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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate

Definitions

• This invention describes a novel method for conjugating oligonucleotides to other molecular entities exclusively at the 5'-position of the oligonucleotide.
• the method of this invention takes advantage of an enzymatic method of synthesizing RNA via an RNA polymerase.
• SELEX Systematic Evolution of Ligands by Exponential Enrichment
• nucleic acid ligands also referred to in the art as "aptamers”
• aptamers each ligand having a unique sequence and property of binding specifically to a desired target compound or molecule.
• the SELEX method involves selection from a mixture of candidate oligonucleotides and step- wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
• the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding. partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes.
• the SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands containing Modified Nucleotides," abandoned in favor of United States Patent Application Serial No. 08/430,709, now United States Patent No.
• the SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,” now United States Patent No. 5,637,459 and United States Patent Application Serial No. 08/234,997, filed April 28, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,” now United States Patent No. 5,683,867, respectively.
• These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
• the SELEX method encompasses complexes of oligonucleotides.
• Nucleic acid ligands derived by the SELEX process have been used in diagnostic applications.
• oligonucleotides and oligonucleotide analogs are used as diagnostic and research reagents and as potential therapeutic agents.
• antisense oligonucleotides are used to bind to certain coding regions in an organism to prevent the expression of proteins or to block various cell functions.
• ribozymes the discovery of RNA species with catalytic functions — ribozymes — has led to the study of RNA species that serve to perform intracellular reactions that will achieve desired effects.
• SELEX process the discovery of the SELEX process
• antisense oligonucleotides as a means for controlling gene expression and the potential for using oligonucleotides as possible pharmaceutical agents has prompted investigations into the introduction of a number of chemical modifications into oligonucleotides to increase their therapeutic activity and stability. Such modifications are designed to increase cell penetration of the oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA and/or to improve the pharmacokinetic properties of the oligonucleotides.
• RNA secondary and tertiary structures can have important biological functions (Tinoco et al (1987) Cold Spring Harb. Symp. Quant. Biol. 52: 135; Larson et al. (1987) Mol. Cell. Biochem. 74:5; Tuerk et al. (1988) Proc. Natl. Acad. Sci. USA 85:1364; Resnekov et al. (1989) J. Biol. Chem. 264:9953).
• RNA Mimicry describes oligonucleotides or oligonucleotide analogs which mimic a portion of RNA able to interact with one or more proteins.
• the oligonucleotides contain modified internucleoside linkages rendering them nuclease-resistant, have enhanced ability to penetrate cells, and are capable of binding target oligonucleotide sequences.
• oligonucleotides as therapeutic and diagnostic agents is growing rapidly with many compounds in preclinical and human clinical trials.
• the oligonucleotide is derivatized or conjugated with another molecular entity.
• conjugations are typically performed for the purpose of attaching fluorescent dyes or other diagnostic reporter groups or for attaching compounds that modulate the activity or the pharmacokinetic behavior of the oligonucleotide.
• Smith et al. describe the synthesis of fluorescent dye- conjugated primers for use in fluorescence-based DNA sequence analysis (Smith et al. (1987) Methods Enzymol. L55: 260-301).
• Oligodeoxynucleotides containing a terminal amino functionality have been utilized for the construction of bioconjugates with novel properties.
• a primary aliphatic amine group is incorporated at the 5 '-terminus of the oligonucleotide in the final step of the assembly of a synthetic oligonucleotide (Tung et al. (1991) Bioconjugate-Chem. 2:464-465; Smith et al. (1987) Methods Enzymol. 155:260-301).
• a commercial reagent for linking to the 5' terminus of an oligonucleotide is 5'-Amino-Modifier C6.
• These reagents are available from Glen Research Corp (Sterling, VA). These compounds have been used by Krieg (Krieg et al. (1971) Antisense Res. and Dev.L 161 ) to link fluorescein to the 5'-terminus of an oligonucleotide. Since many macromolecules of interest are hydrophilic, these reactions are generally done in water, requiring large excesses of reagent to overcome the competing hydrolysis.
• the amine on the oligonucleotide is added to the terminus of the molecule and must compete with free amine and alcohol on the fully deprotected oligonucleotide if this modification is done post-synthetically.
• the molecular entity is converted into a phosphoramidite, which is then added to the free alcohol of the full length oligonucleotide which is attached to a solid support. This method is less than ideal due to the air and water sensitivity of the phosphoramidite, as well as the fact that the molecule can only be added to the terminus of the oligonucleotide.
