WO1994021825A1 - Tests combinatoires pour la determination de facteurs de transcription et d'autres biomolecules par immunoabsorption d'oligomeres - Google Patents

Tests combinatoires pour la determination de facteurs de transcription et d'autres biomolecules par immunoabsorption d'oligomeres Download PDF

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WO1994021825A1
WO1994021825A1 PCT/US1994/002166 US9402166W WO9421825A1 WO 1994021825 A1 WO1994021825 A1 WO 1994021825A1 US 9402166 W US9402166 W US 9402166W WO 9421825 A1 WO9421825 A1 WO 9421825A1
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competitor
oligonucleotides
sets
transcription factor
oligonucleotide
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PCT/US1994/002166
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David J. Ecker
Timothy A. Vickers
Peter W. Davis
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Isis Pharmaceuticals, Inc.
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Publication of WO1994021825A1 publication Critical patent/WO1994021825A1/fr

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    • 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
    • C12N15/1131—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 against viruses
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    • 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
    • C12N15/1131—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 against viruses
    • C12N15/1132—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 against viruses against retroviridae, e.g. HIV
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    • 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
    • C12N15/1131—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 against viruses
    • C12N15/1133—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 against viruses against herpetoviridae, e.g. HSV
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    • 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
    • C12N15/1135—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 against oncogenes or tumor suppressor genes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12N2310/315—Phosphorothioates

Definitions

  • This invention relates to the identification of 5 oligomers useful for therapeutic, diagnostic, and research applications.
  • oligomers which bind to specific transcription factors and modulate their function in cells.
  • RNA binding proteins which regulate the expression of genes. These key proteins thereby control the production of all the other proteins in the cell. Viruses which infect cells and cause disease encode their own transcription factors which
  • virally encoded transcription factors which regulate viral gene expression are the human papillomavirus #2 protein, Hegde, et al. , Nature 1992, 354 , 505, the herpes simplex virus ⁇ .4 protein, Kristie, et al. ,
  • transcription factors such as the fos/jun proteins, Konig, et al . , EMBO Journal 1989, 8, 2559, or the C-MYC protein, Blackwell, et al . , Science 1990, 250, 1149 are links in a chain of signal transduction gone awry. Interference with specific transcription factors might be used as a means to restore a more normal cellular phenotype.
  • Oligomers may be designed which are useful for therapeutic, diagnostic and research applications, such as binding to specific transcription factors.
  • development of biologically active oligomer substances was often limited to the modification of known sequences, unit by unit, until a desired characteristic or efficacy was achieved.
  • Androphy, et al. U.S. Serial No. 302,758 issued 1989, have described methods to bind to the papilloma E2 transcription factor using DNA decoys which have homology to the natural E2 recognition sequence
  • Bielinska, et al. Science 1990, 250, 997, have also described regulation of gene expression with double stranded phosphorothioate oligonucleotides targeted to HIV. Holcenberg, et al.
  • combinatorial nucleic acid selection methods generally select for a specific nucleic acid sequence from a pool of random nucleic acid sequences based on the ability of selected sequences to bind to a target protein. The selected sequences are then amplified and the selection process repeated until a few strongly binding sequences are identified. These methods generally employ enzymatic steps within the protocol. T7 RNA polymerase and Taq I associated with polymerase chain reaction amplification methods are commonly employed. One group recently identified a target sequence to the RNA-binding protein gp43.
  • Tuerk and Gold Science 1990 249, 505.
  • Tuerk and Gold's "systematic evolution of ligands by exponential enrichment” (SELEX) method identified specifically bindable RNA sequences using four cycles of amplification of RNA sequences having variable portions therein and which were specifically bindable to gp43.
  • Another group designed DNA molecules which recognized the protease thrombin. Bock, et al. , Nature 1992, 355, 564. This method involves the preparation of a population involving a random region flanked by known primer regions followed by PCR amplification and selection. Small molecule mimics of metabolic cofactors have been selectively recognized by RNA sequences in this manner by Ellington and Szostak, Nature 1990, 346, 818.
  • Weintraub has described methods for the identification of protein specific oligonucleotide sequences.
  • Weintraub, et al . U.S. Serial No. 9,205,285 issued x 1992, teaches binding of a complex mixture of oligonucleotide containing random sequences and using PCR to identify the best sequence.
  • Oliphant et al. Nucleic Acids Research, 1988, 16, 7673 and Oliphant, et al . , Mol . Cell . Biol . , 1989, 9, 2944 used in vivo selection methods to define the sequence recognition properties of DNA binding proteins.
  • Figure 1 is a schematic representation showing binding affinity of four sets of phosphorothioate oligonucleotides for the transcription factor c-rel.
  • the phosphorothioate oligonucleotide set having the sequence TNNNCNNNT-B had the greatest activity, indicated by amount of binding.
  • the phosphorothioate oligonucleotide set having the sequence TNNNANNNT-B had the least activity, exhibited little to no binding of c-rel.
  • Figure 2 is a schematic representation showing binding affinity of four sets of RNA oligonucleotides for the HIV-tat element at l ⁇ M, lO ⁇ M and lOO ⁇ M concentrations.
