US20090162845A1 - Affinity tag nucleic acid and protein compositions, and processes for using same - Google Patents

Affinity tag nucleic acid and protein compositions, and processes for using same Download PDF

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US20090162845A1
US20090162845A1 US12/004,842 US484207A US2009162845A1 US 20090162845 A1 US20090162845 A1 US 20090162845A1 US 484207 A US484207 A US 484207A US 2009162845 A1 US2009162845 A1 US 2009162845A1
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Prior art keywords
peptide
affinity
seq
nucleic acid
antibody
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Elazar Rabbani
Jannis G. Stavrianopoulos
James J. Donegan
Wayne Patton
Juan Carcamo
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Enzo Biochem Inc
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Individual
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Priority to US12/004,842 priority Critical patent/US20090162845A1/en
Priority to PCT/US2008/087713 priority patent/WO2009086125A2/fr
Priority to EP08869194.4A priority patent/EP2235200B1/fr
Publication of US20090162845A1 publication Critical patent/US20090162845A1/en
Assigned to ENZO BIOCHEM, INC. reassignment ENZO BIOCHEM, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PATTON, WAYNE, STAVRIANOPOULOS, JANNIS G., CARCAMO, JUAN, DONEGAN, JAMES J., RABBANI, ELAZAR
Priority to US15/209,906 priority patent/US10196672B2/en
Priority to US16/225,244 priority patent/US11066694B2/en
Priority to US17/140,257 priority patent/US20210164017A1/en
Priority to US17/215,182 priority patent/US20210214767A1/en
Abandoned legal-status Critical Current

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    • 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
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • This invention relates to affinity tag compositions including affinity tag nucleic acids and proteins, and processes useful for isolating and detecting or quantifying species of a nucleic acid of interest, and other processes for modifying, isolating, detecting or quantifying proteins and analytes of interest.
  • the first important step is isolation of the nucleic acids from other cellular material.
  • the earliest methods were relatively crude methods using ethanol precipitation followed by phase partitioning with organic reagents.
  • phenol has been widely used to separate DNA from cellular material while RNA is more commonly isolated using a guanidinium isothiocyanate/phenol/chloroform mixture.
  • hybrid capture where a nucleic acid complementary to the sequence or sequences of interest is used to specifically hybridize to one or more target nucleic acids.
  • a tag on the capture probe is used to separate material that has hybridized to the capture probe from material that remained unhybridized.
  • Examples of formats that exploit this methodology include beads with oligo T segments for isolation of polyA RNA, and strepavidin-coated microtitre plates that can bind biotinylated primers after amplification reactions.
  • a moiety capable of binding the tag is fixed to a solid support, thus enabling a series of simple washing steps to remove nucleic acids lacking the sequences of interest.
  • a nucleic acid sequence is added to the capture probe, and in the other case, one of the nucleotides is modified by the addition of a ligand.
  • these methods are disadvantaged by the slower kinetics of mixed phase hybridization in the first case and the low capacity engendered by the attachment of large bulky proteins to a solid matrix in the aforementioned biotin/strepavidin method.
  • oligohistidine is the best known example of an oligo peptide that can bind to an immobilized metal, other petides have been described as well, including one that has the amino acid sequence HGGHHG (Cheng et al. 2004 Bio-organic & Medicinal Chemistry Letters 14; 1987-1990)
  • non-nucleic acid affinity tags has also been used in conjunction with nucleic acids.
  • Min and Verdine (1996 Nucleic Acids Research 24:3806-3810) have described a nucleic acid primer with modified bases at the 5′ end with histidine moieties attached to the bases.
  • their primer does not contain an oligopeptide tag as described above, but rather the 5′ end has been modified with a series of histaminyl purine residues.
  • Extension of these primers in a PCR reaction allows collection of the PCR products by means of a chelated resin. No application is described in this publication, however, for using these constructs for either signal detection or analyte isolation.
  • Soderlund et al. (U.S. Patent Appl. No. 20040053300) describe a method of determining the quantity of discrete polynucleotide analytes by the use of a pool of nucleic acid probes of various sizes.
  • the probes hybridize to analytes that have been modified by the addition of an affinity tag (such as oligo histidine) to the base portion.
  • an affinity tag such as oligo histidine
  • complexes are isolated by virtue of the presence of the affinity agent in the analyte allowing binding to a matrix.
  • the bound probes are released and quantified, thus giving an indirect measurement of the amount of analytes present in a sample.
  • the analytes themselves have been covalently attached to an affinity agent.
  • RNA molecules have also been used in conjunction with RNA molecules in Krause and Simmons (U.S. Patent Appl. No. 20060105341).
  • RNA “fusion” molecule with “RNA tags” is described.
  • the “fusion” is not RNA linked to a non-nucleic acid but rather the molecule is a fusion of different nucleic acid sequences resulting in a homogenous nucleic acid where a first RNA segment with a protein binding sequences is appended to a second RNA segment with a selected nucleic acid sequence.
  • This second RNA segment may bind, in turn, to a fusion protein with two domains where one domain binds the RNA tag and the other domain can be an affinity partner, such as an oligo-His tag, that can be used to bind the RNA protein complex to a matrix.
  • This composition has been used for identification and purification of RNA protein complexes and it has not been used for signal generation or isolation of nucleic acid analytes.
  • Histidine has also been used for other purposes besides an affinity label.
  • Van Ness et al. U.S. Pat. No. 7,247,434
  • Sequences are derived from the association of a different tag for each nucleotide base incorporated into nucleic acids synthesized from analyte templates.
