ligand and protein quantitation.
  • Fig. 1 depicts a synthetic scheme for the preparation of ligand-PNA-signal (LPS) species
  • Fig. 2A depicts a scheme for ligand/protein discovery or protein quantitation. After LPS 2 is removed by ultrafiltration, protein/LPSi is dissociated, and LPSi is detected via a hybridization reaction; Fig. 2B depicts a scheme for detecting positive LPS species by PCR (continuation of step 3b in Figure 2A) ; and
  • LPS ligand-PNA-signal
  • PNA peptide nucleic acid
  • S signal group
  • B biotin or biotin-analog
  • Protein/LPS complexes then form between complementary proteins and ligands, which can be referred to as "positive” proteins and "positive” LPS species for each pair. These complexes are then separated from non-complexed LPS species, those that have failed to find a complementary protein partner, and dissociated. Decoding of each positive LPS species enables the identity of its ligand part to be revealed.
  • PNAs Peptide nucleic acids
  • PNAs are DNA mimics in which the backbone, which is substituted with DNA nucleobases, is composed of N- (2-aminoethyl) glycine rather than sugar-phosphate units.
  • PNAs hybridize to complementary DNA and RNA molecules, and to each other. Usually, the hybridization products are stronger with PNAs. Since PNAs can be synthesized on the same instruments that are used for automated synthesis of peptides, and PNA synthons (monomers for the synthesis), for this purpose are available commercially, PNAs are readily available. Compounds have been synthesized in this way which are part peptide and part PNA (Bau, S.
  • glycine spacers can be introduced at positions in the LPS adjacent to the PNA or functional group.
  • serine rather than glycine spacers can be used. Lysine, aspartic acid, glutamic acid and related residues can be employed to establish branch points.
  • LPS species for use in methods of the invention can be prepared as shown in Fig. 1.
  • the starting compound, W-Lys- X 10 is prepared by a procedure similar to that described in Basu, S., and Wickstrom, E., Synthesis and Characterization of a Peptide Nucleic Acid Conjugated to a D-Peptide Analog of Insulinlike Growth Factor 1 for Increased Cellular Uptake, Bioconjugate Chem.
  • the biotinylated lysine synthon can be prepared by reacting N ⁇ -Fmoc-N ⁇ -Boc-lysine (Sigma) with biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma) , and then converting the product, after acidic removal of the Boc group, to an active ester via an activator such as 0- (benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate .
  • an activator such as 0- (benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate .
  • N-hydroxysuccinimide-Y- maleimide 12 which can be prepared by reacting ⁇ -maleimidocaproic acid NHS ester (Sigma, St. Louis, MO) with ⁇ -aminocaproic acid followed by reaction with N-hydroxysuccinimide in the presence of N, N-dicylohexylcarbodiimide, to form Z-maleimide 14, where Z is the combination of W-Lys-X with Y. Reaction of each Z-maleimide compound (having a unique PNA sequence) with a unique ligand that bears a sulfhydryl (thiol) group yields a series of LPS species 16.
  • Such thiol-ligands can be obtained from combinatorial synthesis products as described (MacBeath, G., Koehler, A. N., Schreiber, S. L., Printing Small Molecules as Microarrays and Detecting Protein-Ligand Interactions en Masse, J. Am. Chem. Soc, 121, 7967-7968, 1999) .
  • the lysine side chain of W-Lys-X can be capped with acetic anhydride, forming W-N-acetyl-Lys-X, and the carboxyl group of W then can be coupled to ethylenediamine followed by reaction with ⁇ -maleimidocaproic acid NHS ester.
  • the LPS products in this case could be substituted at opposite ends with biotin and a thiol ligand. If combinatorial ligands are available with amino functional groups, the carboxyl end of W-N-acetyl-Lys-X can be activated with a carbodiimide for coupling to such ligands .
