WO1999004042A1 - Conjugues oligonucleotide-peptide synthetiques et procedes d'utilisation - Google Patents

Conjugues oligonucleotide-peptide synthetiques et procedes d'utilisation Download PDF

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Publication number
WO1999004042A1
WO1999004042A1 PCT/US1998/014782 US9814782W WO9904042A1 WO 1999004042 A1 WO1999004042 A1 WO 1999004042A1 US 9814782 W US9814782 W US 9814782W WO 9904042 A1 WO9904042 A1 WO 9904042A1
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oligonucleotide
peptide
target nucleic
nucleic acid
nucleic acids
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PCT/US1998/014782
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English (en)
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David W. Burden
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Burden David W
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Priority to AU84092/98A priority Critical patent/AU8409298A/en
Publication of WO1999004042A1 publication Critical patent/WO1999004042A1/fr

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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
    • 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
    • C12Q1/6813—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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • This invention relates to the field of diagnostics and separations. More specifically, the invention provides novel synthetic oligonucleotide- peptide conjugates for use in detection of specific nucleic acid molecules and separation of such molecules from a heterogeneous mixture.
  • modified oligonucleotides as probes for detecting specific nucleic acid molecules has advanced the filed of DNA diagnostics in recent years.
  • Detectably labeled oligonucleotides have been used to detect specific nucleic acids in a mixed population of nucleic acids and other biological molecules. This type of detection is currently performed with biotinylated DNA, digoxigenin-labeled nucleic acids and FITC-labeled DNA.
  • biotinylated DNA the biotin is detected indirectly with streptavidin-labeled conjugates.
  • Digoxigenin-labeled nucleic acids are detected with antibodies immunologically specific for digoxigenin.
  • FITC acts as either a fluor ⁇ chrome or an antigen.
  • a limitation to the biotin, digoxigenin and FITC detection systems is the lack of diversity among the labels. This prevents the use of multiple probes for hybridization assays because it is not possible to differentiate between two different probes that are labeled identically.
  • nucleic acid detection system that can provide a wide diversity among labels. It would be of further advantage to develop a system capable of separating or purifying target nucleic acids from a mixed population of nucleic acids or other biological molecules. Compositions and methods comprising such a system would enable multiple nucleic acid detection and separation procedures, which heretofore have been limited or unavailable.
  • compositions and methods that enable detection and/or separation of one or more selected nucleic acids in a test sample containing a larger population of nucleic acids or a heterogeneous mixture of nucleic acids and other molecules.
  • a method for determining the presence or quantity of one or more target nucleic acid molecules in a test sample suspected of containing one or more of the target nucleic acid molecules.
  • the method comprises the steps of: (a) providing a set of oligonucleotide-peptide conjugates, each member of the set comprising (i) an oligonucleotide moiety that specifically hybridizes to one of the target nucleic acid molecules; and (ii) a peptide moiety that binds to one of a set of antibodies, each member of the antibody set being immunologically specific for only one member of the oligonucleotide- peptide conjugate set; (b) contacting the test sample with the set of oligonucleotide-peptide conjugates under conditions that permit the specific hybridization of the oligonucleotide moieties to the one or more target nucleic acids, if present; (c) contacting the test sample with the antibodies, under conditions permitting binding of each member
  • the step of contacting the test sample with the antibodies further comprises separately contacting an aliquot of the test sample with each member of the antibody set, thereby forming a complex in each aliquot comprising the target nucleic acid, if any, one member of the set of oligonucleotide-peptide conjugates, and the antibody immunologically specific for the member of the oligonucleotide-peptide conjugate set.
  • the respective antibodies are attached to a solid support and the step of contacting the test samples with the antibodies results in separately capturing each member of the set of complexes on the solid support.
  • the target nucleic acids (or, alternatively, the oligonucleotide moieties of the set of oligonucleotide- peptide conjugates) are labeled with a reporter substance and the step of detecting the nucleic acid molecules, if any, associated with each member of the set of separated complexes is accomplished by detecting the reporter substance.
  • the selected target nucleic acids comprise RNA and the step of detecting the nucleic acid molecules, if any, associated with each member of the set of separated complexes is accomplished by contacting the complexes with a detectable antibody immunologically specific for DNA/RNA hybrid molecules.
  • each member of the antibody set is labeled with a reporter substance unique to that member of the antibody set.
  • unique means, for example, that if the antibody set has three members, each will be labeled with a different reporter substance.
  • the step of detecting the nucleic acid molecules, if any, associated with each member of the set of complexes is accomplished by detecting the presence or amount of each unique reporter substance in the set of separated complexes. It is preferred that the step of separating the complexes from the test sample comprises capturing the complexes on a solid support, and, optionally, thereafter releasing the captured complexes from the solid support.
  • a method for separating one or more target nucleic acid molecule from a test sample suspected of containing one or more of the target nucleic acid molecules.