  • a single histidine moiety is used as one of the base-specific tags where identification is carried out by mass spectrometry after the nucleic acids have been separated by length.
  • the histidine is not being used as an affinity agent but only as an identifier tag.
  • composition which comprises a nucleic acid and one member of an affinity binding pair, wherein the member is attached to one or more nucleotides of the nucleic acid through a phosphate or sugar of the nucleotide or nucleotides.
  • This invention also provides a composition just described wherein the affinity binding pair comprises: (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group.
  • This invention additionally provides a chimeric nucleic acid comprising at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising a metal binding peptide, wherein the metal binding peptide is attached to one or more nucleotides of the nucleic acid in the first portion through a sugar or phosphate of the nucleotide or nucleotides.
  • a chimeric nucleic acid comprising at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising one member of a peptide affinity group, wherein the member is attached to one or more nucleotides of the nucleic acid in the first portion.
  • the present invention provides a process for isolating one or more species of a nucleic acid of interest.
  • Various steps are used including the first step of providing a sample containing or suspected of containing the nucleic acid of interest, a composition which comprises a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid species of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein the first member of the affinity binding pair is attached to one or more nucleotides in the nucleic acid portion; and a matrix comprising a second member of the affinity binding pair.
  • composition hybridizes with any nucleic acid of interest contained in the sample to form a first complex.
  • the first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair. Bound material is separated from unbound material, thereby isolating the nucleic acid species of interest.
  • the present invention also provides a process for detecting the presence or quantity of a nucleic acid of interest.
  • the following elements are provided: a sample containing labeled nucleic acids, a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, and a matrix comprising a second member of the affinity binding pair; wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein the first member of the affinity binding pair is attached to one or more nucleotides of the nucleic acid portion.
  • composition is allowed to hybridize with any nucleic acid of interest contained in the sample to form a first complex.
  • This first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair.
  • the matrix is washed to remove unhybridized nucleic acids from the matrix. Detecting or quantifying the nucleic acid of interest is carried out by means of detecting or quantifying a signal from the labels.
  • the present invention provides yet another process for detecting the presence or quantity of a nucleic acid of interest.
  • Various steps are performed including the initial step of providing the following elements: a sample containing or suspected of containing the nucleic acid of interest; a labeled probe complementary to the nucleic acid of interest; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein the first member of the affinity binding pair is attached to one or more nucleotides of the nucleic acid portion through a sugar, phosphate or base of the nucleotide or nucleotides; and a matrix comprising a second member of the affinity binding pair.
  • nucleic acids of interest in the sample are allowed to hybridize with labeled probe and the composition to form a first complex.
  • This first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair.
  • the matrix is washed to remove unbound materials from the sample.
  • the nucleic acid of interest is detected or quantified by means of detecting or quantifying a signal from the labels.
  • Yet another process provided by the present invention is one for detecting the presence or quantity of a nucleic acid of interest.
  • Various steps are performed including the initial step of providing a sample containing or suspected of containing nucleic acid of interest; a probe complementary to the nucleic acid of interest and comprising two portions, wherein a first comprises sequences complementary to the nucleic acid of interest, and a second portion comprising a signal sequence; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein the first member of the affinity binding pair is attached to one or more nucleotides of the nucleic acid; and a matrix comprising a second member of the affinity binding pair.
  • nucleic acids of interest in the sample are hybridized with labeled probe and the composition to form a first complex.
  • the first complex is contacted with the matrix to form a second complex by means of a binding interaction between the one or more binding partners and the affinity peptide.
  • the matrix is washed to remove unbound materials from the sample.
  • the nucleic acid of interest is detected or quantified by hybridizing labeled oligonucleotides complementary to the signal sequence.
  • the present invention also provides a fusion protein comprising a biologically active polypeptide or protein and at least one affinity peptide attached to the amino-terminus or the carboxyl-terminus of the biologically active polypeptide or protein, wherein the affinity peptide comprises at least a portion of the amino acid sequence of kininogen, such portion comprising a metal binding peptide.
  • the present invention additionally provides a fusion protein comprising a biologically active polypeptide or protein and an affinity peptide attached at the amino-terminus and an affinity peptide attached at the carboxyl-terminus of the biologically active polypeptide or protein, wherein the affinity peptides comprise at least a portion of the amino acid sequence kininogen, such portion comprising a metal binding peptide.
  • composition provided by this invention is a fusion protein comprising an antibody linked by its amino- and/or carboxyl-terminus to one or two affinity peptides, wherein the affinity peptide binds to a metal, and wherein the antibody has an affinity to an epitope on a different antibody.
  • the invention herein provides a process for modifying a protein of interest, this process comprising the steps of first providing (i) a nucleic acid that codes for the protein of interest; (ii) a nucleic acid that codes for a portion of the amino acid sequence of kininogen, wherein that portion codes for a metal binding peptide; and (iii) an expression vector.
  • the nucleic acid (ii) is added to said nucleic acid (i) to generate a nucleic acid coding for a fusion protein.
  • the nucleic acid coding for the fusion protein is inserted into the expression vector (iii), thereby generating a vector that expresses the modified protein of interest.
  • the invention herein provides a process for isolating a protein of interest, and this process comprises an initial step of providing: (i) a nucleic acid that codes for the protein of interest; (ii) a nucleic acid that codes for a portion of the amino acid sequence of kininogen, wherein the portion codes for a metal binding peptide; (iii) an expression vector; and (iv) a metal-modified matrix.
  • Other steps include adding the nucleic acid (ii) to the nucleic acid (i) to generate a nucleic acid coding for a fusion protein, and inserting the nucleic acid coding for the fusion protein into the expression vector (iii), thereby generating a vector that expresses the protein of interest.