  • the carboxyl end of W-Lys-X can be capped by similar activation in the presence of excess ethanolamine, and then the uncapped lysine can be reacted with succinic anhydride followed by carbodiimide activation of the resulting new carboxyl moiety for reaction with ligands containing a primary a ine functional group.
  • Reaction of the lysine side chain of W-Lys-X with S-acetylthioacetate followed by hydroxylamine would provide a thiol group at this site for reaction with combinatorial ligands bearing a maleimide functional group.
  • Combinatorial ligands with carboxyl functional groups e.g., Tan, D. S., Foley, M.
  • W-Lys-X species are highly versatile molecules, which one can subject to the great diversity of peptide and protein conjugation and cross-linking reactions as known in the art (e.g., G. T. Hermanson, Bioconjugate Techniques,- Academic Press, San Diego, 1996) to fabricate a diversity of LPS species .
  • Biotin analogs such as haptens (e.g., digoxin, digoxigenin, peptides) that are recognized by antibodies can be used instead of biotin, as can a common PNA sequence that every LPS species would contain in addition to its unique PNA sequence.
  • the resin-attached precursor of W-Lys-X in which the free end terminates in a primary amino group, can be reacted with N-succinimidyl-3- (2- pyridyldithio) propionate instead of succinic anhydride.
  • the resulting pyridyldithiopropyl products can be array-spotted onto a thiol-substituted solid surface, such as thiol-substituted resin beads .
  • the residual thiol groups on the surface can be blocked with iodoacetamide, and then ligands can be coupled through various linkages onto the lysine group of the immobilized PNAs.
  • the resulting surface-attached LPS species can be released from the surface by dithiothreitol followed by blocking of the thiol group on these released species with iodoacetamide .
  • Solid phase reactions can be convenient since residual reagents from the reactions can be removed by washing.
  • a collection of pre-assembled molecules, each containing a carboxyl group, a biotin, and a unique ligand can be attached to a resin-attached PNA precursor bearing a free amino group by activating the carboxyl group of the pre-assembled molecules with carbodiimide, or by forming a related active ester, for coupling onto the precursor PNA followed by release of the product from the resin.
  • These pre-assembled molecules can be synthesized as follows.
  • N ⁇ -Boc-lysine (Sigma) is reacted with biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma) , the Boc group is removed under acidic conditions, and the product is reacted with ⁇ - maleimidocaproic acid NHS ester (Sigma) followed by reaction with thiol-substituted ligands.
  • These kinds of compounds can also be prepared by solid phase synthesis.
  • Combinatorial ligands with carboxyl functional groups can be reacted directly with N -Boc- lysine, and then, after Boc removal, biotin can be attached.
  • LPSi 22 and LPS 2 24 LPS species 22 and LPS 2 24 are present.
  • LPS is shown as a PNA molecule with a ligand, and biotin as a signal species, attached to the same end of the PNA, but other configurations can be used.
  • the ligand and biotin moieties can be attached to opposite ends of a PNA molecule, for example, by using the dual-labeling strategy reported elsewhere (Liu, X., Balasubramanian, S., Strategies for the synthesis of fluorescently labeled PNA, Tetrahedron Letters, 41, 6153-6156, 2000) .
  • step 2a LPSi 22 and LPS 2 24, prepared as described earlier, are exposed to a collection of proteins, where LPSi alone is able to form a complex.
  • step 2a the reaction mixture is exposed to a separation step, e.g., ultrafiltration separation, where protein/LPSi is retained because of the large size of the protein, whereas LPS 2 24, since it is an unbound small molecule, passes through the membrane.
  • a separation step e.g., ultrafiltration separation
  • dissociating conditions are applied to the membrane-retained protein/LPSi complex so that further ultrafiltration, in step 2b, brings LPSi 22 through the membrane, leaving protein behind.
  • step 3 there are two options for step 3, in which the positive LPS species (LPSi 22) is detected.
  • LPSi 22 hybridizes onto a sequence-complementary DNA (or PNA) arrayed spot on a solid surface.
  • a complementary DNA (or PNA) spot on the spot- arrayed surface for every LPS that is combined with the original protein sample.