  • RNA oligonucleotide set having the sequence CCANNA NNNGCCUGGGAGCNNNNUGG (SEQ ID NO:24) bound to HIV-tat most specifically at all three concentrations as evidenced by the reduced amount of the TAR element bound by tat.
  • Figure 3 is a schematic representation showing binding affinity of four sets of oligonucleotides for the HIV-tat protein using a competition assay.
  • the oligonucleotide having the sequence TNNN(non C)NNNT exhibited the highest binding activity.
  • the oligonucleotide having the sequence TNNN(A Me)NNNT exhibited the least binding activity.
  • Figure 4 is a schematic representation showing binding affinity of four sets of oligonucleotides for the HIV-tat protein using a capture assay.
  • the phosphorothiate oligonucleotide having the sequence TNNNGNNNT-B exhibited the highest binding activity.
  • the phosphorothioate oligonucleotide having the sequence TNNNCNNNT-B exhibited the least binding activity.
  • Figure 5 is a schematic representation showing binding affinity of four sets of 2'-0-methyl oligonucleotides for the c-myc nuclear protein.
  • the 2'-0-methyl oligonucleotide set having the sequence GCGNNNCNNNNNNCGC (SEQ ID NO:33) bound with c-myc most specifically as evidenced by the reduced amount of c-myc binding to a double stranded c- myc binding site.
  • Figure 6 is a schematic representation showing binding affinity of four sets of oligonucleotides for c-myc protein using a competition assay.
  • the oligonucleotide having the sequence TNNN(biIM A) NNT exhibited the highest binding activity.
  • the oligonucleotide having the sequence TNNN(G Me)NNNT exhibited the least binding activity.
  • Figure 7 is a schematic representation showing binding affinity of four sets of oligonucleotides for the c- rel protein.
  • the oligonucleotide having the sequence TNNN(B ImA)NNNT competed most effectively with the NF-kB binding site exhibiting the highest binding activity.
  • the oligonucleotide having the sequence TNNN(nonylC)NNNT exhibited the least binding activity.
  • Figure 8 is a schematic representation showing binding affinity of four sets of oligonucleotides for c-rel using a competition assay.
  • the oligonucleotide having the sequence TNNN(nonyl C)NNNT exhibited the highest binding activity.
  • the oligonucleotide having the sequence TNNN(G Me)NNNT exhibited the least binding activity.
  • Figure 9 is a schematic representation showing binding of fos and jun to Ab-1 transcription factor.
  • Combinatorial strategies offer the potential to generate and screen extremely large numbers of compounds and to identify individual molecules with a desired binding specificity or pharmacological activity.
  • This invention is directed to substantially non-enzymatic methods of determining oligomers which are specifically active for transcription factors and other target molecules. Methods of the present invention are useful for the determination of oligomers which have specific activity for transcription factors from a pool of primarily randomly assembled subunits. Said methods involve repeated syntheses of increasingly simplified sets of oligomers coupled with selection procedures for determining the oligomer set having the greatest activity in an assay for desired activity.
  • Simplification of the pool occurs because, with each additional step of the methods, at least one additional position in the oligomer is determined. As a result, the possible number of different oligomer molecules in the pool decreases sequentially with the number of random positions remaining in the oligomer.
  • oligonucleotides having specific activity for a transcription factor are provided. These methods involve preparing a group comprising a plurality of sets of oligonucleotides, each oligonucleotide comprising at least four base units, by defining a common position in the oligonucleotides of the sets and synthesizing said sets of oligonucleotides such that each set has a different base unit in said common position and the base units which are not in the common position are randomized.
  • each of the sets are then assayed for activity against the transcription factor and the set having the greatest activity for the transcription factor is selected.
  • each group of oligonucleotides may be subfractionated to provide subfractions of the sets of oligonucleotides. Each subfraction may be assayed against the transcription factor and the set from which the subfraction having the highest activity was derived is selected.
  • These methods further comprise preparing a further group comprising a plurality of sets of oligonucleotides, each of the sets having in the previously defined common position the base unit appearing in the previously defined common position in the previously selected set.
  • Each of said further group of sets has a different base unit in an additional, defined common position.
  • the base units in positions of the oligonucleotides which are not in a common position are randomized.
  • this group may subfractionated to provide subfractions of the sets of oligonucleotides.
  • Each of said sets or subfractions of sets may be assayed for activity for the transcription factor and the set having the highest activity, or the set from which the subfraction having the highest activity was derived, is selected.
  • Methods of determining an oligonucleotide cassette having specific activity for a transcription factor are also provided by the present invention. These methods involve preparing a group comprising a plurality of sets of oligonucleotides, each oligonucleotide comprising at least four base units, by defining a common position in the oligonucleotides of the sets and synthesizing said sets of oligonucleotides such that each set has a different base unit in said common position and the base units which are not in the common position are randomized. Each of the sets are then assayed for activity for a transcription factor and the set having the greatest activity for the transcription factor is selected.
  • a further group comprising a plurality of sets of oligonucleotides, each of the sets having in the previously defined common position the base unit appearing in the previously defined common position in the previously selected set .
  • Each of said further group of sets has a different base unit in an additional defined common position.
  • the base units in positions of the oligonucleotides which are not in a defined common position are randomized.
  • Each set of said further group is assayed for specific activity for the transcription factor and the set having the highest activity is selected. The preceding steps are performed iteratively to provide an oligonucleotide cassette having each position defined.