• a third method of conjugating oligonucleotides to other molecules is the coupling of an alkylthio derivatized oligonucleotide with a ⁇ -haloacetyl or with a maleimide containing compound. (Jones et al. (1995) J. Med. Chem. 38:2138). An alternative method for the synthesis of oligodeoxynucleotides terminated by 5'-amino-5'-deoxythymidine has been described (Bruick et al. (1997) Nucleic Acids Res. 25:1309-1310).
• This method uses a DNA template to direct the ligation of a peptide to an oligonucleotide, in which the peptide is presented by a second oligonucleotide in the form of a reactive thioester-linked intermediate.
• Oligodeoxynucleotides have been labeled for potential in vivo diagnostic imaging by two methods. Hnatowich has synthesized oligodeoxynucleotides with a primary amine on the 5 '-terminus then coupled peptidyl Tc chelates via NHS chemistry (Hnatowich (1995) J. Nucl. Med. 36:2306). Hayes et al.
• Conjugates of oligonucleotides with peptides having specific functions can be useful for various applications. Examples include the use of a nuclear transport signal peptide to direct intracellular trafficking (Eritja et al. (1991) Tetrahedron 47: 4113-4120); a hydrophobic peptide (Juby et al. (1991) Tetrahedron Lett. 32:879-822) or polylysine (Leonetti et al. (1991) Bioconjugate Chem. l:149-153) to increase cell penetrability, and polylysine to provide multiple attachment sites for nonradioactive reporting probes (Haralambidis et al. (1987) Tetrahedron Lett. 28:5199-5202; Haralambidis et al. (1990) Nucleic Acids Res. 18:493-499).
• a nuclear transport signal peptide to direct intracellular trafficking
• a hydrophobic peptide Juby et al. (1991) Tetra
• T7 RNA polymerase Transcription from synthetic DNA templates using T7 RNA polymerase is a convenient method for the synthesis of RNA oligonucleotides.
• the transcription of DNA by T7 RNA polymerase begins at a uniquely defined base relative to the promoter DNA sequence (Chamberlin and Ring (1973) J. Biol. Chem. 248:2235- 2244).
• the first nucleotide transcribed is usually a purine.
• the transcription of a DNA template into an RNA is distinct in that it results in a new RNA having a triphosphate at its 5' terminus.
• Martin and Coleman. Martin and Coleman(1989) Biochemistry 28:2760-2762).
• RNA transcript RNA transcript
• 5'- triphosphate is not utilized in a bond-formation step. That is, while Watson/Crick base-pairing is involved, the 5' region of the initial nucleotide is not involved in binding to the protein and/or to the DNA template.
• initiation of DNA transcription by T7 RNA polymerase proceeds effectively whether initiated with guanosine triphosphate (GTP), guanosine monophosphate (GMP) or guanosine.
• GTP guanosine triphosphate
• GFP guanosine monophosphate
• guanosine guanosine
• the present invention describes a novel and highly efficient method for derivatizing or conjugating oligonucleotides with other molecular entities. Specifically, the present invention describes a method for enzymatically generating oligonucleotides derivatized exclusively at the 5'-position of the oligonucleotide. using 5 '-substituted guanosines as initiators in the enzymatic synthesis of RNA.
• the methods disclosed herein allow for the addition of a variety of molecular entities — including but not limited to reactive molecules, reporter molecules, reporter enzymes, lipophilic molecules, peptides and proteins — to the 5'-terminus of nascent RNA oligonucleotides.
• the method of the instant can be described by the following steps: a) providing a DNA template; and b) combining the DNA template with nucleotide triphosphates, a 5 '-substituted guanosine and an RNA polymerase under conditions suitable for transcription.
• the initiating base on the RNA is a guanosine and the RNA polymerase is T7 RNA polymerase.
• the types of nucleotide triphosphates used will depend on the composition of the template and the desired RNA product.
• the method of this invention utilizes a 5'-modified guanosine monophosphate (GAP) as the initiator in an RNA polymerase-catalyzed template-dependent transcription.
• GAP guanosine monophosphate
• the guanosine initiator is modified at the 5'-position with a molecular entity whose chemical nature is compatible with RNA transcription.
• These guanosines can be substituted at the 5 '-position with molecular entities which differ greatly in size from the triphosphate group of a guanosine triphosphate.
• molecular entities that may be coupled to the oligonucleotide include, but are not limited to other macromolecules, such as oligonucleotides, lipophilic compounds, such as cholesterol, phospholipids, diacyl glycerols and dialkly glycerols, proteins, peptides or carbohydrates, polymers or resins, such as polystyrene, diagnostic detector molecules, such as biotin or fluorescein, reporter enzymes, photoaffinity labels, steroids, pharmacokinetic modulators such as PEG, lipids or liposomes, reactive moieties for post-transcriptional conjugation such as a hexylamine or a diene or dienophile, and chelates for binding metals.