  • the method comprises the steps of: (a) providing a set of oligonucleotide-peptide conjugates, each member of the set comprising (i) an oligonucleotide moiety that specifically hybridizes to one of the target nucleic acid molecules; and (ii) a peptide moiety that binds to one of a set of antibodies, each member of the antibody set being immunologically specific for only one member of the oligonucleotide-peptide conjugate set; (b) contacting the test sample with the set of oligonucleotide-peptide conjugates under conditions that result in the specific hybridization of the oligonucleotide moieties to the one or more target nucleic acids, if present; (c) separately contacting an aliquot of the test sample with each member of the antibody set,
  • the preferred practice of this method is wherein the antibodies are attached to a solid support and the step of contacting the test samples with the antibodies results in separately capturing each member of the set of complexes on the solid support.
  • an assembly of compositions for detecting one or more selected target nucleic acids in, or separating one or more selected target nucleic acids from, a test sample suspected of containing the one or more target nucleic acids.
  • This assembly comprises (a) one or more oligoncleotide-peptide conjugates, each comprising a peptide moiety and an oligonucleotide moiety that specifically hybridizes to one of the target nucleic acid molecules; and (b) one or more antibodies, each being immunologically specific for only one of the one or more oligonucleotide-peptide conjugates.
  • this assembly is prepared for detecting or separating two or three or more different nucleic acids.
  • a test kit for detecting one or more selected target nucleic acids in, or separating one or more selected target nucleic acids from, a test sample suspected of containing the one or more target nucleic acids.
  • the kit comprises a container containing: (a) instructions for preparing one or more oligoncleotide- peptide conjugates, each comprising a peptide moiety and an oligonucleotide moiety that specifically hybridizes to one of the target nucleic acid molecules; and (b) one or more antibodies, each being immunologically specific for only one of the one or more oligonucleotide-peptide conjugates.
  • the kit contains the peptide moieties of the one or more oligonucleotide-peptide conjugates (preferably each bound to a solid support) , and instructions for completing preparation of the conjugates.
  • the kit may contain the one or more oligonucleotide-peptide conjugates fully prepared, instead of the instructions for preparing the conjugates.
  • Such test kits also may include, optionally, reagents for preparing the oligonucleotide-peptide conjugates, and instructions and reagents for using the conjugates and antibodies to detect or separate one or more target nucleic acids. Additional features and advantages of the present invention will be better understood by reference to the detailed description and examples set forth below.
  • a system for 1) the detection of a multiplicity of specific sequences located within a target nucleic acid molecule e.g. , PCR product or non- amplified DNA or RNA
  • a target nucleic acid molecule e.g. , PCR product or non- amplified DNA or RNA
  • 2) the detection and differentiation of PCR products from a multiplex reaction or 3) the separation of specific nucleic acids from a heterogeneous solution.
  • a synthetic molecule which is a combination molecule comprising an antigenic peptide moiety and an oligonucleotide moiety (the combination molecule is referred to hereinafter as an "OPC" - oligonucleotide-peptide conjugate) .
  • the oligonucleotide moiety is designed to specifically hybridize with a predetermined target nucleic acid (DNA or RNA) .
  • the peptide moiety either by itself, or in combination with adjuvants, carriers, or the oligonucleotide moiety, serves as an antigen for the production of immunologically specific antibodies.
  • immunologically specific refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • biotinylated nucleic acids where the biotin is detected indirectly with streptavidin-labeled conjugates
  • digoxigenin labeled nucleic acids where the digoxigenin serves as an antigen
  • FITC labeled DNA where the FITC can act as a fluorochrome or as an antigen
  • Those systems are limited by the lack of diversity among the labels, thereby preventing the use of multiple probes for solution based assays (i.e., it is impossible to differentiate between two different probes labeled identically) .
  • oligonucleotide-peptide conjugates as probes, multiple probing can be accomplished since each probe has a unique label.
  • PCR products generated from a primer set may have many potential sequence variations within the internal portion of the molecule (i.e., allelic variations, as with the HLA genes) .
  • allelic variations as with the HLA genes
  • separate assays would have to be performed to detect each sequence variation.
  • these variations can be differentiated by using a battery of oligonucleotide- peptide conjugates, each conjugate being designed to hybridize to a particular allele while possessing a unique antigenic marker. This has important applications in detection and diagnostic testing, as it enables high- throughput solution-based multiple screenings in a single test sample.
  • the system for using the OPCs of the invention revolves around a molecule in which the oligonucleotide is homologous to a DNA sequence to be detected and the peptide has been used as an antigen for the production of peptide antibodies.
  • oligonucleotides or peptides can be used in this conjugate, thus allowing tremendous flexibility and variety within this synthetic molecule. Any peptide which can be used to generate a specific antibody can be applied in this system, as well as any oligonucleotide which will specifically hybridize to a target DNA or RNA sequence.