  • methods for determining an oligonucleotide having specific activity for a transcription factor comprise preparing a group comprising a plurality of sets of oligonucleotides, each of the oligonucleotides comprises at least one oligonucleotide cassette and at least one flanking region.
  • a common position is defined in a flanking region of the oligonucleotides of the sets and the sets of oligonucleotides are synthesized such that each set has a different base unit in said common position and the base units which are not in the common position are randomized.
  • Each of the sets are then assayed for activity for a transcription factor and the set having the greatest activity for the transcription factor is selected.
  • These methods also may comprise preparing a further group comprising a plurality of sets of oligonucleotides, each of the sets having in the previously defined common position the base unit appearing in the previously defined common position in the previously selected set. Each of said further group of sets having a different base unit in an additional, defined common position in the flanking region.
  • the base units in positions of the oligonucleotides which are not in a common position in the flanking region are randomized.
  • Each of the sets of oligonucleotides are assayed for specific activity for the transcription factor and the set having the highest activity is selected.
  • the preceding steps may be and preferably are performed iteratively.
  • oligonucleotide having specific activity for a transcription factor comprising preparing a group comprising a plurality of sets of oligonucleotides.
  • Each oligonucleotide comprises at least four nucleotide units and a common position is defined in the oligonucleotides of the sets.
  • Said sets of oligonucleotides are synthesized such that each set has a different base unit in said common position, the base units which are not in said common position being randomized.
  • the oligonucleotides of the sets of oligonucleotides are modified with linker moieties for attaching the oligonucleotide to a solid support.
  • the sets of oligonucleotides are each incubated with the transcription factor under binding conditions to form oligonucleotide-transcription factor complexes, and the oligonucleotides are attached to a solid support via the linker moiety. Bound and unbound transcription factor molecules are separated and binding of the transcription factor to each set of oligonucleotides is detected wherein the greatest binding is indicative of highest activity. The set having highest activity is selected.
  • oligonucleotide sets are prepared, and oligonucleotides of each set of oligonucleotides are detectably labeled.
  • the transcription factor is modified with a linker moiety for attaching the transcription factor to a solid support and incubated with each of said sets of oligonucleotides under binding conditions to form oligonucleotide-transcription factor complexes.
  • the transcription factor is attached to a solid support via the linker moiety and bound from unbound oligonucleotide is separated.
  • the binding of each oligonucleotide set to the transcription factor is detected wherein greatest binding is indicative of highest activity.
  • the set having highest activity is selected.
  • oligonucleotide sets are prepared and the transcription factor is modified with a linker moiety for attaching the transcription factor to a solid support.
  • the transcription factor is incubated with each of the sets of oligonucleotides under binding conditions to form oligonucleotide-transcription factor complexes.
  • a competitor is added under binding conditions to form transcription factor-competitor complexes.
  • the transcription factor is attached to a solid support via the linker moiety and bound and unbound competitor are separated. Binding of the competitor is detected wherein lowest binding is indicative of highest oligonucleotide activity.
  • the set having the highest oligonucleotide activity is selected.
  • oligonucleotide sets are prepared and a competitor is modified with a linker moiety for attaching the competitor to a solid support.
  • Each of the oligonucleotide sets are incubated with the transcription factor under binding conditions to form oligonucleotide-transcription factor complexes.
  • the competitor is added under binding conditions to form transcription factor-competitor complexes.
  • the competitor is attached to a solid support via the linker moiety and bound and unbound transcription factor molecules are separated. Binding of the transcription factor to the competitor is detected wherein lowest binding is indicative of highest oligonucleotide activity. The set having the highest activity is selected.
  • oligonucleotide sets are prepared and a first competitor is modified with a linker moiety for attaching the first competitor to a solid support.
  • the transcription factor and the first competitor are incubated under binding conditions to form transcription factor-competitor complexes.
  • Each of the oligonucleotide sets are incubated with the transcription factor-competitor complexes under binding conditions to form oligonucleotide-transcription factor-competitor complexes.
  • a second competitor is added under binding conditions to form further complexes.
  • the first competitor is attached to a solid support via the linker moiety and bound from unbound second competitor is separated.
  • Binding of the second competitor to the transcription factor is detected wherein lowest binding is indicative of highest oligonucleotide activity.
  • the set having the highest activity is selected.
  • oligonucleotide sets are prepared and the oligonucleotides of the sets are detectably labeled.
  • a first competitor is modified with a linker moiety for attaching the first competitor to a solid support and incubated with the transcription factor under binding conditions to form transcription factor-competitor complexes.
  • Each of the sets of oligonucleotides are incubated with the transcription factor-competitor complexes under binding conditions to form oligonucleotide-transcription factor-competitor complexes.
  • a second competitor is added under binding conditions to form further complexes and the first competitor is attached to a solid support via the linker moiety. Bound from unbound oligonucleotide is separated and binding of the oligonucleotide to the transcription factor is detected wherein highest binding is indicative of highest oligonucleotide activity. The set having the highest activity is selected.
  • the present invention is directed to non-enzymatic methods for determining oligomers which have specific binding activity for a transcription factor.
  • methods of determining oligonucleotides having specific binding affinity for a transcription factor are provided.