• macromolecules such as oligonucleotides, lipophilic compounds, such as cholesterol, phospholipids, diacyl glycerols and dialkly glycerols, proteins, peptides or carbohydrates, polymers or resins, such
• the molecular entity can be designed to serve in a large variety of functions.
• a reporter group such as biotin or a fluorescent molecule may be incorporated into the bioconjugate to provide reporter bioconjugates for use as diagnostic reagents.
• a macromolecule such as a polyethylene glycol may be incorporated into the bioconjugate to provide a bioconjugate with improved pharmacokinetics.
• Chelates for binding metals particularly radioactive metals such as "m Tc can be attached to the oligonucleotide for diagnostic imaging purposes.
• Other radioactive metals, such as rhenium- 188 can be conjugated for directed radiotherapy applications.
• Bioconjugates may also comprise peptides which are reactive to an active site on a protein.
• haptens such as the Bolton- Hunter reagent can be incorporated to facilitate radio-iodination.
• Structural probes such as fluorescent quenching agents or spin labels can be incorporated to study protein-nucleic acid interactions.
• a photoaffinity label such as a psoralen, acridine, or a like molecule can be conjugated.
• a chemical entity such as a diene or Schiffs base could be incorporated for chemical covalent SELEX.
• the combinatorial small molecule library can be conjugated to the transcript.
• This application further discloses a method for generating bioconjugates comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5'-position of the nucleic acid ligands.
• This particular embodiment takes advantage of the method for identifying nucleic acid ligands referred to as SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment.
• bioconjugates to a target are identified by the SELEX method by the steps comprising:
• preparing a candidate mixture of bioconjugates by the steps comprising (a) providing a DNA template having a sequence to be transcribed and (b) combining the DNA template with nucleotide triphosphates, a 5'-modified guanosine, and an RNA polymerase under conditions suitable for transcription;
• bioconjugate candidate mixture 1) contacting the bioconjugate candidate mixture with a target, wherein bioconjugates having an increased affinity to the target relative to the bioconjugate candidate mixture may be partitioned from the remainder of the bioconjugate candidate mixture ; 3) partitioning the increased affinity bioconjugates from the remainder of the bioconjugate candidate mixture; and
• the 5'-substituted GAP can aid in (1) the SELEX partition step, e.g. BIA-SELEX
• Blended SELEX methodology United States Patent No. 5,683,867, issued November 04, 1997, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended
• This embodiment of the invention provides a method for identifying and synthesizing oligonucleotides derivatized with molecular entities, which are selected based upon the desired properties for the oligonucleotide, examples of which are described above.
• the 5 '-derivatized guanosine contains a reactive moiety which can be used for post-transcription conjugation of the transcript.
• This embodiment of the invention can be described by the following steps: a) providing a DNA template b) combining the DNA template with nucleotide triphosphates, a 5 '-substituted guanosine, wherein said 5 '-substituent contains a reactive moiety and an RNA polymerase under conditions suitable for transcription; and c) reacting the product from step b) with a molecular entity containing a moiety capable of reacting with the reactive moiety on said 5'- substituent.
• oligonucleotides derivatized with molecular entities which are not compatible with transcription.
• reactive moieties include but are not limited to amines, dienes, dienophiles, thiols, vinylsulfones, photoaffinity labels and interchelators.
• FIGURE 1 illustrates graphically the percent incorporation of GAP and the yield of GAP transcript at concentrations of GAP in the range between 0 and 10 mM.
• FIGURE 2 depicts the results of the GAP-TEG-biotin / ⁇ - 32 P-GTP initiation competition assay described in Example 2.
• the products of the transcription reactions were analyzed by denaturing gel electrophoresis. All lanes are labeled with the ratio of GAP-biotin to GTP, with the exception of lane C which contains 6 mM GTP as a control. As the concentration of GAP-biotin increased the ⁇ - 32 P-GTP decreased.
• FIGURE 3 shows the results of the Streptavidin shift assay described in Example 2.
• the reaction products were combined with Streptavidin and analyzed by denaturing gel electrophoresis. All lanes are labeled with the ratio of GAP-biotin to GTP.
• FIGURE 4A shows the results of the transcription reactions with GAP analogs 11-16.
• Lanes 1 and 2 contain the transcript without GAP
• lane 3 is the GAP- TEG transcript
• lane 4 is the GAP-biotin transcript
• lane 5 is the GAP-TEG-biotin transcript
• lane 6 is the GAP-Tc chelate transcript
• lane 7 is the GAP-TEG-Tc chelate transcipt
• lane 8 is the GAP-fluorescein transcript
• lane 9 is the GAP-TEG- fluorescein transcript.