  • An antibody immunologically specific for the peptide portion of the conjugate, whether monoclonal or polyclonal, is an integral part of the system of the present invention.
  • Such antibodies are produced by standard methods.
  • Monoclonal antibodies may be prepared according to general methods of K ⁇ hler and Milstein, following standard protocols.
  • the advantage of the OPCs of the invention is that multiple detections or purifications (sometimes referred to herein as "multiplex reactions") can be achieved from a single test sample, or from a plurality of similar test samples (e.g., blood samples from several patients being tested for allelic anomalies in a particular gene) . Accordingly, the invention contemplates generating a group or set of OPCs.
  • the term “set” means, for purposes of this invention, two or three or more OPCs.
  • the term “set” is sometimes substituted with the terms “multiplicity”, “plurality”, “series” or “array”, all of which are used interchangeably. It will be understood, however, that each OPC can be used individually in a manner similar to digoxigenin-based techniques, for example.
  • OPCs there is no upper limit to the number of OPCs that can be generated for a particular array.
  • preferred for the practice of the present invention are multiples that are convenient for commercially available assay kits, such as 96-well microtiter plates.
  • a six- OPC array can be generated in which each OPC has a unique peptide attached to an oligonucleotide specific for DNA from a specific mycoplasma contaminant.
  • oligonucleotide moiety of the OPC should be of a size sufficient to effect specific hybridization with the target nucleic acid, without an excess of "noise” or false positives.
  • specific hybridization means the association between two single- stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single- stranded nucleic acids of non-complementary sequence.
  • Oligonucleotides between 10 and 100 bases in length are preferred for practice of the invention. Particularly preferred are oligonucleotides between 15 and 30 nucleotides, and most preferably 18-24 nucleotides long.
  • the peptide moiety should be of a size that is relatively convenient to prepare synthetically, though this is not an absolute requirement. Moreover, it must be of a size to be antigenic, either alone or in combination with an adjuvant, or in a modified form (e.g., carrying protecting groups as discussed in greater detail below) .
  • Peptides between 6 and 30 residues in length are preferred for practice of the invention, and peptides most preferably are between 12 and 14 residues.
  • peptides are generated which are strongly antigenic.
  • weakly antigenic peptides may also be used, since numerous methods have been utilized for stimulating general immune responses for weakly antigenic substances.
  • adjuvants such as complete Freund's and Ribi's, have long been used for this purpose.
  • Another approach to stimulating the immunogenicity of a weakly-antigenic peptide or protein has been to couple the weak antigen to a carrier protein that is known to be a good immunogen.
  • Common carrier proteins include keyhole limpet hemocyanin, fowl gamma- globulin and bovine serum albumin.
  • the immunogencity of a weak antigen may be enhanced by polymerizing it into large aggregates by way of cross- linking agents, such as glutaraldehyde.
  • cross- linking agents such as glutaraldehyde.
  • solid-phase resins and peptide synthetic methods may be employed to synthesize a peptide repeatedly, to form a highly-branched complex.
  • oligonucleotide moiety and the peptide moiety can be synthesized together, or synthesized separately and linked together, in different orientations as follows: 5'-oligo-3' N terminus-peptide-C terminus
  • linkages are contemplated for use in the present invention. It should be understood that the oligonucleotide moiety and the antigenic moiety must be functionally linked to one another.
  • the term "functionally linked” is defined generally as linking of the moieties in such a way that each moiety retains its intended function.
  • oligonucleotide-peptide conjugates As described in accordance with the present invention heretofore has not been described.
  • the synthesis of a hybrid DNA/peptide molecule can be accomplished by several different approaches. For instance, the use of modified oligonucleotides as probes is very common and well documented.
  • One suitable location for the linkage between the oligonucleotide and peptide is the 5' or 3' ends of the oligonucleotide and an available amine, carboxy, or thiol on the peptide.
  • the specific location of the linkage on the peptide will depend on the primary structure of that molecule. Suitable synthesis options are outlined below: l. Organic Solvent Based/Protected Fragments a. Continuous synthesis of DNA-Peptide on resin by solid phase synthesis; b. Synthesis of protected DNA on resin followed by fragment coupling of protected peptide. 2. Native/Semi-Protected Synthesis a. Solid phase coupling of DNA and peptide in aqueous solution; b. Coupling of DNA and peptide in aqueous solution.
  • oligonucleotide- peptide chimeric molecule is synthesized continuously on a solid phase.
  • Fragment coupling involves separate synthesis of the DNA and peptide followed by coupling of the fragments.
  • the chemoselective ligation of peptide and oligonucleotide fragments is preferred.
  • a ino acids are coupled to the oligonucleotide by standard solid phase Fmoc chemistry.
  • the oligonucleotide is the support for peptide synthesis. Assuming the peptide (amide) backbone (this can be determined experimentally) is resistant to the deprotection conditions required to remove the pac- amidite protecting groups (Harlambidis et al., Nucl.