  • an oligomer is a string of units linked together by chemically similar covalent linkages. Nucleic acids linked together via phosphodiester bonds to form polynucleotides, amino acids linked together via peptide bonds to form polypeptides and monosaccharides linked together via glycosidic linkages to form polysaccharides are examples of naturally occurring oligomers. Also encompassed by the term "oligomer" includes two or more oligomer species linked together. For example, polypeptides and polysaccharides may be linked together to form glycoproteins and lipids and polysaccharides may be linked together to form glycolipids. In the context of this invention, the term
  • oligonucleotide refers to a polynucleotide formed from naturally occurring bases and furanosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally occurring species or synthetic species formed from naturally occurring subunits or their close homologs.
  • oligonucleotide may also refer to moieties which have portions similar to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter- sugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species which are known for use in the art.
  • the phosphodiester bonds of the oligonucleotide have been substituted with a structure which functions to enhance the stability of the oligonucleotide or the ability of the oligonucleotide to penetrate into the region of cells where the viral RNA is located. It is preferred that such substitutions comprise phosphorothioate bonds, phosphotriesters, methyl phosphonate bonds, short chain alkyl or cycloalkyl structures or short chain heteroatomic or heterocyclic structures.
  • Methods of preparing such oligonucleotides are well known in the art and may be synthesized by modification of procedures such as described in Goodchild, Bioconjugate Chemistry, 1990, 2:165-187;
  • the phosphodiester backbone of the oligonucleotide may be replace with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone.
  • the phosphodiester bonds are substituted with other structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
  • Oligonucleotides may also include species which include at least some modified base forms.
  • purines and pyrimidines other than those normally found in nature may be so employed such as disclosed in PCT/US91/04681 filed July 1, 1991, incorporated by reference herein in its entirety.
  • modifications on the furanosyl portion of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-0-alkyl- and 2'-halogen- substituted nucleotides such as described in Uhlmann and Peyman, Chemical Reviews, 1990, 90, 543-584.
  • modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH 3 , F, OCN, 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 where n is from 1 to about 10; C x to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl, Br, CN, CF 3 , OCF 3 , 0-, S-, or N- alkyl; 0- , S-, or N-alkenyl; SOCH 3 , S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleot
  • Oligonucleotides may also comprise other modifications consistent with the spirit of this invention. Such oligonucleotides are best described as being functionally interchangeable with yet structurally distinct from natural oligonucleotides. All such oligonucleotides are comprehended by this invention so long as they effectively function as subunits in the oligonucleotide.
  • the methods of the present invention are useful to determine oligomers which are specifically active for a transcription factor.
  • determine refers to concurrent identification of the sequence of an oligomer and the binding activity of the oligomer for a transcription factor.
  • neither the oligomer sequence nor its specific activity is known prior to performance of methods of the present invention.
  • a particular oligomer sequence may be known, those skilled in the art may not recognize its activity for a particular transcription factor.
  • binding affinity refers to the ability of the oligomer to bind to a transcription factor via hydrogen bonds, van der Waals interactions, hydrophobic interactions, or otherwise.
  • an oligomer may bind to a "leucine zipper" transcription factor or a helix-loop-helix transcription factor via positively charged amino acids in one region of the transcription factor.
  • Transcription factors refers to DNA- or RNA-binding proteins which regulate the expression of genes. HIV tat and c-rel are examples of transcription factors which regulate the expression of genes . Also encompassed by the term are DNA and RNA binding proteins which are not strictly considered transcription factors, but which are known to be involved in cell proliferation.
  • transcription factors include c-myc, fos, and jun.
  • Methods of the present invention are particularly suitable for use with transcription factor as target molecules since transcription factors generally occur in very small cellular quantities. Methods of the present invention require relatively small amounts of transcription factor which may, but need not, be purified.
  • Target molecules of the present invention may include any of a variety of biologically significant molecules.
  • Target molecules may be nucleic acid strands such as significant regions of DNA or RNA.
  • Target molecules may also be carbohydrates, glycoproteins or other proteins.
  • said target molecule is a protein such as an immunoglobulin, receptor, receptor binding ligand, antigen or enzyme and more specifically may be a phospholipase, tumor necrosis factor, endotoxin, interleukin, plasminogen activator, protein kinase, cell adhesion molecule, lipoxygenase, hydrolase or transacylase.
  • said target molecules may be important regions of the human immunodeficiency virus, Candida, herpes viruses, papillomaviruses, cytomegalovirus, rhinoviruses, hepatitises, or influenza viruses.
  • said target molecules may be regions of an oncogene.
  • said target molecule is ras 47-mer stem loop RNA, the TAR element of human immunodeficiency virus or the gag-pol stem loop of human immunodeficiency virus (HIV) .
  • Still other targets may induce cellular activity. For example, a target may induce interferon.
  • the transcription factor need not be purified, but may be present, for example, in a crude cell lysate containing the transcription factor, serum or extract.
  • purified transcription factor is also useful in some aspects of the invention.
  • synthetically prepared transcription factor may be useful.
  • the transcription factor may also be modified, such as by biotinylation or radiolabeling.
  • synthetically prepared transcription factor may incorporate one or more biotin molecules during synthesis or may be modified post-synthesis.
  • a group of sets of random oligomers is prepared. Oligomers may be prepared by procedures known to those skilled in the art.