• the analysis was performed on an 8% polyacrylamide gel containing 7 M urea.
• FIGURE 4B depicts the transillumination of the fluorescein-GAP initiated transcripts in lanes 8 and 9.
• FIGURE 5 shows the results of the "m Tc labeling of the GAP-Tc chelate initiated transcript.
• the "m Tc labeled transcript was analyzed by 8% polyacrylamide denaturing gel electrophoresis in the absence of EDTA.
• Lane 1 contains the 32 P full length transcript control and lane 2 contains the "m Tc labeled GAP-Tc chelate.
• the present invention includes a novel method for enzymatically generating oligonucleotide bioconjugates.
• this invention describes a novel method for enzymatically generating bioconjugates comprising RNA oligonucleotides derivatized specifically at the 5'-position with a molecular entity.
• This method utilizes 5'-substituted guanosines as initiators in RNA polymerase catalyzed template- directed synthesis of bioconjugates.
• the method of this invention can be used to conjugate an oligonucleotide prior to transcription or to incorporate a reactive moiety into the transcript which can then be used to bioconjugate the oligonucleotide post- transcription.
• This method may be applied to the synthesis of a variety of conjugated ribonucleotides including nucleic acid ligands, ribozymes and antisense RNA.
• the molecular entity can be any molecule, including another macromolecule, which is compatible with transcription.
• Examples of molecular entities that may be coupled to the oligonucleotide include, but are not limited to other macromolecules, such as oligonucleotides, lipophilic compounds, proteins, peptides or carbohydrates, polymers or resins, such as polystyrene, diagnostic detector molecules, such as biotin or fluorescein, reporter enzymes, photoaffinity labels, steroids, pharmacokinetic modulators such as PEG, lipids or liposomes, reactive moieties for post- transcriptional conjugation such as a hexylamine or a diene or dienophile, and chelates for binding metals.
• the molecular entity can be designed to serve in a large variety of functions.
• a reporter group such as biotin or a fluorescent molecule may be incorporated into the bioconjugate to provide reporter bioconjugates for use as diagnostic reagents.
• a macromolecule such as a polyethylene glycol may be incorporated into the bioconjugate to provide a bioconjugate with improved pharmacokinetics.
• Chelates for binding metals particularly radioactive metals such as "m Tc can be attached to the oligonucleotide for diagnostic imaging purposes.
• Other radioactive metals such a rhenium- 188, can be conjugated for directed radiotherapy applications.
• Bioconjugates may also comprise peptides which are reactive to an active site on a protein. Bioconjugates can also be used to attach the oligonucleotide to columns, solid support matrices, or surfaces such as microtiter plates.
• nucleoside means either a deoxyribonucleoside or a ribonucleoside or any chemical modifications thereof. Modifications of the nucleosides include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8- position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, and the like.
• Nucleotide as used herein is defined as a modified or naturally occurring deoxyribonucleotide or ribonucleotide. Nucleotides typically include purines and pyrimidines, which include thymidine, cytidine, guanosine, adenine and uridine. "Oligonucleotide” refers to a polynucleotide formed from a plurality of linked nucleotide units as defined above. The nucleotide units each include a nucleoside unit linked together, typically via a phosphate linking group. The term oligonucleotide also refers to a plurality of nucleotides that are linked together via linkages other than phosphate linkages. The oligonucleotide may be naturally occurring or non-naturally occurring. In a preferred embodiment the oligonucleotides of this invention have between 1-1,000 nucleotides.
• Nucleic acid ligand as used herein is a nucleic acid having a desirable action on a target.
• a desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating a reaction between the target and another molecule.
• the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.
• the nucleic acid ligand is a non-naturally occurring nucleic acid.
• the nucleic acid ligands are identified by the SELEX methodology.
• Nucleic acid ligands includes nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand- enriched mixture of nucleic acids.
• Nucleic acid means either DNA, RNA, single-stranded or double-stranded and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or the nucleic acid ligand as a whole.
• modifications include, but are not limited to, 2'-position sugar modifications, 5 -position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
• DNA template refers to a deoxyribonucleotide which provides instructions for an RNA polymerase to assemble a complementary ribonucleotide copy in a process termed "transcription.”
• the strand of DNA copied is called the "sense strand.”
• the DNA template strand also provides signals to initiate the copy synthesis by the enzyme at specific locations before the start of the sense strand and to terminate the copy synthesis at specific locations shortly after the end of the sense strand.
• the DNA template may be single-stranded or double-stranded. In a preferred embodiment, the DNA template is double-stranded.
• “Non-immunogenic, high molecular weight compound” is a compound of approximately 1000 Da or more that typically does not generate an immunogenic response.