  • the modified protein of interest is purified by binding the protein of interest to the metal-modified matrix (iv).
  • compositions including a fusion protein comprising an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody.
  • Yet another fusion protein provided by this invention is one comprising an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody.
  • the invention herein also provides a process for isolating an analyte of interest, the process comprising the initial step of providing (i) a sample containing or suspected of containing the analyte of interest; (ii) a first antibody having an affinity for the analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; and (iv) a matrix comprising a metal or a second member of the affinity
  • the sample is contacted with the first antibody, thereby forming a first complex between the first antibody and any analyte present in the sample.
  • the first complex is complexed with the fusion antibody, thereby forming a second complex between the first complex and the fusion antibody.
  • the second complex is contacted with the matrix to bind the second complex to the matrix. Unbound material is removed from the matrix.
  • the analyte of interest is released from the second complex, thereby isolating the analyte of interest.
  • the first step provides (i) a labeled sample containing or suspected of containing labeled analyte of interest; (ii) a first antibody having an affinity for the analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; and (iv) a matrix comprising a metal or
  • the sample is contacted with the first antibody, thereby forming a first complex between the first antibody and any analyte present in the sample.
  • the first complex is contacted with the fusion antibody, thereby forming a second complex between the first complex and the fusion antibody.
  • the second complex is contacted with the matrix to bind the second complex to the matrix. Unbound material is removed from the matrix. Labeled analytes bound to the matrix are detected or quantified by means of detecting or quantifying a signal from the labels.
  • Yet another process provided herein is one for detecting or quantifying an analyte of interest, the process comprising various steps including the first step of providing (i) a labeled sample containing or suspected of containing labeled analytes of interest; (ii) a first antibody having an affinity for the analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; and (iv) a matrix comprising a metal or
  • a first complex is formed among the matrix, the fusion antibody and the first antibody.
  • the first complex is contacted with the labeled sample, thereby forming a second complex between the first complex and any labeled analytes that may be present in the sample. Unbound material is removed from the matrix.
  • the labeled analytes bound to the matrix are detected or quantified by means of detecting or quantifying a signal from the labels.
  • FIG. 1 depicts various format that could be used with chimeric primers.
  • FIG. 1A illustrates a chimeric construct with a first portion consisting of a nucleic acid complementary to a chosen nucleotide sequence and a second portion with an oligohistidine portion used to bind nucleic acids with the chosen sequences to a solid matrix thereby allowing isolation of either nucleic acids that bind to the matrix or nucleic acids that lack complementarity to the construct.
  • FIG. 1B depicts a format where a chimeric construct similar to the one in FIG. 1A is used to detect the presence of the complementary sequence when a collection of labeled analytes are allowed to hybridize to the construct.
  • FIG. 1C shows a chimeric construct similar to the one in FIG. 1A that is used to detect the presence of unlabeled analytes by by means of a probe complementary to the sequence of interest.
  • FIG. 1D illustrates a chimeric construct having energy transfer elements where hybridization of an analyte labeled with energy transfer elements provides signal generation that is dependent upon hybridization of the analyte to the construct.
  • FIG. 1E is a depiction of a format where energy transfer takes place between a labeled analyte and a signal probe.
  • FIG. 1F is a depiction of a format where the analyte is unlabled and analyte specific energy transfer takes place between a signal probe and a chimeric construct.
  • FIG. 2 is illustrative of binding of chimeric constructs to matrices.
  • FIG. 2A shows binding and elution of labeled chimeric constructs with Ni column.
  • FIG. 2B shows binding of labeled chimeric constructs with a 96 well plate.
  • FIG. 3 illustrates the effects of various reagents on binding of chimeric constructs.
  • This invention provides a composition which comprises a nucleic acid portion that provides specific hybridization to a nucleic acid analyte of interest and a non-nucleic acid portion that comprises at least one member of an affinity binding pair that allows capture of the composition to a solid matrix wherein the member is attached to one or more nucleotides of the nucleic acid and this attachment can be through the phosphate, sugar or base of the nucleotide or nucleotides.
  • affinity binding pairs contemplated by this invention are pairs comprising an immobilized metal and a peptide or oligopeptde that has an affinity for such a metal.
  • the present invention provides a composition which comprises a nucleic acid and one member of an affinity binding pair, wherein the member is attached to one or more nucleotides of the nucleic acid through a phosphate or sugar of the nucleotide or nucleotide, and such attachment to such nucleotide or nucleotides can be through a linker arm as described further below.
  • the affinity binding pair comprises: (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group.
  • the metal is immobilized by chelation.
  • a peptide or oligopeptide is defined as a succession of amino acids joined through peptide bonds.
  • Examples of metals that may be bound by such peptides are nickel, copper, cobalt and zinc.
  • Examples of such peptides are oligohistidine and an oligopeptide with the sequence HGGHHG that have been referred to earlier.
  • Other such oligopeptides that may be of use can include SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG and HPHHGGSPHHG described by Pasquinelli et al., 2000 (Biotechnol. Prog. 16, 86-91), KDHLIHNVHKEEHAHAHNK described by Chaga et al., 1999 (J. Chromatog A.
  • the metal binding peptide can comprise any of the aforementioned amino acid sequences: oligohistidine, HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG, HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK or HGLGHGHEQQHGLGHGH.
  • affinity binding pairs that may find use with the present invention can include peptide affinity pairs.