  • the oligonucleotide is synthesized using pac-amidites on a standard resin followed by amidating the 5' hydroxyl and extending the chain using carbodiimide, or carbodiimide/HOBt, or carbodiimide/active ester chemistry (e.g., NHS active esters) .
  • the protecting groups on the peptide portion are removed using TFA followed by deprotection and cleavage from the resin of the oligonucleotide-peptide chimeric using ammonia. If the oligonucleotide portion of the molecule suffers depurination as a result of TFA exposure, an alternative step is to build peptide portion with no side chain protection, therefore, no TFA treatment is necessary.
  • Trityl protection is already available for several amino acids and could be used to protect lysine, histidine, and cysteine, arginine, glutamine, asparagine, and possibly serine, threonine, and tyrosine. If unavailable, synthesis should not be difficult.
  • a more orthogonal (chemoselective) approach is used.
  • the oligonucleotide is extended in the usual fashion, subsequently amidated, and then deprotected.
  • the peptide is then extended in the normal fashion using active esters to form the peptide bonds.
  • Active esters are selective molecules and should not cross react with the deprotected bases on the oligonucleotide. This would obviate the need of exposing the peptide to the ammonia conditions of oligonucleotide deprotection.
  • the final step is removal of the oligonucleotide- peptide conjugate from the resin support.
  • a linker not requiring ammonia cleavage is necessary.
  • N-Fmoc strategy with Merrifield protecting groups (benzyl based side chain protection) .
  • the benzyl protecting groups are esters, and as such are labile to base hydrolysis. They may actually be an improvement when compared to the lability to ammonia hydrolysis of t-butyl protection used in the FMOC chemical strategy.
  • Benzyl or butyl protected peptides are tested for base hydrolysis lability by mixing protected amino acid with ammonia (oligo deprotection conditions) ; analyze on reverse phase at 220 nm or following derivatization with PITC. It should be noted that this combination of benzyl protecting groups and N-Fmoc synthesis yields a new type of amino acid synthesis process.
  • Synthesizing the peptide first also requires building the peptide on a resin labile to base hydrolysis, such as the Merrifield Resin, where the linkage between peptide and resin is a benzyl ester.
  • the peptide is constructed so that the only primary amine is limited to the N terminal amino acid. This primary amine acts as the reacting group for the coupling of activated nucleotide phosphoramidites in a standard DNA synthesis. In this manner, the peptide is synthesized followed by the direct addition of the protected nucleotides.
  • An oligonucleotide with a peptide located on the 3' end of the molecule results. Again, problems with TFA exposure and Ammonia deprotection would be the same as described above.
  • Blocking the 3' end of the oligonucleotide is useful for preventing mispriming if the conjugate is added to PCR reactions.
  • a multiple chain peptide can be used as an antigen, thus increasing the immunogenicity of the peptide and hence the signal.
  • both the oligonucleotide-peptide conjugate combined can be used as an immunogen to further enhance antibody binding.
  • Synthesizing the peptide followed by DNA provides several additional options. In this methodology, the peptide requires protecting groups which can be cleaved by base hydrolysis. This is fundamentally important since the phosphoramidites used for DNA synthesis must be coupled to only one primary amine or hydroxyl on the peptide.
  • the continuous solid phase method is a practical method of synthesis for large scale production of the conjugate. Since researchers will presumably be synthesizing their own unique oligonucleotide probes, with the continuous solid phase technique it would be necessary to provide the peptide sequence for synthesis along with the corresponding antibody used for detection. Alternatively, a series of solid resins to which the respective peptides are attached, prepared for oligonucleotide synthesis thereon, is produced. The users of the OPCs simply follow instructions on initiation of oligonucleotide synthesis, thereby preparing OPCs customized in their laboratories for a particular assay or purification.
  • Solid Phase Chemoselective Ligation of Fragments Coupling fragments where one fragment is immobilized on a solid phase possesses all the advantages of simplified sample handling associated with solid phase methods in general. Immobilizing DNA (either covalent or passive) to a resin allows for coupling of the peptide followed by efficient removal of unreacted peptide. The reverse is also true.
  • One reason why the chemoselective ligation technique is preferred over the continuous (solid phase stepwise) approach is the already substantial body of work published in the last five years or so concerning the synthesis of multiple antigenic peptides (MAPs) using, among other things, chemoselective ligation of unprotected peptides.
  • MAPs are synthetic proteins showing promise in both biochemical and biomedical applications.
  • Chemoselective ligation also referred to as segment or fragment condensation, has been a topic for research in synthetic peptide chemistry for many years.
  • the technique of constructing large peptides by ligating shorter, protected peptide fragments has not been particularly successful, partly due to the limited solubility of the protected peptide fragments.
  • the use of unprotected peptides as ligation components has improved the situation since these molecules are water soluble and coupling is conducted in aqueous solutions at high concentrations.
  • the thiol and weak base chemistries appear to offer the most promise.