• An immunogenic response is one that induces the organism to produce antibody proteins.
• non-immunogenic, high molecular weight compounds include polyethylene glycol (PEG); polysaccharides, such as dextran; polypeptides, such as albumin; and magnetic structures, such as magnetite.
• macromolecule refers to a large organic molecule.
• macromolecules include, but are not limited to nucleic acids, oligonucleotides, proteins, peptides, carbohydrates, polysaccharides, glycoproteins, lipophilic compounds, such as cholesterol, phospholipids, diacyl glycerols and dialkyl glycerols, hormones, drugs, non-immunogenic high molecular weight compounds, fluorescent, chemiluminescent and bioluminescent marker compounds, antibodies and biotin, etc without limitation.
• Bioconjugate refers to any oligonucleotide which has been derivatized with another molecular entity.
• the oligonucleotide is derivatized with a macromolecule.
• Bioconjugation or “Conjugation” refers to the derivatization of an oligonucleotide with another molecular entity.
• the "molecular entity” can be any molecule and can include a small molecule or another macromolecule.
• molecular entities include but are not limited to other macromolecules, polymers or resins, such as polyethylene glycol (PEG) or polystyrene, diagnostic detector molecules, such as biotin, fluorescein or coumarin, reporter enzymes, photoaffinity labels, steroids, pharmacokinetic modulators such as PEG, lipids or liposomes, reactive moieties for post-transcriptional conjugation such as a hexylamine or a diene or dienophile, and chelates for binding metals or any other modifying group.
• PEG polyethylene glycol
• diagnostic detector molecules such as biotin, fluorescein or coumarin
• reporter enzymes such as a fluorescent protein
• photoaffinity labels such as a hexylamine or a diene or dienophile
• pharmacokinetic modulators such as PEG, lipids or liposomes, reactive moieties for post-transcriptional conjugation such as a hexylamine or a diene or
• Diagnostic Agent means a bioconjugate which can be used for detecting the presence or absence of and/or measuring the amount of a target in a sample. Detection of the target molecule is mediated by its binding to a nucleic acid component of a bioconjugate specific for that target molecule.
• the bioconjugate can be labeled, for example radiolabeled, to allow qualitative or quantitative detection.
• “Improved pharmacokinetic properties” means that a bioconjugate shows a longer circulation half-life in vivo relative to a nucleic acid that is not part of a bioconjugate, or has other pharmacokinetic benefits such as improved target to non- target concentration ratio.
• Target refers to any compound upon which a nucleic acid can act in a predetermined desirable manner.
• a SELEX target molecule can be a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell, tissue, etc., without limitation. Virtually any chemical or biological effector would be a suitable SELEX target. Molecules of any size can serve as SELEX targets.
• a target can also be modified in certain ways to enhance the likelihood of an interaction between the target and the nucleic acid.
• RNA polymerase catalyzed DNA template-dependent transcription
• the enzyme uses one strand of DNA as a template to assemble a complementary RNA copy.
• the transcription of DNA by T7 RNA polymerase begins at a uniquely defined base relative to the promoter DNA sequence. No primer piece of RNA is required to start the copy synthesis. Successive nucleotide triphosphates are condensed such that the growth of the RNA copy is from the 5'-end to the 3'-end.
• the enzyme positions the first nucleotide (usually GTP or ATP) and the 3'-hydroxyl group of this nucleotide then reacts with the 5'-triphosphate of the incoming nucleoside.
• the 3'-hydroxyl group of the dinucleotide then condenses with the next nucleotide brought into position; and so on.
• the synthesis is driven forward by the hydrolysis of pyrophosphate.
• the present invention provides a method for the enzymatic synthesis of bioconjugates comprising RNA derivatized exclusively at the 5'-position with a molecular entity.
• the method of the instant invention can be described by the following steps: a) providing a DNA template and b) combining the DNA template with nucleotide triphosphates, a 5'-substituted guanosine and an RNA polymerase under conditions suitable for transcription.
• the types of nucleotide triphosphates used will depend on the composition of the template and the desired RNA product.
• the 5'-modified guanosine can only be added at the initiating 5'-end of the transcript during the initiation phase of transcription.
• transcript elongation is driven forward by the hydrolysis of pyrophosphate, therefore it is necessary that the remaining nucleotides be nucleoside triphosphates.
• the 5 '-substituted guanosine does not have a 5'- triphosphate group and as such it can participate in initiation, but not elongation. Therefore, in contrast to other methods of enzymatically incorporating substituted nucleotide triphosphates during RNA synthesis, wherein substituted nucleotide triphosphates are incorporated throughout the RNA transcript, the method of the present invention provides a unique method of synthesizing bioconjugates comprising a molecular entity attached exclusively to the 5'-position of an oligonucleotide.