  • a peptide affinity pair is defined as any binary combination of peptides, oligopeptides or proteins that that are capable of recognizing and binding to each other. Examples of such pairs can include but is not necessarily limited to pairs such as S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo Phe, KSI and oligo Leu, as well as “complementary” pairings such as oligo Arg with oligo Glu, and oligo Arg with oligo Asp.
  • the peptide affinity group can comprise S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
  • One member of the affinity binding pair will comprise part of a chimeric construct joined to a nucleic acid while the corresponding member of the pair is affixed or immobilized to a matrix. It is also understood that either member of a pair may be used as the non-nucleic acid portion such that it can be used with its corresponding member on the matrix.
  • a chimeric nucleic acid can comprise an oligohistidine portion for capture by metal chelates attached to a solid matrix (an IMAC column or plate), or on the other hand, a nucleic acid can be used that has been modified by the presence of one or more metals allowing capture on a matrix comprising oligohistidine or some other metal binding peptide.
  • the chimeric nucleic acid provided by the present invention can comprise at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising a metal binding peptide, e.g., nickel, copper, cobalt or zinc.
  • the metal binding peptide can be attached, desirably through a linker arm as previously described, to one or more nucleotides of the nucleic acid in the first portion through a sugar or phosphate of the nucleotide or nucleotides.
  • the metal binding peptide can comprise any of the amino acid sequences: oligohistidine, HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG, HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK or HGLGHGHEQQHGLGHGH.
  • One or more energy transfer donors or one or more energy transfer acceptors can be incorporated into or attached to the chimeric nucleic acid just described.
  • Another chimeric nucleic acid provided by the present invention comprises at least two portions, a first portion comprising a nucleic acid complementary to a nucleic acid sequence of interest, and a second portion comprising one member of a peptide affinity group.
  • the member can be attached, using a linker arm desirably, to one or more nucleotides of the nucleic acid in the first portion.
  • the peptide affinity binding group includes any of the pairs: S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
  • This additional embodiment of a chimeric nucleic acid can also further comprise one or more energy transfer donors, or one or more energy transfer acceptors.
  • Synthesis of the chimeric composition can be carried out by a variety of means where either the base, sugar or phosphate position of a nucleotide in the nucleic acid portion is used to attach the affinity agent.
  • Examples of means of modifying nucleic acids that may be used for this purpose are described in Ward et al. in U.S. Pat. No. 4,711,955, Engelhardt et al., in U.S. Pat. No. 5,241,060, Stavrianopoulos et al., in U.S. Pat. No. 4,707,440, Pergolizzi et al., in EP 0 611 828 and Engelhardt et al., in U.S. Patent Application No.
  • Attachment can be by means of a covalent attachment of one of the foregoing metals, oligopeptides or proteins to the nucleic acid portion, or it may be by means of noncovalent attachment through a secondary binding pair such as avidin and biotin.
  • a secondary binding pair such as avidin and biotin.
  • one of the proteins described above as a member of an affinity pair can be biotinylated using standard methods and the nucleic acid can be covalently linked to strapavidin. Formation of a complex between these two entities will create chimeric molecule comprising the affinity member and a nucleic acid portion.
  • Covalent attachment may be direct where the affinity agent is attached by itself to the nucleic acid portion or it may involve indirect covalent attachment where there is a linker arm joining the affinity agent to the nucleic acid portion.
  • the position of attachment of the non-nucleotide portion to the nucleic acid can involve any chosen nucleotide; i.e., either internal or terminal nucleotides are suitable for carrying out the present invention.
  • Linker arms are well-known in the art and have been described by a number of authors and researchers. See, for example, Ward et al. in U.S. Pat. No. 4,711,955, Engelhardt et al., in U.S. Pat. No. 5,241,060, Engelhardt et al., in U.S. Pat. No. 4,894,325, and Stavrianopoulos et al., in U.S. Pat. No. 7,186,478, all of which are incorporated herein by reference.
  • the presence of the nucleic acid portion will allow the capture of a nucleic acid and binding of it to a matrix through the affinity agent.
  • the species of interest can be as broad or as narrow as the user desires by the appropriate choice of sequences used for the nucleic acid portion.
  • the sequence can be selective for a single species such as a nucleic acid coding for a particular gene, or it may represent an entire class of molecules. Selectivity can be carried out with a single sequence in a chimeric composition or there may be more than one selective sequence in the chimeric composition. It is also envisioned that selectivity for different sequences may be carried out either sequentially or in parallel by having different selective sequences as part of separate chimeric compositions.
  • sequences in the nucleic acid portion can comprise generic sequences such as oligo T or oligo A that can bind to a wide variety of different nucleic acids or the nucleic acid portion may comprise unique sequences that will bind to specific mRNA or cDNA species.
  • FIG. 1A An illustration of a possible means of carrying this out is shown in FIG. 1A .
  • This aspect of the present invention may be used for either positive or negative selection.
  • the nucleic acid portion may comprise oligo or poly T sequences allowing the subsequent binding of polyA mRNA.
  • RNA generally consists of only 3-6% of total RNA
  • the subsequent removal of RNA unable to bind to a matrix bound chimeric construct results in a powerful enrichment of the poly A sequences that may be then used for a variety of purposes.
  • the majority of total RNA consists of rRNA sequences and these may be removed by the use of chimeric molecules that comprises sequences complementary to rRNA.
  • the portion of the total RNA that contains mRNA, hnRNA, ⁇ RNA and snRNA remains unbound, thereby allowing any and all of these species to be used in further steps.
  • RNA may even be deleterious since it may consume reagents and contribute noise to analytic methods, as seen for example, when total RNA is labeled by photobiotin, 94-97% of the labeled material would be irrelevant to analysis of polyA mRNA.