  • Several methods are available for coupling, the first employing a bifunctional cross-linking reagent and the second involving direct activation of one of the fragments.
  • the practical choices for peptide functional groups for coupling are the N-terminal amine, the lysine sidechain amine, the cysteine thiol, the carboxyl groups of glutamic and aspartic acids, and the terminal carboxyl group.
  • oligonucleotides which may also be modified as stated above, can be modified at both the 5' and 3' ends with the addition of amines, carboxyls and thiols.
  • Modified oligonucleotide bases may also be added to the internal portion of the oligonucleotide, such as, but not limited to, 4-thio-uracil deoxyriboside, which can also serve as sites for peptide coupling
  • modified groups are normally attached to carbon linkers which will serve to separate the conjugated molecules.
  • the simplest coupling of DNA and peptide will make use of either a homobifunctional (e.g., glutaraldehyde) or heterobifunctional cross-linker. With cross-linking, one of the molecules is mixed with excess linker and reacted with one end of the activated agent. A separation is required to remove the unreacted cross-linker from the activated molecule.
  • That molecule is then coupled to the second half of the chimera. Due to the potential for peptide-peptide or DNA-DNA coupling, using a homobifunctional agent is least desirable.
  • a heterobifunctional cross-linking reagent allows for only one conjugated product. It is feasible and practical to activate either the DNA or peptide for the coupling.
  • a free carboxyl group of the peptide can be modified to an active ester by treatment with carbodiimide/HOBt or carbodiimide and N-hydroxysuccinimide.
  • a cyclic anhydride succinic anhydride, for example
  • a peptide will always contain a free c-terminal carboxyl group unless it is a peptide amide, CONH 2 .
  • 2, 2'-dithiodipyridine in an aqueous buffer yields an activated thiol.
  • This is a chemoselective active group specific for thiol groups.
  • DNA in which a free thiol is added will then couple to the peptide containing the activated disulfide. This method is less energetic, but should be quite selective.
  • the DNA thiol is activated and coupled to the peptide containing a free thiol.
  • cysteine-containing peptides it is important to deprotect using TFA containing either ethanedithiol or mercaptoethanol to prevent thiol alkylation thus insuring the presence of free thiol in the deprotected peptide.
  • the first approach for ligation is to chemoselectively couple the unprotected peptide component (either C or N terminus) to the 5' end of the oligonucleotide.
  • Chemoselectivity in this case simply means the peptide contains a nucleophile specific for an electrophile located at the 5' end of the oligonucleotide.
  • the oligonucleotide-peptide conjugate is the only product formed because no oligo-oligo or peptide-peptide coupling is possible. This approach is preferable from a commercial perspective since the unprotected peptide, along with any ancillary reagents required, could be supplied as a kit.
  • the peptide is added as a unit to either the hydroxyl, aminated, phosphorylated, or thiolyated 5' end of the oligonucleotide.
  • a bifunctional cross-linking reagent is reacted with the DNA and results in an activated 5' thiol, amine, carboxylic acid, or aldehyde which can be used to couple to the peptide chemoselectively.
  • a thiolated DNA can be linked to the peptide N terminal amine with the bifunctional cross-linker SPDP.
  • the immobilized DNA is first treated with the bifunctional cross-linker, washed to remove excess reagent, and then reacted with the peptide. Activating the immobilized molecule simplifies the removal of excess cross-linking reagent.
  • the deprotected peptide in solution is attached to a deprotected, resin-bound oligonucleotide, assuming the covalent bond connecting the oligonucleotide to the resin support is not base labile.
  • the oligonucleotide- resin is deprotected, e.g., with ammonia, the unprotected peptide ligated to the deprotected oligonucleotide-resin, followed by removal of the oligonucleotide-peptide conjugate from the solid support. In this process, the peptide is not exposed to the ammonia, which is a desirable feature of the process.
  • Such a linker may comprises, for instance, a disulfide linkage with diimide coupling of the oligonucleotide and the peptide.
  • a post-synthetic approach is for the person utilizing the conjugate to synthesize the oligonucleotide, deprotect in the customary fashion, and then non-covalently (and reversibly) link the oligonucleotide to a membrane or beaded polymer solid support. Both modified 5' and 3' ends, or modified bases, of the oligonucleotide can be made available for ligation, and the unprotected peptide is then chemoselectively ligated to the bound oligonucleotide. The oligonucleotide-peptide conjugate is then released from the solid support.
  • An exemplary protocol for the above strategy is:
  • immobilizing the peptide for the ligation could be a practical approach to conjugate formation.
  • the user of the conjugate synthesizes an oligonucleotide probe in the customary way with the final step being attachment of a functional group to the oligonucleotide (e.g., NH2 , SH, etc.), which is necessary for ligation to the peptide.
  • the unprotected peptide is provided either covalently bound to a resin, or passively adsorbed to a stationary phase.
  • the oligonucleotide probe, deprotecting in the usual fashion, is then ligated to the unprotected peptide.