  • SAv streptavidin
  • a streptavidin analog such as avidin or neutravidin may also be used.
  • the specific location on the arrayed solid surface where the signal accumulates, and the known DNA (or PNA) oligomer at each site initially establishes the identity of the LPSi species in terms of its PNA part.
  • this information allows reverse tracing of the synthetic history of LPSi, leading to the identification of the ligand part.
  • Such reverse tracing of synthetic history to learn the structures of the compounds that test positive in a ligand assay is a standard procedure in many combinatorial synthesis techniques. More detail concerning the procedure of Fig. 2A is provided in Example 1.
  • the LPSi 22 can be complexed via its biotin moiety onto a solid surface containing streptavidin or streptavidin-analog.
  • the PNA code is detected via binding of a complementary double-stranded DNA probe, which, in turn, via a polymerase chain reaction (PCR) (steps 3c, d) , yields complementary coded amplicons.
  • PCR polymerase chain reaction
  • step 3c the PCR reaction begins with the application of heat to dissociate the target DNA molecules to single strands, and then standard PCR amplification conditions in step 3d yield products called amplicons.
  • one of the PCR primers is labeled with biotin, so one- half of each amplicon duplex product is biotin-labeled.
  • biotin-labeled products are referred to as PCR Amplicons in Fig. 2B.
  • the biotin- labeled strands of these amplicons can be specifically hybridized onto a complementary array of PNA (or DNA) spots on a solid surface followed by detection with signal-labeled streptavidin (step 3e) .
  • PNA spots are advantageous because they can provide intense binding for the biotin-labeled strand of the amplicons.
  • the now-discovered ligands of the combinatorial synthesis library can be scaled up in step 4 for preparation of affinity columns to isolate sufficient amounts of corresponding positive proteins for identification as by mass spectrometry. While detection via step 3b is more complex than detection by step 3a, the 3b option, via the PCR reaction, provides ultrasensitivity, which can be useful, for example, to study the proteome from a single biological cell, or from a relatively small number of such cells.
  • the method of the invention can also be practiced using techniques other than ultrafiltration to separate protein/ligand complexes from residual, unbound ligands.
  • chromatographic techniques such as size exclusion, ion exchange, immobilized metal, reversed-phase and hydrophobic interaction chromatography can be used in addition to ultrafiltration, as can electrophoretic techniques such as .zone electrophoresis and isoelectric focusing, including capillary electrophoresis and capillary electrochromatography forms of these techniques.
  • Isopycnic centrifugation can also be used by preparing LPS species that have a different buoyant density from that of proteins so that free and protein-complexed LPS species give rise to different bands upon centrifugation.
  • alkyl-substitution of LPS species can reduce their buoyant density while iodo-substitution, e.g., with diatrizoic acid, can increase their buoyant density.
  • proteins tend to have the same buoyant density aside from special classes such as lipoproteins .
  • PNA molecules are particularly attractive as the coding labels in the assay technique of the invention for several reasons.
  • the coding ability of PNA molecules is high.
  • a 10-mer offers more than one million different codes.
  • these species are made up of noncharged, moderately polar substituents in a linear configuration, they can have good solubility in water and exhibit minimal nonspecific binding properties .
  • DNA labels would make poor substitutes for PNAs since the intense charge of DNA labels would tend to cause much nonspecific binding in a multiplexed ligand/protein assay.
  • the peptide backbone of the PNA species provides high chemical and physical stability. These molecules are easy to synthesize and decorate with substituents in various geometries to create a variety of LPS species. A large number of LPS species, for example, more than 100, or even more than 10000, can be employed together for the simultaneous detection of an equivalent number of complementary proteins .
  • Some of the proteins of proteomes are present in very small numbers in cells.
  • the small numbers generally reflect their special importance in the cell. These proteins are particularly difficult to discover with prior technology due to the sensitivity challenge.
  • the methods of the invention particularly in the PCR mode, open up a new way to reach the detection of such special proteins .