  • nucleic acids of the present invention may also be used in a number of different ways: as part of a detection system; where a label may be included as part of the composition itself; when the analyte is being detected or quantified; or when a probe recognizes the analyte or combinations thereof.
  • nucleic acid analytes from biological samples may be labeled directly by modifying the base, sugar or phosphate moieties.
  • analytes may also be labeled during the course of copying or in amplification procedures where labeled nucleotides are provided during the course of such procedures, thereby synthesizing labeled complementary or identical copies of the original analytes.
  • a chimeric composition could be used as a primer to generate a labeled complementary copy that may be subsequently isolated afterwards by means of the second member of the affinity pair or a normal primer could be used preparation of the labeled complementary copies where a hybridization with a chimeric composition is carried out afterwards.
  • An example of a copying reaction that may find use in the present invention could be the use of samples containing mRNA where labeled cDNA copies are prepared by means of reverse transcriptase or a DNA polymerase with reverse transcriptase activity. A general depiction of this type of format is shown in FIG. 1B .
  • amplification systems that may be useful in the present invention can include but are not necessarily limited to the polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription mediated amplification (TMA), Strand diplacement amplification (SDA), Nucleic acid sequence based amplification (NASBA) and Secondary Structure Amplification (Rabbani et al., in U.S. Pat. No. 6,743,605) all of which are incorporated by reference.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TMA transcription mediated amplification
  • SDA Strand diplacement amplification
  • NASBA Nucleic acid sequence based amplification
  • Rabbani et al., in U.S. Pat. No. 6,743,605 all of which are incorporated by reference.
  • Amplifications may be directed towards specific nucleic acid sequences as is generally used in the preceding methods, or there may be a more global amplification of multiple sequences from a library that includes the preceding methods as well as methods such as those taught by Van Gelder et al., in U.S. Pat. No. 5,545,522, Kurn in U.S. Pat. No. 6,251,639 and Stavrianopoulos et al., in U.S. Pat. No. 7,163,796, all of which are incorporated by reference.
  • the synthesis of nucleic acids may take place after the nucleic acid(s) of interest have been isolated from a biological sample and released from a matrix or the reactions may take place while the nucleic acids are still bound to the matrix.
  • the nucleic acids of the present invention may be used in a passive manner where they are only used to immobilize a nucleic acid in an environment where nucleic acid synthesis reactions may take place.
  • it may be an active participant where the chimeric nucleic acid comprises a promoter or acts as a primer in reactions such as those cited above.
  • Detection of an analyte may also take place with unlabled analytes by means of the additional use of a labeled probe that is complementary to the nucleic acid(s) of interest. This may be used with nucleic acids in their native forms, or complementary copies derived form copying or amplification procedures. A depiction of a format with this process is shown in FIG. 1C .
  • compositions of the present invention can comprise one or more energy transfer donors, or one or more energy transfer acceptors.
  • compositions can be used to isolate one or more species of a nucleic acid of interest.
  • various elements would be provided including a sample containing or suspected of containing the nucleic acid of interest, a composition which comprises a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid species of interest, and the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group; and wherein the first member of the affinity binding pair being attached, for example, through a linker arm, to one or more nucleotides in said nucleic acid portion; and a matrix comprising a second member of the affinity binding pair.
  • the composition hybridizes with any nucleic acid of interest contained in the sample to form a first complex.
  • This is followed by contacting the first complex with the matrix provided to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair.
  • the material bound to the matrix could then be separated from unbound material, thereby isolating said nucleic acid species of interest.
  • the portion of the sample that remains bound to the matrix can or may comprise the nucleic acid species of interest.
  • the portion of the sample that remains unbound to the matrix may or could comprise the nucleic acid species of interest. It should be understood to those skilled in the art that one or more washing steps could be used in the process just described above.
  • the metal binding peptide, the immobilized metal, the peptide affinity group, linker arms have been described above with respect to other descriptions of the present compositions and processes.
  • a process for detecting the presence or quantity of a nucleic acid of interest.
  • various elements are provided. These include a sample containing labeled nucleic acids, a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, and a solid support or matrix comprising a second member of the affinity binding pair.
  • the affinity binding pair can comprise: (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group.
  • the first member of the affinity binding pair is attached to one or more nucleotides of said nucleic acid portion, and this attachment can be through a linker arm as described in further detail above.
  • the above composition is hybridized with any labeled nucleic acid of interest contained in the sample to form a first complex.
  • This first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair.
  • the matrix is washed one or more times to remove unhybridized nucleic acids from the matrix. Detection or quantification of the nucleic acid of interest is carried out by means of detecting or quantifying a signal from the labels.
  • Such labels are detectable fluorescently, chemiluminescently, colorimetrically or enzymatically.
  • the just described process can include a further step of releasing the second complex from the matrix prior to detecting or quantifying the nucleic acid of interest.
  • the nature of the metal binding peptide, the immobilized metal, the peptide affinity group, the linker arm, energy transfer donors and energy transfer acceptors have been described earlier in this disclosure and need not be reiterated here.
  • a sample containing or suspected of containing the nucleic acid of interest a labeled probe complementary to the nucleic acid of interest; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein the first member of the affinity binding pair is attached, e.g., through a linker arm, to one or more nucleotides of said nucleic acid portion through a sugar, phosphate or base of the nucleotide or nucleotides; and a matrix comprising a second member of the affinity binding pair.
  • any nucleic acids of interest in the sample are hybridized with labeled probe and the composition to form a first complex.