  • oligonucleotides, polypeptides, polysaccharides, glycoproteins and glycolipids may be prepared by solid state synthesis or by other means known to those skilled in the art.
  • oligonucleotides may be prepared using standard phosphoramidite chemistry.
  • oligomer groups may further be labeled, such as by radiolabeling or fluorescent labeling.
  • an oligonucleotide group may be labeled at the 5' termini of the oligonucleotides using [ ⁇ - 32 P] ATP and T4 polynucleotide kinase. Labeled oligomer groups may be useful in a number of assays which can not be performed using unlabeled oligomer groups.
  • Oligomers of each set may be of predetermined length. It is preferred that such oligomers be from about 4 to about 50 units in length. It is more preferred that such oligomers be from 4 to about 40 units in length. It is also preferred for some embodiments of the present invention that less than about 10 units of an oligomer are randomized. In some cases, it may be desirable to provide an oligomer which initially comprises 6, 7, or 8 random units.
  • the length of said oligomer need not be constant throughout the procedure.
  • an 8-mer may be assayed to determined the sequence having highest binding affinity for a transcription factor. Subsequently, the 8-mer may be extended and tested as a 15-mer to determine the 15-mer sequence having the highest binding affinity for the transcription factor.
  • Groups of the present invention are made up of a plurality of sets which may remain constant throughout the procedure. From about three to about twenty sets can make up each group. In one preferred embodiment of the present invention four sets make up each group. In another embodiment of the present invention twenty sets make up each group. Alternatively, three sets may make up each group. The number of sets that make up each group is dependent upon the number of possible distinct chemically similar units which exist for any one species of molecule. For example, an oligonucleotide group may be comprised of four sets since there are four similar units making up the nucleic acid species, i.e. guanine, adenine, cytosine, thymine or adenine, guanine, cytosine and uracil. Alternatively, an oligonucleotide group may be comprised of more than four sets representing for example, the four commonly occurring bases and additional modified bases.
  • Twenty sets may make up a polypeptide group, representing the twenty commonly occurring amino acids, lysine, arginine, histidine, aspartic acid, glutamic acid, glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan. Greater than twenty sets may also make up a polypeptide group if uncommon or modified amino acids are included in the assay.
  • Subgroups of basic units may also determine the number of sets in any one group. For example, in procedures to determine a particular polypeptide, sets may represent acidic, basic and neutral amino acid units, i.e. three sets. The number of sets in groups in any one procedure need not remain constant throughout, but may fluctuate. For example, in a one group there may be three sets representing three types of polypeptides and in a next group there may be twenty sets representing each commonly occurring
  • the use of additional units such as nucleotide or amino acid analogs may be preferred in some instances where it is desirable to increase the complexity of the group of oligomers.
  • the complexity of a group may be calculated by the formula P X P N where P is the number of different units used and N is the number of positions in an oligomer which are randomized.
  • the complexity of a set (Q) is represented by the formula P N .
  • Table 1 illustrates the change in group complexity as a result of the increase in the number of analogs used. Of course, the number of different units used also determines the number of sets prepared.
  • Each of the sets in a group has a different unit in a common position of said oligomer. For example, in determining an oligonucleotide, only one of four sets will contain an adenine in a common position, only one set will contain a guanine in a common position, etc.
  • the remaining positions in each set of oligonucleotides are comprised of any combination of random basic units.
  • common positions are comprised of multiple oligomer positions.
  • one common position may be the third position of the 9-mer, or the common position may be comprised of the third position and the fourth position of the 9-mer.
  • an oligonucleotide may be sterically constrained by providing complementary ends at the 3' and 5' termini of the region of interest, which region comprises randomized positions. The complementary ends will hybridize to form a secondary structure.
  • An oligomer cassette is a oligomer for which a sequence has been determined.
  • the cassette may be comprised of a sequence of known significance, or may be determined such as by the procedures of the present invention.
  • an oligonucleotide cassette is a defined oligonucleotide sequence.
  • an oligomer may comprise at least one oligomer cassette and at least one flanking region of unrandomized positions.
  • an oligomer may be comprised of more than one cassette wherein each cassette is flanked by at least one region of randomized positions.
  • a oligonucleotide cassette of known sequence may be flanked at the 3' terminus, the 5' terminus, or both the 3' and 5' termini.
  • each set of oligomers is assayed for desired activity.
  • identical empirical assays of subfractions of oligomer sets described above are performed in order to identify those subfractions having the strongest activity as indicated by a strong signal to noise ratio.
  • the set having the highest activity or the set from which the subfraction having the highest activity is derived is selected and further unrandomization may be performed if desired.
  • binding conditions simulate physiological conditions. In other preferred embodiments of the present invention binding occurs in a buffer of from about 80 mM to about 110 mM sodium chloride and from about 10 to about 15 mM magnesium chloride. Oligomers may also generally be assayed for catalytic or enzymatic activity. Gel shift assays may be used to visualize binding of an oligomer to a transcription factor.
  • radiolabelled transcription factor bound to oligomer of the present invention may be run on a gel such as a polyacrylamide gel .
  • Bound transcription factor has less mobility than unbound transcription factor, and therefore will not migrate as far on the gel .
  • the radioactive label allows visualization of the "shift" in mobility by standard procedures for example, by means of X-ray radiography or by using a phosphorimager (Molecular Dynamics) .