  • ligand-based chips for measuring proteins in which many spots of ligands with known specificity are set up for capture of their complementary proteins. This includes the option of sandwiching a given protein between two ligands, one of which is surface attached, and the second of which bears a signal group for detection purposes.
  • the other sandwich-binding partner instead of a second ligand, can be a labeled antibody or antibody fragment. The small size of the ligands can help to minimize nonspecific binding.
  • bound proteins are detected, e.g., by staining with dyes, or by covalent reaction with biotin NHS ester followed by streptavidin signal labeling.
  • the protein samples are labeled before exposure to a protein chip.
  • the bound proteins in the non- sandwich case can also be detected by mass spectrometry, e.g., by adding matrix and carrying out MALDI-TOF-MS analysis.
  • adsorbed protein spots can be digested with trypsin to peptides before analysis by MS.
  • binding proteins such as antibodies, antibody fragments, phage display products and receptors are used in competitive and noncompetitive ligand assays to quantify analytes such as drugs, hormones, metabolites, toxins and other proteins.
  • Multiplexing is achieved by using PNAs as the labels in these assays, attached either to ligands that compete with the binding proteins, or attached to the binding proteins, depending on the assay principle.
  • PNAs as the labels in these assays, attached either to ligands that compete with the binding proteins, or attached to the binding proteins, depending on the assay principle.
  • such assays can be divided into at least three types (I, II, and III) .
  • a type IV assay in which a protein analyte is detected via sandwich binding between a ligand and a binding protein or between two ligands .
  • one or more analytes are quantified together in a sample by adding, for each analyte, a distinctive, complementary protein and an LPS species consisting of a distinctive peptide nucleic acid part covalently attached to a distinctive ligand part L and a signal part S.
  • a distinctive, complementary protein and an LPS species consisting of a distinctive peptide nucleic acid part covalently attached to a distinctive ligand part L and a signal part S.
  • Each target analyte and corresponding LPS compete for binding to their complementary protein.
  • the corresponding protein-bound form of LPS is decreased and the free form of LPS increases .
  • Either form of the LPS can be measured to quantify the amount of the corresponding analyte.
  • the free and bound forms of LPS are separated before this measurement.
  • Detection takes place via hybridization of the free or bound form of LPS onto an array of PNA or DNA spots on a solid surface, based on the PNA and signal groups of the LPS tag.
  • one or more analytes are quantified together in a sample by adding, for each analyte, a distinctive complementary protein, usually in excess relative to its target analyte.
  • This protein previously was covalently labeled with a molecular species PS where P is a distinctive peptide nucleic acid and S is a signal group.
  • PS molecular species
  • the given protein binds its corresponding analyte, forming a soluble complex.
  • a solid-phase distinctive ligand substance then is added for each analyte to bind residual protein that has not formed a complex, and at least the PS part of each complex is detected by a hybridization reaction via binding to a complementary PNA or DNA spot on a solid surface.
  • this detection step it is preferred to cleave the PNA-signal from the binding protein (release PS from Ab) to enhance the detection of PS via hybridization.
  • This step can be implemented by incorporating a trypsin-cleavable group into the PNA (Weiler, J. , Gausepohl, H., Hauser, N. , Jensen 0. N., Hoheisel, J. D., Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays, Nucleic Acids Research, 25, 2792-2799, 1997) .
  • a sample containing one or more analytes of interest is combined with one or more analyte- complementary protein pairs, one pair for each analyte, wherein one member of each protein pair is PNA-labeled; at least one of the analytes becomes sandwiched between an analyte complementary protein pair, and at least one protein pair/analyte sandwich is detected via hybridization of its PNA part to an array of complementary PNA (or DNA) spots on a solid surface.
  • Analyte is necessary for the sandwich complex to form, and, in turn, this complex must exist in order for the signal group of this complex to localize on the surface at the DNA or PNA spot designated for a given analyte based on the distinctive PNA that is part of the complex for that analyte.