  • This first complex is contacted with the matrix to form a second complex by means of a binding interaction between the first member and the second member of the affinity binding pair.
  • the matrix can be washed one or more times to remove unbound materials from the sample.
  • Detection or quantification of the nucleic acid of interest can be carried out by means of detecting or quantifying a signal from the labels.
  • Such labels are detectable fluorescently, chemiluminescently, colorimetrically or enzymatically.
  • An additional step of releasing the second complex from the matrix can be carried out or included in this process prior to carrying out detection or quantification.
  • the labeled probes can comprise one or more energy transfer donors and the composition can comprise one or more energy transfer acceptors.
  • the labeled probes can comprise one or more energy transfer acceptors and the composition can comprise one or more energy transfer donors.
  • the labeled probes can comprise one or more energy transfer donors and the nucleic acids in the sample provided can be labeled with one or more energy transfer acceptors.
  • the labeled probes can comprise one or more energy transfer acceptors and the nucleic acids in the sample provided can be labeled with one or more energy transfer donors.
  • the present invention and compositions can be directed to another process for detecting the presence or quantity of a nucleic acid of interest.
  • a sample containing or suspected of containing the nucleic acid of interest a probe complementary to the nucleic acid of interest and comprising two portions, wherein a first comprises sequences complementary to the nucleic acid of interest, and a second portion comprising a signal sequence; a composition comprising a nucleic acid portion and a first member of an affinity binding pair, wherein the nucleic acid portion comprises sequences complementary to the nucleic acid of interest, wherein the affinity binding pair comprises (a) a metal binding peptide and an immobilized metal, or (b) a peptide affinity group, and wherein the first member of the affinity binding pair is attached, through a linker arm, for example, to one or more nucleotides of the nucleic acid; and a matrix comprising a second member of the affinity binding pair.
  • nucleic acids of interest which are in the sample are allowed to hybridize with the labeled probe and the composition to form a first complex.
  • Such first complex is contacted with the matrix to form a second complex by means of binding interactions between one or more binding partners and the affinity peptide.
  • the matrix is washed in a single step or a series of washing steps to remove unbound materials from the sample.
  • Detection or quantification of the nucleic acid of interest is carried out by hybridizing labeled oligonucleotides complementary to the signal sequence.
  • the labeled oligonucleotides are detectable fluorescently, chemiluminescently, colorimetrically or enzymatically.
  • the nature of the metal binding peptide i.e., the amino acid sequences used therein, the immobilized metal, the peptide affinity group, and the like, have been previously described in this disclosure and will not be repeated here.
  • signal sequence that may be used for this purpose have been described previously, including methods and compositions described by Pergolizzi et al., in European Publication No. 0 128 332 A1, based on U.S. patent application Ser. No. 06/491,929, filed May 5, 1983; and Urdea et al., U.S. Pat. No. 5,124,246.
  • signal sequence can comprise a homopolymeric sequence, or it can comprise a heterologous sequence where the heterologous sequence is neither identical or complementary to the nucleic acid of interest.
  • the use of the kininogen peptide sequence is disclosed as being useful for incorporation into nucleic acids coding for proteins of interest.
  • the provision of this novel affinity peptide may increase the range of fusion proteins that may be successfully designed with an affinity sequence.
  • even the flexibility of being able to use either the carboxy or amino terminus as an insertion site may be insufficient and both locations may interfere in either production or activity of the recombinant protein of interest.
  • the availability of an alternative peptide sequence may allow generation of recombinant proteins that overcome this problem.
  • compositions including one or more energy transfer donors or one or more energy transfer acceptors.
  • this invention is also directed to and provides a fusion protein comprising a biologically active polypeptide or protein and at least one affinity peptide attached to the amino-terminus or the carboxyl-terminus of the biologically active polypeptide or protein, wherein the affinity peptide comprises at least a portion of the amino acid sequence of kininogen, the portion comprising a metal binding peptide.
  • the invention also provides a fusion protein comprising a biologically active polypeptide or protein and an affinity peptide attached at the amino-terminus and an affinity peptide attached at the carboxyl-terminus of the biologically active polypeptide or protein, wherein the affinity peptides comprise at least a portion of the amino acid sequence kininogen, the portion also comprising a metal binding peptide.
  • a preferred sequence for the kininogen used as the affinity peptide in such fusion proteins is GHGLGHGHEQQHGLGHGHK, or a portion thereof.
  • the kininogen can be human kininogen if desired.
  • Other aspects of the fusion proteins just described above should be noted.
  • One aspect relates to the amino acid sequence between the biologically active polypeptide or protein and the affinity peptides, and this sequence is or can be recognizable by a protease, such as enterokinase or coagulation factor X a .
  • the affinity peptide may bind nickel, copper, cobalt or zinc.
  • a format may be used where the affinity tagged antibody is specific for a unique target of interest where the target may be a protein or some other molecule of interest.
  • This approach entails construction of a unique antibody for each antigen of interest and it has been previously described in the context of protein arrays by Wingren et al., (2005 Proteomics 5; 1281-1291) where a library of single-chain Fv antibodies were fixed to a matrix by either a metal or an anti-tag antibody.
  • Other antibodies that have been modified this way have been described by Johnson et al. in U.S. Patent Application No. 2004/0197866 and Wu et al., in U.S. Patent Application No. 2006/0094062.
  • the present invention can be used to provide a process for modifying proteins of interest.
  • three elements are provided including: (i) a nucleic acid that codes for the protein of interest; (ii) a nucleic acid that codes for a portion of the amino acid sequence of kininogen, wherein the portion codes for a metal binding peptide; and (iii) an expression vector.