  • solution synthesis The characteristics of solution synthesis are: (1) the coupling reaction occurs in a homogeneous solution; (2) the molecules are coupled using same chemistries as solid phase methods; and (3) separation of the chimeric molecules from unreacted materials is required.
  • solution based coupling is a viable alternative for practicing the present invention, solid phase coupling of fragments will likely be more attractive to most practitioners of the invention, due to the simplified means for obtaining the conjugates.
  • the OPC system comprises a plurality of OPCs, for use in detecting multiple nucleic acid targets in, and/or separating (sometimes referred to herein as “capturing") such targets from, a heterogenous mixture of biological or other molecules (e.g., biological fluids, water or food samples, and the like) .
  • detection means sometimes used interchangeably with the terms “label”, “detectable label” or “reporter substance” refers to any substance whose detection or measurement, either directly or indirectly, by a physical or chemical means, is indicative of the presence of an OPC complex.
  • reporter substances include, but are not limited to: molecules or ions directly or indirectly detectable based on light absorbance (“chromophores") , fluorescence (“fluorophores”) , reflectance, light scatter, phosphorescence or luminescence properties; molecules or ions detectable by their radioactivity properties; and molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties; and molecules detectable by their enzymatic properties. Referring to capturing or separating, in some embodiments this is facilitated by immobilizing a capture agent on a solid support. As described in greater detail below, the capture agent can be the specific anti-OPC antibody. In other embodiments, the capture agent may comprise streptavidin, protein A, or another antibody, such as an anti-DNA/RNA hybrid antibody.
  • solid support can also mean an insoluble fluid support.
  • solid supports useful for the practice of the present invention include, but are not limited to: membranes such as nitrocellulose, polystyrene or nylon membranes; microbeads or microparticles, such as agarose or latex beads, or magnetic or paramagnetic beads; and magnetic or paramagnetic fluids.
  • a series of OPCs is designed, each member having a different oligonucleotide and peptide component, respectively.
  • the oligonucleotide component of the OPC is made detectable or capturable (e.g. by labeling with biotin, which can be detected or captured via its interaction with streptavidin) .
  • the population of nucleic acid molecules containing (or suspected of containing the target) can be made detectable or capturable.
  • Antibodies that immunospecifically bind to each OPC in the series are generated. These can be used to detect or capture the OPC in an OPC-specific manner.
  • each OPC is detectable or capturable by either the oligonucleotide moiety or the peptide moiety.
  • the system is used as follows:
  • a test sample containing (or suspected of containing) one or more of the nucleic acids to which one of the OPC series hybridizes is provided.
  • the series of OPCs is added to the test sample (s) , under conditions that permit the specific hybridization between the OPCs and their respective target nucleic acids, if present. 3.
  • the OPCs are contacted with the series of antibodies immunologically specific for the respective members of the OPC series, under conditions permitting the immunological reaction to occur. Steps 2 and 3 can occur in either order, but that the result is a three- part complex comprising the target nucleic acid, the OPC, and its respective antibody.
  • NA nucleic acid
  • the OPC antibodies are immobilized on a solid support (e.g., each different antibody is bound to a well of a icrotiter plate) , such that Step 3 above results in capture and sequestration of respective OPCs in the series.
  • the presence or amount of OPC complex thus immobilized and sequestered may then be determined.
  • Table 2 below sets forth several preferred ways of detecting the captured nucleic acids.
  • RNA DNA/RNA Anti-DNA/RNA Such antibodies Hybrid Antibody are available
  • a separate Probe Conjugate labeled probe is hybridized to a common sequence on the selected molecule Using one of these approaches to detect selected nucleic acids, the steps of the method would be: (1) hybridize the OPCs to the respective targets; (2) immunospecifically capture the hybridized OPCs; and (3) detect the selected targets via one of the labels described above.
  • the nucleic acid detection means set forth in Table 2 can also be used as a capture means. Accordingly, in another embodiment of the invention, the system could be used as follows: (1) hybridize the OPCs to the respective targets; (2) capture the entire population of targets (i.e. as a way to separate the nucleic acids from other types of molecules in the sample) ; (3) optionally, release the population of targets; and (4) differentially label individual targets by immunospecifically binding the respective antibodies, each of which has been differentially labeled, e.g., with a unique reporter substance, such as a chromophore or fluorophore. Steps 1, 2 and 3 could be performed in any order.
  • the end product is a solution of OPC complexes, each hybridized to a different target and labeled with a unique label.
  • a single sample of the solution could be assessed for the presence or amount of a selected target nucleic acid; e.g., spectrophotometrically or spectrofluro etrically at the appropriate wavelengths for the chromophores or fluorophores used.
  • nucleic acid/OPC/antibody complexes can be immunoprecipitated, then resuspended and measured as described above.
  • test kits for use in detection and purification of nucleic acids, or for therapy in delivering certain nucleic acids. Described below are several preferred embodiments for test kits.