• a 5'-derivatized guanosine (referred to herein as GAP) will compete with a GTP as the first component of the nascent RNA transcript.
• GAP 5'-derivatized guanosine
• a mixture of RNA oligonucleotides containing bioconjugates comprising 5'-substituted RNA oligonucleotides and 5'-unsubstituted RNA oligonucleotides will be obtained.
• concentration of the 5 '-derivatized guanosine in the transcription reaction relative to the GTP concentration, however, proportionally more derivatized guanosine will be incorporated into the transcript.
• a ratio of GAP:GTP of 10:1 results in 92 % of the transcript being initiated with GAP. Theoretically, if GAP and GTP are used as initiating nucleotides with equal efficiency, GAP should be present 90.91 % of the time. Depending on the required level of purity of the 5'-substituted transcript, the GAP-conjugate:GTP ratio can be varied.
• a 5 '-derivatized guanosine as a substrate in the enzymatic synthesis of an oligonucleotide bioconjugate offers significant advantages over currently available methods or synthesizing these compounds.
• this method offers the ability to specifically incorporate a macromolecule at the 5'-position of the RNA oligonucleotide during enzymatic synthesis of the RNA oligonucleotide.
• One embodiment of the present invention includes a method for generating high affinity bioconjugates to specific target molecules.
• the nucleic acid ligand is identified by the SELEX method.
• the SELEX method is described in United States Patent Application Serial No. 07/536,428, filed June 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned; United States Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled “Nucleic Acid Ligands," now United States Patent No. 5,475,096; United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled “Methods of Identifying Nucleic Acid Ligands," now United States Patent No. 5,270,163 (See also PCT Application Publication No. WO 91/19813).
• These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
• the SELEX process may be defined by the following series of steps:
• a candidate mixture of nucleic acids of differing sequence is prepared.
• the candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences.
• the fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the target, or (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture.
• the randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
• the candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid- target pairs between the target and those nucleic acids having the strongest affinity for the target.
• the nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with a lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning. 4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.
• the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase.
• the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
• the SELEX Patent Applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixtures.
• the SELEX Patent Applications also describe ligands solutions obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.
• the SELEX Patent Applications describe a number of uses for nucleic acid ligands including numerous therapeutic and diagnostic uses.
• bioconjugate is prepared by the SELEX method as described in the SELEX Patent Applications. Briefly, bioconjugates to a target are identified by the SELEX method by the steps comprising:
• preparing a candidate mixture of bioconjugates by the steps comprising (a) providing a DNA template having a sequence to be transcribed and (b) combining the DNA template with nucleotide triphosphates, a modified guanosine, and an RNA polymerase under conditions suitable for transcription; 2) contacting the bioconjugate candidate mixture with a target, wherein bioconjugates having an increased affinity to the target relative to the bioconjugate candidate mixture may be partitioned from the remainder of the bioconjugate candidate mixture;
• the 5 '-substituted GAP can aid in (1) the SELEX partition step, e.g. BIA-SELEX (see United States Application Serial No. 08/792,075, filed January 31, 1997, entitled “Flow Cell SELEX, which is incorporated herein by reference), plate SELEX (Conrad et al. (1996) Methods of Enzymol.
• Blended SELEX United States Patent No. 5,683,867, issued November 04, 1997, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX”
• nucleic acid ligands derivatized exclusively at the 5'- position of the nucleic acid ligand with virtually any molecular entity which is compatible with transcription can be prepared and identified.
• Molecular entities that can be coupled to nucleic acid ligands include, but are not limited to lipophilic molecules, proteins, peptides, reporter molecules, reporter enzymes and steroids.
• the 5 '-derivatized guanosine contains a reactive moiety which can be used for post-transcription conjugation of the transcript.
• This embodiment of the invention can be described by the following steps: a) providing a DNA b) combining the DNA template with nucleotide triphosphates, a 5 '-substituted guanosine, wherein said 5'-substituent contains a reactive moiety and an RNA polymerase under conditions suitable for transcription; and c) reacting the product from step b) with a molecular entity containing a moiety capable of reacting with the reactive moiety on said 5 '-substituent.
• oligonucleotides derivatized with molecular entities which are not compatible with transcription.
• reactive moieties include but are not limited to amines, dienes, dienophiles, thiols, vinylsulfones, photoaffinity labels and interchelators.
• the molecular entity may provide certain desirable characteristics to the nucleic acid ligand, such as, increasing RNA hydrophobicity and enhancing binding, membrane partitioning and/or permeability.