  • the type IV assay is a sandwich assay just like the type III assay, except that one or both of the pairs of binding partners for the analyte is a ligand. This restricts the type IV assay to protein analytes, since high affinity binding is necessary in a sandwich complex for quantifying analytes reliably, and ligand/ligand interactions usually would be too weak for quantification.
  • each protein analyte is complexed between a complementary signal-labeled ligand and a complementary PNA-labeled ligand or between a complementary protein and a complementary ligand, where the protein and the ligand are labeled with either signal or a PNA.
  • the sandwich complexes are detected via hybridization to a complementary PNA or DNA arrayed spot on a solid surface, based on the signal group of each complex.
  • a library of LPS species is obtained as shown in Fig. 1.
  • a proteome is obtained as described (Sreekumar, A., Nyati, M. K., Varambally, S., Barrett, T. R. , Ghosh, D. , Lawrence, T. S., Chinnaiyan, A. M., Profiling of Cancer Cells Using Protein Microarrays: Discovery of Novel Radiation-regulated Proteins, Cancer Res., 61, 7585-7593, 2001) or by related technique.
  • Fractions of this proteome ⁇ can be obtained by chro atography such as size exclusion or ion exchange chromatography, or by isoelectric focusing.
  • the proteome of a single cell can be obtained by pulling a cell of interest into a micropipet under a light microscope, and then lysing the cell hypotonically or with detergent.
  • the LPS library and proteome (or proteome fraction) are combined and incubated in buffer at neutral pH, and subjected to ultrafiltration (e.g., using an ultrafiltration membrane from Amicon or Millipore) by means of centrifugation, pressure or vacuum.
  • the retained, complementary protein/LPS pairs (containing positive LPS species) are subjected to washing steps with buffer to thoroughly remove noncomplexed LPS species.
  • Dissociation of the protein/LPS complexes is accomplished by adding a different solution such as high pH buffer, low pH buffer, aqueous-organic solvent mixture, aqueous detergent, aqueous chaotropes, aqueous urea or a combination of these.
  • the temperature also can be raised.
  • Ultrafiltration then yields the dissociated, positive LPS species in the ultrafiltrate.
  • this solution is then exposed to a solid surface containing an array of PNA or DNA spots such that there is a complementary coded spot for each species in the original LPS library. Nonbound LPS species are washed away with buffer or one of the above types of dissociation solutions.
  • Spots that contain complexed LPS species can be detected by adding, e.g., streptavidin or streptavidin analog labeled with a signal group such as an enzyme (e.g., alkaline phbsphatase) , fluorophore (e.g., phycoerythrin, Cy5) , or radioisotope (e.g., 125 I, 32 P) .
  • a signal group such as an enzyme (e.g., alkaline phbsphatase) , fluorophore (e.g., phycoerythrin, Cy5) , or radioisotope (e.g., 125 I, 32 P) .
  • LPS species obtained after ultrafiltration also can be identified in some cases by mass spectrometry. If the LPS species contains a reversible linkage (e.g., disulfide) between the ligand and the PNA, the PNA part can be dissociated and identified alone by mass spectrometry.
  • a reversible linkage e.g., disulfide
  • Antibodies are raised or obtained commercially against analytes of interest, or receptors for 'the analytes are obtained.
  • Each of the analytes of interest is conjugated with a unique PNA.
  • a ligand that cross-reacts with the antibody or receptor for a given analyte is conjugated with a PNA.
  • Each LPS species is referred to as a tracer, and the antibody or receptor as the binder.
  • a standard curve is set up for each analyte by incubating increasing amounts of the analyte as solutions in a series of containers with a fixed amount of complementary tracer and binder, wherein the amounts of the tracer, binder and analyte are similar (e.g., within a factor of 50).
  • the multiple tracers and corresponding multiple binders can then all be combined, and a multiplexed standard curve is obtained similarly via incubation of the cocktail of tracers and binders with the corresponding standard analytes of interest throughout a range of concentrations, to set up a corresponding collection of standard curves, one for each analyte.