  • a test kit will comprise (1) the relevant series of peptide antigens, or instructions (i.e., sequence information) for synthesizing the antigens; and (2) the respective immunologically specific antibodies.
  • the antibodies provided in the kit are used for the detection or purification.
  • the antibodies are provided immobilized to a substrate, so that the OPC/target nucleic acid complex can be affinity captured.
  • the antibodies will be provided already differentially labeled, e.g., with a series of chromophores or fluorophores.
  • the test kit in a situation where a continuous synthesis of the conjugate, starting with the oliognucleotide, is to be performed, the test kit will contain the antibodies (optionally labeled or immobilized on a solid support) and sequence information for the array of peptide antigens and instructions on how to conduct the synthesis.
  • the user of the OPC will design appropriate oligonucleotides for a selected separation or detection and will synthesize the entire set of OPCs according to the instructions provided.
  • the test kit contains the antibodies and either (1) instructions and sequence information for the peptides or (2) the peptides themselves, immobilized on a resin suitable for continued synthesis of the oligonucleotide moiety.
  • the user of the OPCs designs appropriate oligonucleotides for a selected separation or detection.
  • the kit may contain the antibodies and (1) the pre-made peptides, either in solution or on a solid support; and (2) the linking agent and instructions for use.
  • the user designs and synthesizes the oligonucleotides, then links them to the peptides according to the instructions provided in the kit.
  • the kit contains the antibodies and a set of ready-made OPCs each with a unique peptide moiety linked to an oligonucleotide probe designed to identify a particular target nucleic acid (e.g. an established allelic anomaly) .
  • OPCs will find numerous applications in addition to those described above and exemplified below.
  • OPCs can be used as primers for PCR, and products which are amplified from OPC primers can be affinity purified using an immobilized antibody column, much the same as any antigen. This could be very useful in isolating differential display products and expressed sequence tags.
  • the conjugate will also find use as an anti-sense therapeutic with the peptide serving as a ligand for a specific cell based receptor.
  • Binding of the ligand to the receptor may be a means of targeting a specific antisense oligonucleotide for a given cell. If the chimeric oligonucleotide-peptide is linked with a disulfide bond, release of the oligonucleotide would occur up assimilation of the molecule into the reduced cellular environment.
  • PCR Located within a PCR Product
  • the analysis of genetic variation is fundamental to many diagnostic tests, including cystic fibrosis, HLA, and Alzheimer's testing. Often PCR can be performed on specific regions of DNA with the primer set being designed to flank and amplify the allelic variations. In this manner, one conserved primer set can amplify many or all of the genetic variations of a gene.
  • the region located within the PCR product, i.e., between the primers, is the target for the analysis.
  • biotinylated primer e.g., the forward primer
  • the plus strand of the product can be labeled.
  • This 5' biotinylation is a very common technique used to label PCR products.
  • this discussion will examine biotinylated PCR products, other labels are available as well, including digoxigenin and fluorescent tags, such as FITC.
  • a set of oligonucleotide-peptide conjugates are designed to hybridize specifically to the variations of the biotinylated PCR strand.
  • Each oligonucleotide-peptide conjugate will have specificity to a particular allele while possessing the unique antigen used to raise a specific peptide antibody.
  • the oligonucleotide-peptide conjugates will hybridize to their homologous sequences, i.e., if those sequences are present, and indirectly label those sequences with a unique antigen.
  • the resulting hybrid will possess a biotinylated plus strand hybridized to a oligonucleotide which is linked to a specific antigenic marker. Consequently, each sequence variation in the PCR product will be associated with a specific antigen.
  • biotin-PCR product/oligonucleotide-peptide conjugate complex Several strategies and formats can be used to detect this biotin-PCR product/oligonucleotide-peptide conjugate complex.
  • One approach could simply involve adsorbing each peptide antibody to a well of a microtiter plate, so to make each well a specific immunoassay for a specific nucleic acid sequence.
  • the biotin-PCR product/oligonucleotide-peptide conjugate complex would be added to each well, where the complex would bind to the immobilized antibodies.
  • the complex could be detected in a standard enzyme immunoassay format in which a streptavidin- enzyme is used to detect the biotin label.
  • An alternative method may involve capturing the biotinylated strand with immobilized streptavidin and then adding differentially labeled antibodies. This would be particularly applicable to antibodies differentially labeled with fluorescent tags, with each fluorophor having a distinct emission.
  • Magnetic beads with antibodies against pathogenic microorganisms are mixed with a test sample and vortexed. Using a magnet, the beads are pulled from the solution. The beads are washed to remove residual debris, suspended in a reverse transcriptase buffer, and heated to 95°C for 10 min. This heating step will lyse bacteria bound to the beads, thus releasing RNA molecules.