• reporter molecules such as biotin, fluorescein, or peptidyl metal chelates for incorporation of diagnostic radionuclides may be added, thus providing a bioconjugate which may be used as a diagnostic agent.
• Example 1 describes the synthesis of a variety of 5 '-modified guanosine monophosphates. For commonly used functional groups it is more efficient to conjugate the moiety of interest to the GAP molecule prior to transcription. This allows for large scale synthesis of the initiator, pre-transcription purification of the initiator, and negates the need for post-transcriptional conjugations.
• the modified guanosines synthesized are set forth in Schemes 1, 2, 4 and 5 and include GAP (5), GAP-fluorescein (11), GAP-biotin (12), GAP-Tc chelate (13), GAP-TEG (10), GAP- TEG-fluorescein (14), GAP-TEG-biotin (15) and GAP-TEG-Tc chelate (16).
• GAP (5) GAP-fluorescein
• GAP-biotin (12)
• GAP-Tc chelate 13
• GAP-TEG 10
• GAP- TEG-fluorescein 14
• GAP-TEG-biotin 15
• GAP-TEG-Tc chelate 16
• Biotin and fluorescein are very common conjugates which provide very useful properties for RNAs.
• the GAP -TEG analogs were synthesized because they are less expensive, less hydrophilic and potentially less immunogenic than the GAP analogs. When conjugating GAP to more hydropho
• Example 2 illustrates the feasibility of using transcription with 5'-modified guanosines to synthesize oligonucleotides modified exclusively at the 5'-position.
• This example demonstrates that both GAP and GAP conjugates can compete with GTP for the initiation of RNA synthesis.
• a ratio of GAP:GTP of 10: 1 results in 92 % of the transcript being initiated with GAP.
• Example 2 also illustrates that the yield of full length product does not decrease as a result of GAP- Biotin inhibiting the transcription reaction.
• Example 3 illustrates the post transcription conjugation of a GAP initiated transcript.
• Post transcription conjugation is necessary to obtain oligonucleotides derivatized with molecular entities that are not compatible with transcription.
• Transcription with primary amine initiators allows for the post-transcriptional conjugation of RNA with a wide variety functional groups through easily available NHS chemistry.
• a GAP initiated RNA was reacted with a biotin NHS ester.
• Example 4 demonstrates that GAP (5) and GAP-TEG (10) incorporate to the same extent resulting in the same amount of full length 5'-modified oligonucleotide product.
• Example 5 ( Figure 4A) demonstrates that GAP conjugate compounds 11-16 incorporate to the same extent resulting in the same amount of full length product as GTP (see Table below).
• This example compares the transcription reactions run with GAP analogs 11-16 and a control run without GAP. The results are set forth in the table below.
• Figure 4B shows that the full length transcripts initiated with GAP- fluorescein (11) and GAP-TEG-fluorescein (14) result in a fluorescent signal upon irradiation with ultra violet light.
• This example clearly demonstrates that virtually any linker or conjugate attached to guanosine, which ultimately is compatible with the transcription enzymes, could be used to enzymatically derivatize the 5'- terminal end of an RNA molecule.
• Example 6 describes the labeling of a GAP-Tc chelate (13) initiated transcript with "m Tc.
• RP-HPLC was performed on a Waters' Delta Pak 5 ⁇ C18 300 A, 3.9 x 150 mm column.
• Buffer A 100 mM TEAA at pH 7.0; Buffer B: AC ⁇ .
• the temperature was 30°C and the flow rate was 0.50 mL/min.
• ⁇ MR spectra were recorded on a Bruker ARX 300 spectrometer using CDC1 3 and (CD 3 ) 2 SO as solvents with TMS as an internal standard.
• Electrospray mass spectrometry was performed on a Fissions Quattro II (Beverly, MA) using negative ion mode. The samples were delivered in a 1 :1 MeOH/H 2 O (v/v) containing 0.1% TEA at 10 ⁇ L/min to the mass spectrometer.
• T7 R ⁇ A polymerase was purchased from Enzyco, Denver, Colorado.
• 2'-F-CTP and -UTP were purchased from USB Biochemicals.
• the transcriptional template was a 104-bp D ⁇ A amplified by PCR from a linearized pUC plasmid with the sequence:
• GAP 5'-(O'-hexylamino)guanosine monophosphate
• GAP (5) was synthesized with commercially available reagents using a DNA/RNA synthesizer. Starting with acetate protected guanosine CPG (Glen Research) a single coupling step was used to add 5'-Amino-modifier C6 phosophoramidite (Glen Research). The product was cleaved from the solid support, deprotected with NaOH and purified by reverse phase chromatography to yield GAP (5). The success of this experiment stimulated the larger scale production of the GAP molecule, discussed below. Large scale solution phase synthesis of 5'-(O'-hexylamino)guanosine monophosphate (5). Scheme 1 sets forth the large scale solution phase synthesis of GAP (5).