  • the multiplexed ligand assay is then ready for the determination of the concentrations of a mixture of analytes, by using the standard curves to convert the amounts of the free tracers into the amounts of the unknown analytes .
  • the bound form of the tracers prevents the PNAs of such tracers from hybridizing to a complementary PNA or DNA arrayed spot on a solid surface, then this can be used to advantage since it means that the separation step for the free and bound tracers can be omitted prior to measuring the free tracers on a PNA or DNA array.
  • a library of solid-phase ligands (e.g., ligands on beads) is prepared as by combinatorial synthesis, or the library members of this synthesis are released from the solid surface and re-attached to a set of beads well suited for ligand assays such as magnetic particles form DYNAL, yielding so-called "tracer particles.”
  • ligand assays such as magnetic particles form DYNAL, yielding so-called "tracer particles.”
  • bioconjugation reagents e.g., G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996)
  • the technique for incorporating a trysin-cleavage site into a PNA Weiler, J. , Gausepohl, H., Hauser, N., Jensen 0.
  • PNAs are prepared having the composition maleimide-trypsin cleavage site- PNA-biotin, termed "MTPB,” and are conjugated, one-by-one, to a set of ligand/analyte binding proteins (such as antibodies, antibody fragments or receptors) to form PS-labeled products termed X binders," while keeping track of which MTBA is attached to which binder.
  • MTPB maleimide-trypsin cleavage site- PNA-biotin
  • An assay is set up by combining binders and analytes, and then later the tracer particles are added to remove uncomplexed binders, e.g., by magnetic separation, filtration or centrifugation.
  • the residual, dissolved analyte/binder complexes are measured by digestion with trypsin to release the PS moieties, which in turn are reacted onto an array of PNA or DNA spots on a surface followed by detection with a streptavidin-signal reagent.
  • complementary protein, protein pairs are set up (e.g., by preparing or buying antibodies, antibody fragments, receptors) that form a sandwich complex consisting of protein/analyte/protein.
  • a signal group such as biotin, an enzyme or a fluorophore is attached in a noninterfering way to one complementary protein, one for analyte pair, and a PNA is attached similarly to the other protein member.
  • a collection of these analytes then can be detected simultaneously by incubating with the complementary protein pairs, followed by exposure to an array of complementary PNA or DNA spots on a solid surface, washing and detection of the signal group.
  • a trypsin-releasable PNA can be used in this assay, so that PNA molecules released from the sandwich complexes are detected.
  • This assay is conducted in the same way as Example 4, except that the analytes are necessarily proteins and the pair of sandwich reagents for each protein are either a PNA-labeled ligand and signal-labeled binding protein, or a PNA-labeled ligand and a signal-labeled ligand, where the two sandwich reagents for each protein analyte do not compete in their binding to the analyte.
  • An assay can comprise a mixture of the two kinds of sandwich pairs, where each protein analyte is targeted by one pair or another.
  • the PNA-based ligand/protein binding assays of the invention have advantages both in their discovery and quantitation modes.
  • discovery mode complementary ligands and proteins are mutually discovered at the same time.
  • individual proteins must be isolated and labeled before they are ready for screening against natural products or combinatorial synthesis compounds for ligand discovery.
  • the large numbers of proteins, all of which are important, in all forms of life, make this approach to global ligand/protein discovery very onerous, to say the least.
  • the binding properties and stability of a protein may be degraded when it is labeled. Some proteins inherently are not very stable, and are especially difficult to label and purify. Also, relatively large quantities of a target protein may be needed with current technology.
  • the methods of the invention permit the replacement of this complex process by a scheme in which many proteins, even an entire proteome, can be combined directly, without any need for prior protein purification or labeling, in the solution phase with a candidate ligand library, based on the convenient preparation of ligand-PNA-signal (LPS) conjugates as a tag on the ligand.
  • LPS ligand-PNA-signal
  • Positive LPS species can be isolated by a step as simple as ultrafiltration. Ultrafiltration membranes are widely employed to remove small, unbound molecules from proteins and protein complexes.