  • the RT buffer contains primers sets which are all labeled with a single peptide antigen and which are also specific for the microorganisms under investigation. Each primer set is targeted to an identifying sequence within the associated genome of that organism. These primers set will be used for both cDNA synthesis and PCR. Upon cooling the mixture, the beads and solution are separated using a magnet, with the solution being transferred to a PCR tube.
  • Reverse transcriptase is added to the solution, followed by an incubation during which cDNA is synthesized.
  • the solution is then adjusted for PCR by the addition of buffers, dNTPs, and Taq polymerase.
  • the tubes are opened and a solution of EDTA and oligonucleotide- peptide conjugated probes are added. These OPC probes are targeted against sequences located within the PCR product.
  • the tube is sealed, heated to denature the PCR products, and then cooled to an annealing temperature suitable for the hybridization of the OPC probes.
  • the addition of the EDTA is to chelate Mg ions and thus prevent the continued activity of the Taq polymerase.
  • the solution is added to a small glass tube containing a lyophilized antibody against the peptide label on the primers. This antibody is coupled to colored latex microbeads.
  • the antigenic PCR products and residual OPC primers are allowed to bind with the antibody-latex bead conjugate.
  • a dipstick with the sequence specific anti-OPC antibodies immobilized to distinct locations is then added to the glass tube. With dipstick assays, a wick draws the test solution up the dipstick. As the antigens flow past the immobilized antibodies, the latex bead-PCR-OPC probes bind to their specific antibodies. As the complexes concentrate on the immobilized antibodies, the latex beads produce a visible color. This type of dipstick immunoassay is well documented and has been used to detect and differentiate numerous antigens in a simple low cost approach.
  • each primer set could contain a common label (e.g., biotin) which will bind to the antibody-latex bead conjugate, and a peptide label specific for that PCR product.
  • biotin e.g., biotin

Abstract

L'invention concerne des compositions et des procédés permettant de détecter ou de purifier une multiplicité de molécules d'acide nucléique sélectionnées. Les compositions comprennent des conjugués oliogonucléotide-peptide (OPC) et des anticorps immuno-spécifiques à ces conjugués. La fraction oligonuclotide de chaque conjugué s'hybride spécifiquement avec une molécule d'acide nucléique cible sélectionnée. La fraction peptide agit comme antigène pour la production d'un anticorps spécifique qui se lie exclusivement à son OPC respectif. Les OPC sont utilisés avec avantage en groupes, ce qui permet la détection ou la séparation de multiples acides nucléiques cibles dans un seul échantillon grâce à l'interaction antigène/anticorps spécifique qui rend possible une détection ou une capture différenciée des OPC. L'invention concerne également des procédés et des assortiments de matériel permettant l'utilisation des systèmes OPC décrits.
PCT/US1998/014782 1997-07-16 1998-07-16 Conjugues oligonucleotide-peptide synthetiques et procedes d'utilisation WO1999004042A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2000056926A2 (fr) * 1999-03-19 2000-09-28 Valigen (Us), Inc. Detection de polymorphismes d'acides nucleiques au moyen d'oligocucleotides marques peptides et de batteries d'anticorps
JP2019523000A (ja) * 2016-07-18 2019-08-22 セル アイディーエックス， インコーポレイテッド 抗原をカップリングさせたハイブリダイゼーション試薬
US11867696B2 (en) 2015-02-06 2024-01-09 Cell Idx, Inc. Antigen-coupled immunoreagents

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US5470705A (en) * 1992-04-03 1995-11-28 Applied Biosystems, Inc. Probe composition containing a binding domain and polymer chain and methods of use
US5525465A (en) * 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
US5604097A (en) * 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags

Patent Citations (3)

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US5525465A (en) * 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
US5470705A (en) * 1992-04-03 1995-11-28 Applied Biosystems, Inc. Probe composition containing a binding domain and polymer chain and methods of use
US5604097A (en) * 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056926A2 (fr) * 1999-03-19 2000-09-28 Valigen (Us), Inc. Detection de polymorphismes d'acides nucleiques au moyen d'oligocucleotides marques peptides et de batteries d'anticorps
WO2000056926A3 (fr) * 1999-03-19 2002-01-03 Valigen Us Inc Detection de polymorphismes d'acides nucleiques au moyen d'oligocucleotides marques peptides et de batteries d'anticorps
US6403309B1 (en) 1999-03-19 2002-06-11 Valigen (Us), Inc. Methods for detection of nucleic acid polymorphisms using peptide-labeled oligonucleotides and antibody arrays
US11867696B2 (en) 2015-02-06 2024-01-09 Cell Idx, Inc. Antigen-coupled immunoreagents
JP2019523000A (ja) * 2016-07-18 2019-08-22 セル アイディーエックス， インコーポレイテッド 抗原をカップリングさせたハイブリダイゼーション試薬
EP3485275A4 (fr) * 2016-07-18 2020-06-17 Cell IDX, Inc. Réactifs d'hybridation couplés à un antigène

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