  • the nucleic acid (ii) is added to the nucleic acid (i) to generate a nucleic acid coding for a fusion protein.
  • the fusion protein coding nucleic acid is inserted into the expression vector (iii), thereby generating a vector that expresses the modified protein of interest.
  • the expression vector (iii) can comprise a number of different types, including a mammalian expression vector, a bacterial expression vector, an insect cell expression vector and a yeast expression vector.
  • the expression vector (iii) can be plasmid or a viral vector.
  • this invention when applied to the isolation of a protein of interest, provides the following process.
  • Several elements are provided including (i) a nucleic acid that codes for the protein of interest; (ii) a nucleic acid that codes for a portion of the amino acid sequence of kininogen, wherein that portion codes for a metal binding peptide; (iii)an expression vector; and (iv) a metal-modified matrix.
  • the nucleic acid (ii) is added to the nucleic acid (i) to generate a nucleic acid coding for a fusion protein.
  • the nucleic acid coding for the fusion protein is inserted into the expression vector (iii), resulting in the expression of the protein of interest.
  • the expression vector (iii) can comprise a mammalian expression vector, a bacterial expression vector, an insect cell expression vector or a yeast expression vector.
  • the expression vector (iii) can also be a plasmid or a viral vector.
  • an antibody to a protein can be engineered to have an amino sequence that comprises an affinity peptide, thus allowing capture of the antibody onto a solid matrix as well as any complex formed between the modified antibody and its target. This will be of special use and significance when the target is a protein that is desired to be isolated.
  • a more universal reagent can be made by construction of a tagged antibody that has an affinity for other antibodies.
  • an anti-goat antibody that is derived from mouse cells can be redesigned to comprise an affinity peptide and used to collect complexes that are made of goat antibodies that are bound to their particular analyte targets.
  • This system uses a universal reagent in that only the anti-goat antibody needs to be modified and this reagent should be able to recognize a wide variety of complexes formed between goat antibodies and their antigen targets.
  • the need for individually modifying each antibody used for as an antibody/antigen pair is obviated.
  • antibody or “antibodies” are used in the present invention, such terms include, without limitation, antibody fragments, single chain antibodies, and the like.
  • This invention further provides a fusion protein comprising an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group.
  • the antibody has an affinity for a different antibody in this case.
  • a different embodiment provided by the present invention is a fusion protein comprising an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody.
  • the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody.
  • the peptide affinity group can comprise S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
  • the metal binding peptide can comprise oligohistidine or an oligopeptide comprising any of the following sequences: HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG, HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK or HGLGHGHEQQHGLGHGH.
  • Processes for analyte isolation and detection or quantification are also provided by the present invention.
  • an analyte of interest the following process can be used in accordance with this invention.
  • Four elements are initially provided, including (i) a sample containing or suspected of containing the analyte of interest; (ii) a first antibody having an affinity for the analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different
  • the sample is allowed to contact the first antibody, thereby forming a first complex between the first antibody and any analyte present in the sample.
  • the first complex is allowed to contact with the fusion antibody, thereby forming a second complex between the first complex and the fusion antibody.
  • the second complex is contacted with the matrix to bind the second complex to the matrix. Unbound material is removed from the matrix, and the analyte of interest is released from the second complex, thereby isolating the analyte of interest.
  • the peptide affinity group can comprise S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
  • the metal binding peptide can comprise oligohistidine or an oligopeptide comprising any of the sequences: HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG, HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK or HGLGHGHEQQHGLGHGH.
  • Analyte detection or quantification can also be carried out in accordance with this invention.
  • an analyte of interest e.g., a protein or a polypeptide
  • the following elements are provided: (i) a labeled sample containing or suspected of containing labeled analyte of interest; (ii) a first antibody having an affinity for the analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the affinity peptide comprises
  • the sample is contacted with the first antibody, thereby forming a first complex between the first antibody and any analyte present in the sample.
  • the first complex so formed is contacted with the fusion antibody, thereby forming a second complex between the first complex and the fusion antibody.
  • the second complex is contacted with the matrix to bind the second complex to the matrix. Unbound material is removed from the matrix. Detection or quantification of the labeled analytes bound to the matrix is performed by means of detecting or quantifying a signal from the labels.
  • the labels are detectable fluorescently, chemiluminescently, colorimetrically or enzymatically.
  • the peptide affinity group can comprise S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
  • the metal binding peptide can comprise oligohistidine or an oligopeptide comprising any of the sequences: HGGHHG, SPHHG, SPHHGGSPHHG, HPHHG, HPHHGGHPHHG, SPHHGGHPHHG, HPHHGGSPHHG, KDHLIHNVHKEEHAHAHNK, GHGLGHGHEQQHGLGHGHK or HGLGHGHEQQHGLGHGH.
  • This invention also provides a process for detecting and quantifying an analyte of interest, such as a protein or polypeptide.
  • an analyte of interest such as a protein or polypeptide.
  • the following elements are provided: (i) a labeled sample containing or suspected of containing labeled analytes of interest; (ii) a first antibody having an affinity for the analyte; (iii) a fusion antibody comprising: (a) an antibody and at least one affinity peptide attached to the amino terminus or the carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein the antibody has an affinity for a different antibody; or (b) an antibody and an affinity peptide attached to the amino terminus and an affinity peptide attached to carboxyl terminus of the antibody, wherein the affinity peptide comprises a metal binding peptide or is one member of a peptide affinity group, wherein
  • a first complex is formed among the matrix, the fusion antibody and the first antibody.
  • the first complex is contacted with the labeled sample to form a second complex between the first complex and any labeled analytes that may be present in the sample. Unbound material is removed from the matrix.