  • a gel shift assay may be performed wherein an unlabeled transcription factor may be bound to radiolabelled oligomer.
  • Radiolabeled oligomer may also be useful for the streptavidin capture of a biotin-transcription factor bound to an oligomer.
  • a transcription factor may be biotinylated prior to incubation with radioactively labeled random oligomer sets. Such biotinylation can be performed by those skilled in the art using well know reagents and procedures. Each set is thereafter incubated with the transcription factor under identical conditions and the transcription factor is captured on streptavidin-coated beads. Alternatively, the transcription factor may be captured prior to incubation with the oligomer. Consequently any oligomer which bound to the transcription factor will also be captured.
  • Streptavidin-coated beads are available commercially such as for example, streptavidin-coated manganese particles available from Promega. The beads are washed and the reaction may be reequilibrated to further enrich the "winning" sequence. The percent of oligomers from each set which bound is determined by the amount of radioactivity remaining after wash. Measuring radioactivity in a sample may be performed by a number of methods known in the art. For example, the amount of radioactivity may be determined directly by counting each sample, using for example a scintillation counter. Samples may also be run on a polyacrylamide gel, the gel may be placed under x-ray film and a densitometric reading of the autoradiogram may be taken.
  • a competitor may be co-incubated with transcription factor and oligomer sets to determine which oligomer set best competes for a binding site against a molecule known to bind to the transcription factor.
  • a competitor in the context of this invention, is a molecule which is known to recognize the transcription factor.
  • the competitor may be, for.example, a double or single stranded oligonucleotide.
  • the competitor may, in other embodiments of the present invention, be a protein known to bind to the transcription factor.
  • a truncated version of the TAR hairpin loop naturally recognizes the HIV tat protein and may compete with oligomers of the present invention, preventing binding of the oligomer to the HIV tat transcription factor.
  • a DNA myc binding site may compete with oligomers of the present invention for the myc nuclear protein and the NF-kB binding site may compete with oligomers of the present invention for the rel transcription factor.
  • a "capture" assay may also be performed without the use of radioactivity.
  • each set of random oligomers may be prepared with a linker moiety.
  • the linker moiety is a moiety which is useful for attaching the oligomer, and anything appended thereto, to a solid support by chemical bond, or otherwise.
  • well known linker moieties include chemical handles such as biotin and polyT. Such linker moieties may be incorporated during synthesis of the oligomer, or may be incorporated following synthesis.
  • each set of oligomers is incubated with the transcription factor under identical conditions and the oligomers are attached to a solid support via the linker moiety.
  • the transcription factor and oligomer sets are incubated following attachment of the oligomers to a solid support.
  • biotin- labeled oligomers may be captured on a streptavidin-coated solid support such as streptavidin-coated beads or streptavidin-coated plates.
  • polyT-labeled oligomer may be attached to a polyA-labeled solid phase. Consequently, any transcription factor which has bound to oligomers will also be attached.
  • the solid support is washed and the reaction may be reequilibrated to further enrich the "winning" sequence. Thereafter binding of the transcription factor is detected such as by the use of an antibody which is specific for the transcription factor.
  • the antibody may detect and quantitate the set which bound the most transcription factor either by attached reporter or a secondary antibody containing a reporter. In such an assay, the oligomer set having the most binding is the "winner" set.
  • a competitor may be co- incubated with transcription factor and oligomer sets to determine which oligomer set best competes for a binding site against a molecule known to bind to the transcription factor.
  • Another assay which may be useful for the detection of oligomer binding is a "competition” assay. This assay selects for molecules which bind to a transcription factor specifically at the site that the protein naturally binds a molecule of interest (a competitor) . Thus, this assay selects for a competitive inhibitor.
  • each set of oligomers may be incubated with the transcription factor under binding conditions. To this incubating mixture is then added a competitor.
  • a competitor in the context of this invention, is a molecule which is known to recognize the transcription factor.
  • the competitor may be, for example, a double or single stranded oligonucleotide.
  • the competitor may, in other embodiments of the present invention, be a protein or peptide known to bind to the transcription factor.
  • the competitor further comprises a linker moiety which can be used to attach the competitor to a solid support.
  • the competitor may be modified or synthesized with a chemical handle such as biotin or polyT.
  • the competitors are attached to a solid support via the linker moiety either before or after incubation with the transcription factor and oligomer sets.
  • biotin-labeled competitor may be attached to a streptavidin-coated solid support such as streptavidin-coated beads or streptavidin-coated plates.
  • poly T-labeled competitors may be captured on a poly A-labeled solid support.
  • any transcription factor which is bound to the competitor will also be bound while transcription factor bound to oligomers will not be bound. Thereafter the solid support is washed.
  • the percent of binding of the transcription factor to the competitor is determined by the use of an antibody which is specific for the transcription factor.
  • the antibody may detect and quantitate the set which bound the most transcription factor either by attached reporter or a secondary antibody containing a reporter.
  • oligomer sets which bind the transcription factor prevent binding to the competitor which is also present in the assay. Since only the competitor can be attached to the solid support, only transcription factor molecules which are able to bind the competitor will remain on the solid support after washing.