• 2'.3'-diacetyl- 2 N-isobutyrylguanosine (3) The 2',3'-diacetyl-5'-dimethoxytrityl- 2 N- isobutyrylguanosine (2) (5.5 g, 7.44 mmol) was brought up in 10 mL of DCM and loaded onto a Biotage Flash 40 silica gel column. The dimethoxytrityl was removed on the column using a of 3 % solution of trichloroacetic acid (TCA) and 0.5 % MeOH in DCM..
• TCA trichloroacetic acid
• the solution was concentrated in vacuo to approximately one fifth of its volume and then brought up in EtOAc (500 mL) and washed with 5 % ⁇ aHSO 3 (2 x 300 mL) and saturated NaHCO 3 (2 x 300 mL). The aqueous washes were back extracted with EtOAc (500 mL) and the
• Tetraethylene glycol phthalimide phosphoramidite (8) was synthesized as outlined in Scheme 3.
• Tetraethylene glycol monotosylate (6) Tetraethylene glycol (100 mL, 575 mmol) was dissolved in 250 mL of pyridine and cooled to 0°C and treated with 11.0 g (0.1 eq., 57.5 mmol) ?-toluenesulfonyl chloride. When solution was complete, the reaction was stored in the refrigerator overnight. The reaction was complete as determined by TLC (19:1 EtOAc/MeOH). The reaction mixture was then concentrated in vacuo. The residue was dissolved in 600 mL of EtOAc and extracted with H 2 O (3 x 200 mL).
• Tetraethylene glycol monophthalimide (7) Tetraethylene glycol monophthalimide (7).
• the solution was heated at 70°C for 18 hours and then concentrated in vacuo. The reaction was determined complete by TLC (19: 1 EtOAc/MeOH).
• DMSO 21.85 mL, 20 mg/mL
• TEA 1.15 mL, 5%
• biotinamidocaproate N-hydroxysuccinimidyl ester 909 mg, 2 equiv.
• the reaction was complete in one hour as determined by RP-HPLC. Purification by RP-HPLC afforded a yield of 719.8 mg (90%) of pure product compound 14.
• GAP / ⁇ - 32 P-GTP initiation competition assay A series of transcription reactions were performed using ⁇ - 32 P-GTP to determine the extent to which GAP (5) would compete with GTP for initiation of transcription reactions. The reactions were run under the standard conditions set forth above. The reactions were performed with the GAP molecule added to a final concentration of 0 to 10 mM while GTP (1 mM) and gamma labeled 32 P-GTP were held at fixed concentrations. Since the 32 P is in the gamma position, only those GTP molecules which initiate transcription, will result in the incorporation of a radiolabel into the transcript. The reaction products were analyzed by denaturing gel electrophoresis. Full length transcript bands were excised from the gel, the RNA was eluted from the gel slices and was quantitated by UV absorbance at 260 nm. Percent incorporation was calculated with the following equation:
• GAP- biotin were combined with 10 ⁇ M streptavidin in 37.5 mM Tris, pH 7.5. The products of the reaction were analyzed on a denaturing gel and quantified by phosphoimager. The amount of Streptavidin shift was correlated to the theoretical amount of GAP-biotin that should have been incorporated ( Figure 3). GAP-biotin incorporation The presence of the 5' primary amine was assessed by the ability to conjugate
• Transcription reactions were performed under standard conditions as set forth above, with GAP (5) or GAP-TEG (10) added at a ratio of 10 to 1 over the concentration of GTP.
• the reaction products were analyzed on a denaturing gel, which showed that transcription with GAP and GAP-TEG resulted in the same yield of full length transcript.
• This example describes the incorporation of GAP conjugates (11-13) and GAP-TEG conjugates (14-16). Transcription reactions were performed in parallel under standard conditions as set forth above using a 104-bp transcriptional template with the addition of ⁇ - 32 P-ATP. The GAP conjugates were added at a ratio of 10 to 1 over the concentration of GTP. A control reaction was run in which no modified guanosine was added to the standard RNA transcription reaction. The reaction products were analyzed on an 8% polyacrylamide gel containing 7 M urea. The gel was visualized by autoradiography. ( Figure 4A). The bands corresponding to full- length transcript were cut out of the gel, eluted and quantitated by UV spectroscopy.
• Example 6 Labeling of GAP-Tc chelate (13) transcript
• the labeling reaction was initiated by the addition of 10 ⁇ L 5 mg/mL SnCl 2 .
• the reaction mixture was incubated for 15 minutes at 90 °C.

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