  • detection is based on array detection on PNA or DNA spots, another technology that is also well established.
  • the invention pulls together convenient, well- established chemical and physical tools and techniques in a new way to make a major advance in technology for discovering ligand/protein binding pairs.
  • Every protein is significant, and having a ligand for every protein can not only speed up drug discovery, but increase our ability to study and control proteins including their quantitation.
  • efficient methods of ligand discovery will be required to provide, in the limit, a small molecule partner for every gene product.”
  • Another advantage of the invention is that it provides several options for quantifying proteins. These options exist in both the first and second embodiments. It is well-known that the properties of proteins cover a wide range in size, shape, charge, polarity and stability. This makes it difficult for any single assay technique to provide optimum conditions including ligands and binding reagents for quantitation of all of the proteins. Also, there may be trade-off in speed, cost, accuracy and degree of multiplexing among different assays. By offering a variety of assay principles and conditions, the methods of the invention help to cope with the diversity of proteins and analytical requirements in different studies. Which assay option is best for a given situation depends on the above considerations and is sorted out empirically and by experience.
  • the methods of the invention help to cope with the problem of protein diversity in a second way. This is due to the use of solution-phase conditions for the ligand/protein binding reactions. It is difficult to create a solid surface, especially where binding proteins themselves are on the surface, that is compatible (inert) towards all nontarget proteins. Indeed, this is a major problem that faces the development of protein chips (Service, R. F., Searching for Recipes for Protein Chips, Science, 294, 2080-2082, 2001) . In the methods of the invention, an array of hybridization reactions on a solid surface can be used instead of an array of protein binding reactions on a solid surface.
  • Nonspecific binding is much less of a problem with hybridization than with protein-binding solid surfaces, especially when the hybridization reagents involve PNAs, because of the small size, lack of charge, moderate polarity and high affinity hybridization reactions. It is novel to detect proteins via PNA or DNA chips, and very practical because the technology for using these chips is so well established.

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Abstract

DOSAGES DE PROTEINE/LIGAND DE FIXATION MULTIPLEXES AVEC MARQUEURS A PROTEINE PNA La presente invention concerne des techniques multiplexags destinees A decouvrir et quantifier des ligands, des proteines et des interactions ligand-proteine. Dans un mode de realisation de l'invention, un ou plusieurs ligands candidat, de preference issus d'une bibliotheque combinatoire et marques chacun avec une seule proteine PNA (acide nucleique peptidique), sont combines simultanement avec une ou plusieurs proteines, et on decouvre des combinaisons ligand/proteine complementaires. Les techniques de cette invention presentent. les avantages d'une ultra-mutiplicite, permettant pratiquement par exemple, de tester une bibliotheque de syntheses combinatoires immediatement sur un proteome entier, dans la mesure oiu on peut tester des proteines non marquees, et de 1'ultra-sensibilite, un proteome d'une seule cellule pouvant etre etudie. Les techniques de cette invention permettent de progresser dans le domaine des puces proteiniques et dans le multiplexage par competition ou non de dosages de ligand qui utilisent des proteines connues comme reactifs de fixation. Elles permettent aussi de realiser des dosages sandwich d'analytes proteiniques A base de ligand.