  • oligomer sets which retain or bind more transcription factor should lower the amount of transcription factor available to bind the competitor and therefore lower the overall signal in a "competition” assay.
  • Still another assay which selects for molecules which bind to a transcription factor specifically at the site that the protein naturally binds a molecule of interest (a competitor) is the “heterodimer targeting” assay.
  • This assay also selects for a competitive inhibitor.
  • the transcription factor is incubated with a first competitor which functions to attach the transcription factor to a solid support.
  • a first competitor may be a double or single stranded oligonucleotide known to recognize and bind to the transcription factor.
  • the first competitor further comprises a linker moiety which can be used to attach the first competitor to a solid support.
  • the first competitor may be modified or synthesized with a chemical handle such as biotin or polyT.
  • the first competitor is attached to a solid support via the linker moiety.
  • biotin-labeled first competitor may be captured on a streptavidin-coated solid support such as streptavidin-coated beads or streptavidin-coated plates.
  • poly T labeled first competitors may be attached to a poly A labeled solid support.
  • the transcription factor is directly attached to the solid support via a linker moiety such a biotin or poly T as described above. Following attachment of the transcription factor, Each set of oligomers may be incubated with a sample of the transcription factor under binding conditions. Thereafter, a second competitor is added.
  • attachment may follow incubation.
  • the solid support is washed and presence of the second competitor is detected by the use of an antibody which is specific for the second competitor.
  • the antibody may detect and quantitate the oligomer set which bound the most transcription factor either by attached reporter or a secondary antibody containing a reporter. The set which competes most effectively with the natural recognition site of the competitor to the transcription factor will produce the lowest signal.
  • each of said further groups have a selected number of sets of oligomers.
  • Sets of further groups have in a previously defined common position, units appearing in the previously defined common position in previously selected set .
  • Each set of the further sets has a different unit in an additional defined common position.
  • the units in the positions of the oligomer that are not in a common position are randomized.
  • the previously selected set may be comprised of an adenine in the previously defined common position.
  • a further group may retain said adenine in said previously defined common position, and at another defined common position each set in said further group may be comprised of a different unit, either adenine, guanine, thymine or cytosine.
  • common positions are comprised of multiple oligomer positions as described above.
  • the one common position may be the third position of the 9-mer, or the common position may be comprised of the third position and the fourth position of the 9-mer.
  • oligomer groups may be comprised of multiple units, may be sterically constrained and may be subfractionated prior to assaying for specific activity.
  • oligomers of further groups may comprise one or more cassettes. Sets are again assayed for desired activity. The steps described above may be performed iteratively.
  • Unmodified DNA oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 38OB) using standard phosphoramidite chemistry with oxidation by iodine.
  • S-cyanoethyldiisopropyl phosphoramidites may be purchased from Applied Biosystems (Foster City, CA) .
  • Unmodified RNA oligonucleotides having random base sequences were synthesized on an automated DNA synthesizer (Applied Biosystems model 38OB) using modified standard phosphoramidite chemistry synthesis with oxidation by iodine.
  • the standard synthesis was modified by increasing the wait step after the pulse delivery of tetrazole to 900 seconds, j ⁇ -cyanoethyldiisopropyl phosphoramidites were purchased from Applied Biosystems (Foster City, CA) .
  • the bases were deprotected by incubation in methanolic ammonia overnight. Following base deprotection, the oligonucleotides were dried in vacuo .
  • RNA oligonucleotide was further purified on C 18 Sep-Pak cartridges (Waters, Division of Millipore Corp., Milford, MA) and ethanol precipitated.
  • Phosphorothioate oligonucleotides represent a class of oligonucleotide analog that is substantially nuclease resistant.
  • Phosphorothioate RNA oligonucleotides and phosphorothioate DNA oligonucleotides were synthesized according to the procedure set forth in Examples 1 and 2 respectively, replacing the standard oxidation bottle by a 0.2M solution of 3H-1, 2-benzodithiole-3-one 1,1-dioxide in acetonitrile for stepwise thiation of phosphite linkages.
  • the thiation cycle wait step was increased to 68 seconds and is followed by the capping step.
  • 2'-0-methyl phosphorothioate oligonucleotides were synthesized according to the procedures set forth in Example 3 substituting 2'-0-methyl 3-cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham, MA) for standard phosphoramidites and increasing the wait cycle after the pulse delivery of tetrazole and base to 360 seconds. Similarly, 2'-0-propyl, 2'-0-phenyl and 2'-0-nonyl phosphorothioate oligonucleotides may be prepared by slight modifications of this procedure.
  • Oligonucleotides were prepared by incorporating 2' aminopentoxyadenosine at desired sites. The oligonucleotides were dissolved in 0.2M NaHC0 3 buffer and treated with 50 fold excess of N-hydroxysuccinimide ester of pyrene-1-butyric acid dissolved in dimethylformamide. The resultant mixture is incubated at 37°C for 4-5 hours and the conjugate is purified by reverse phase HPLC followed by desalting in a G-25 Sephadex column.
  • Example 1 through 6 may be radiolabeled using [ ⁇ - 32 P] ATP and T4 polynucleotide kinase as described in Maniatis, et al . "Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor, NY) .