  • Labeled analytes bound to the matrix are detected or quantified by means of detection or quantification of the the signal generated from the labels. Such labels are detectable fluorescently, chemiluminescently, colorimetrically or enzymatically.
  • the matrix can comprise an array of different first antibodies, thereby allowing for detection or quantification of multiple analytes of interest.
  • a 5′ amino modified oligonucleotide was ordered from Sigma Genosys (Sigma-Aldrich, St. Louis, Mo.) with the following sequence: 5′ amine-tcaaccaac 3′.
  • the oligonucleotide was labeled by terminal transferase using a 3′-Oligonucleotide labeling system (Enzo Life Sciences Inc, Farmingdale, N.Y.) and rhodamine labeled dUTP (Enzo Life Sciences Inc, Farmingdale, N.Y.).
  • 100 g of the the oligo prepared in step 2 was phenol extracted, ethanol precipitated and dissolved in 200 l of 0.2M Sodium Borate, 5 mM EDTA, pH 8.5 followed by addition of 300 I of DMF and 50 I of the N-trifluoroheptahistidine-NHS ester synthesized in step 1.
  • the mixture was stirred overnight and the derivatized DNA was then precipitated by the addition of 10 volumes of n-butanol.
  • the pellet was dissolved in 200 ul of 1M Lithium Hydroxide solution and left at room temperature for 30 minutes to remove the trifluoroacetyl groups.
  • the heptahistidine modified DNA was then precipitated with 10 volumes of ethanol and redissolved in binding buffer (20 mM Phosphate, 500 mM NaCl, pH 7.4)
  • the heptahistidine modified rhodamine oligonucleotide from Example 1 was diluted in binding buffer (20 mM Phosphate, 500 mM NaCl, pH 7.4) and added to a Ni-column (Ni Sepharose high performance, GE Healthcare, Piscataway, N.J.).
  • a control rhodamine labeled oligonucleotide without the addition of the heptahistidine was also added to a Ni-column.
  • the columns were then washed with 8 volumes of binding buffer followed by a 5 volumes of binding buffer containing 0.5 M Imidazole to release the oligonucleotide histidine groups and the eluants collected. Quantification was carried out using a spectrofluorometer (Ex: 556 nm, Em: 580) for both the Effluent (Ft) that did not bind and for the Eluent (Elu) that was released after binding.
  • FIG. 2(A) The results of this experiment are shown in FIG. 2(A) as represented by the percentage of the rhodamine signal of the input material.
  • the lack of quantitative binding by the oligohistidine modified preparation is likely to be an indication that not all of the oligonuclotides were conjugated to the peptide.
  • Binding of the oligohistidine modified oligonucleotide to a matrix was also tested by binding to 96 well plates instead of the columns used in Example 2. Identical dilutions of histidine modified and unmodified rhodamine labeled oligonucleotides from Example 2 were added to Nickel-coated plates (HisGrab Nickel coated 96-well plates, Pierce, Rockford, Ill.) and incubated for 3 hours at room temperature, followed by washing 3 times with 200 ul binding buffer. The bound DNA was measured by detecting rhodamine with a plate reader (filters: Ex550, Em610, Fluostar Optima, BMG Labtech). Results of this experiment are shown in FIG. 2(B) .
  • the Histidine-modified DNA bound to the Ni-plates more effectively than the control DNA.
  • an input of 10 ul of control and His(7)-DNA showed 90% of the histidine modified DNA being bound to the wells, while only 10% of the control DNA was detected.
  • the results shown in FIG. 2 (B) also indicate that with the highest input level (50 ul), the wells were overloaded.
  • nucleic acids are commonly taken up in the presence of chelators such as EDTA or SSC (standard saline citrate). Since it is possible that these could be competitors for a peptide/chelate interaction, the oligonucleotides from Example 1 were tested for the ability to be bound in their presence.
  • the histidine modified oligos were incubated with Ni beads in the presence of binding buffer (control), or binding buffer with either 0.5 mM EDTA or 1 ⁇ SSC .
  • binding buffer control
  • binding buffer with either 0.5 mM EDTA or 1 ⁇ SSC

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US12/004,842 US20090162845A1 (en) 2007-12-20 2007-12-20 Affinity tag nucleic acid and protein compositions, and processes for using same
PCT/US2008/087713 WO2009086125A2 (fr) 2007-12-20 2008-12-19 Compositions d'acides nucléiques et de protéines à étiquettes d'affinité, et procédés pour leur utilisation
EP08869194.4A EP2235200B1 (fr) 2007-12-20 2008-12-19 Compositions d'acides nucléiques et de protéines à étiquettes d'affinité, et procédés pour leur utilisation
US15/209,906 US10196672B2 (en) 2007-12-20 2016-07-14 Affinity tag nucleic acid and protein compositions, and processes for using same
US16/225,244 US11066694B2 (en) 2007-12-20 2018-12-19 Affinity tag nucleic acid and protein compositions, and processes for using same
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EP3633047A1 (fr) 2014-08-19 2020-04-08 Pacific Biosciences of California, Inc. Compositions et procédés d'enrichissement d'acides nucléiques
US10435685B2 (en) 2014-08-19 2019-10-08 Pacific Biosciences Of California, Inc. Compositions and methods for enrichment of nucleic acids
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US20170029868A1 (en) 2017-02-02
US20210164017A1 (en) 2021-06-03
US10196672B2 (en) 2019-02-05
US11066694B2 (en) 2021-07-20
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EP2235200B1 (fr) 2016-03-09

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