PCT/US2002/011411 2001-04-10 2002-04-10 Dosage de proteine/ligand de fixation multiplexes avec marques a proteine pna WO2002083951A1 (fr)

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WO2004042030A2 (fr) 2002-11-01 2004-05-21 Leucadia Technologies, Inc. Immuno-pcr sandwich par deplacement
WO2005026387A1 (fr) * 2003-09-18 2005-03-24 Nuevolution A/S Procede permettant d'obtenir des informations structurelles concernant une molecule codee et procede permettant de selectionner des composes
EP2411807A2 (fr) * 2009-03-23 2012-02-01 Biotex, Inc. Procédés de génération simultanée de ligands fonctionnels
US8338579B2 (en) 2004-11-03 2012-12-25 Iris Molecular Diagnostics, Inc. Homogeneous analyte detection
US8513032B2 (en) 2004-11-03 2013-08-20 Iris International, Inc. Microbubbles for affinity separation
US8932992B2 (en) 2001-06-20 2015-01-13 Nuevolution A/S Templated molecules and methods for using such molecules
US9096951B2 (en) 2003-02-21 2015-08-04 Nuevolution A/S Method for producing second-generation library
US9109248B2 (en) 2002-10-30 2015-08-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US9121110B2 (en) 2002-12-19 2015-09-01 Nuevolution A/S Quasirandom structure and function guided synthesis methods
US9574189B2 (en) 2005-12-01 2017-02-21 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
US10731151B2 (en) 2002-03-15 2020-08-04 Nuevolution A/S Method for synthesising templated molecules
US11225655B2 (en) 2010-04-16 2022-01-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes

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EP0781853A2 (fr) * 1995-12-12 1997-07-02 Eli Lilly And Company Méthode de mesure d'acides nucléiques
US6060242A (en) * 1997-02-27 2000-05-09 Lorne Park Research, Inc. PNA diagnostic methods
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10669538B2 (en) 2001-06-20 2020-06-02 Nuevolution A/S Templated molecules and methods for using such molecules
US8932992B2 (en) 2001-06-20 2015-01-13 Nuevolution A/S Templated molecules and methods for using such molecules
US10731151B2 (en) 2002-03-15 2020-08-04 Nuevolution A/S Method for synthesising templated molecules
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
US9109248B2 (en) 2002-10-30 2015-08-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US11001835B2 (en) 2002-10-30 2021-05-11 Nuevolution A/S Method for the synthesis of a bifunctional complex
US10077440B2 (en) 2002-10-30 2018-09-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US9284600B2 (en) 2002-10-30 2016-03-15 Neuvolution A/S Method for the synthesis of a bifunctional complex
EP1563100A2 (fr) * 2002-11-01 2005-08-17 Leucadia Technologies, Inc. Immuno-pcr sandwich par deplacement
EP1563100A4 (fr) * 2002-11-01 2006-12-27 Leucadia Technologies Inc Immuno-pcr sandwich par deplacement
WO2004042030A2 (fr) 2002-11-01 2004-05-21 Leucadia Technologies, Inc. Immuno-pcr sandwich par deplacement
US9121110B2 (en) 2002-12-19 2015-09-01 Nuevolution A/S Quasirandom structure and function guided synthesis methods
US9096951B2 (en) 2003-02-21 2015-08-04 Nuevolution A/S Method for producing second-generation library
US11965209B2 (en) 2003-09-18 2024-04-23 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US11118215B2 (en) 2003-09-18 2021-09-14 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
WO2005026387A1 (fr) * 2003-09-18 2005-03-24 Nuevolution A/S Procede permettant d'obtenir des informations structurelles concernant une molecule codee et procede permettant de selectionner des composes
US8338579B2 (en) 2004-11-03 2012-12-25 Iris Molecular Diagnostics, Inc. Homogeneous analyte detection
US8513032B2 (en) 2004-11-03 2013-08-20 Iris International, Inc. Microbubbles for affinity separation
US8835186B2 (en) 2004-11-03 2014-09-16 Iris International, Inc. Microbubbles for affinity separation
US9234890B2 (en) 2004-11-03 2016-01-12 Iris International, Inc. Homogeneous analyte detection
US9574189B2 (en) 2005-12-01 2017-02-21 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US11702652B2 (en) 2005-12-01 2023-07-18 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
EP2411807A4 (fr) * 2009-03-23 2013-02-20 Biotex Inc Procédés de génération simultanée de ligands fonctionnels
EP2411807A2 (fr) * 2009-03-23 2012-02-01 Biotex, Inc. Procédés de génération simultanée de ligands fonctionnels
US11225655B2 (en) 2010-04-16 2022-01-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes

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