  • Example 8
  • NNNANN Set 13 NNNANN Set 14
  • NNNCNN Set 15 NNNGNN Set 16
  • NNNTNN Set 17 NNNNAN Set 18
  • NNNNCN Set 19 NNNNGN Set 20
  • NNNNTN Set 21 NNNNNA Set 22
  • NNNNNC Set 23 NNNNNG Set 24
  • Each of the sets is tested for activity against a target molecule to determine which order of unrandomization gives the highest initial specific activity.
  • An oligonucleotide group having the sequence TNNNXNNNTB, wherein N is any of A, G, C, or U, X is one of A, G, C and U and B is biotin, is prepared in accordance with Examples 3 and 6.
  • Biotin may be incorporated at the 3' or 5' end of the oligonucleotide by placement of the biotin moiety as the first or last subunit during synthesis.
  • the sequence is designed with flanking thymidines to provide sites for radiolabeling.
  • a control having the sequence TNNNXNNNT is also prepared in accordance with Examples 3 and 6.
  • Oligonucleotide groups having the sequence NNNXNNNU are prepared in accordance with Example 1 and 6 incorporating one or more of the nucleoside analogs 2'-0-nonyl adenosine, N6-imidazoylpropyl guanosine, 2' -O-aminopentyl cytidine, 2'- O-pentyl-adenosine, 2' -O-pentyl-guanosine, 2' -O-pentyl- cytidine, 3' -terminal 2'-0-methyl uridine and 6-amino-2- hydroxylmethyl-1-hexanol .
  • nucleosides 2'-0-nonyl adenosine, N6-imidazoylpropyl guanosine, 2' -O-aminopentyl cytidine, 2' -O-pentyl-adenosine, 2' -O-pentyl-guanosine, 2'-0- pentyl-cytidine, 3' -terminal 2'-0-methyl uridine were prepared by modification of the methods described in PCT US91/00243 filed 1/11/91. 6-amino-2-hydroxylmethyl-l-hexanol is available commercially. The nucleosides are modified to provide the corresponding phosphoramidite by methods known to those skilled in the art.
  • RNA was enzymatically synthesized, 32 P end-labeled according to standard procedures, and gel-purified.
  • 2 ' -O-Methyl oligonucleotide analog libraries comprising four sets were prepared in accordance with Examples 4 and 6. Each set was tested for binding against the RNA target and a "set K D " was determined in accordance with the following procedure.
  • the ras RNA target was incubated at a concentration of approximately 10 pM with each of the four random 2'-0-methyl oligonucleotide sets, at concentrations of 5, 10, 50 and 100 ⁇ M in a buffer consisting of 100 mM NaCl and 10 mM MgCl 2 .
  • the hybridization was carried out for four hours at 37°C, followed by electrophoretic separation of bound vs. unbound material on a 20% acrylamide gel in Tris- Borate buffer (TBE) plus 50 mM NaCl, run at 25 W for four hours. The gel was dried and the radioactive bands were visualized on a phosphorimager (Molecular Dynamics) .
  • the ras stem/loop target alone is the lowest band visible on the gel (highest mobility) .
  • this target binds oligonucleotide (non-radioactive)
  • the mobility of the ras target is decreased, shifting the band to a higher position on the gel (complex) .
  • NNNNANNNN shows a slight shift at 100 ⁇ M
  • NNNNCNNNN shifts more than 50% of the target to the bound form at 50 ⁇ M oligonucleotide concentration.
  • the protocol was then repeated in Round 2.
  • the ras RNA target was incubated at a concentration of approximately 10 pM with each of the four random oligonucleotide sets synthesized according to the method described above, at concentrations of 1 and 10 ⁇ M to provide the gel image of
  • the second gel shows that oligonucleotide sets NNNNCNANN, NNNNCNGNN and NNNNCNUNN show minimal binding.
  • NNNNCNCNN shows a shift of more than 25% of the target at l ⁇ M and about 50% of the target at lO ⁇ M.
  • N is any of A, C, G or T; ** Q is set complexity.
  • positions near the center of the oligonucleotide had a greater effect on the K D than positions on the extreme 5 ' or 3 ' ends. For example, an attempt to fix the 3' position in round 4 did not yield results that distinguished the sets. An alternative position was selected for round 4 which yielded a clear winner, and then the sequence was elucidated from the center of the oligonucleotide to the ends.
  • the final oligonucleotide selected by the procedure is complementary to the single stranded loop region of the target RNA.
  • HSV-1 envelope glycoprotein B gB
  • NHDF normal dermal fibroblast cells
  • KOS HSV-1
  • oligonucleotide were tested in triplicate wells at four concentrations. Cells were fixed 48 hours postinfection and assayed for the presence of HSV-1 gB antigen by ELISA. Standard deviation were typically within 10%.
  • Phosphorothioate Oligonucleotide Sets A group of 65,536 unique 8-mers in 4 sets of 16,348 was prepared in accordance with Examples 3 and 6 each was screened for activity against human herpes simplex virus type 1 (HSV-1) in cell culture in accordance with the procedure described in Example 9. As illustrated in Table 4, antiviral activity was observed with increasing potency at each round of synthesis and screening, with no difficulty discerning the most active set (in bold) in each round.
  • HSV-1 human herpes simplex virus type 1
  • N is any of A, C, G or T; ** where Q is set complexity.
  • the oligonucleotide set containing a fixed guanine had the most activity in every round of HSV screening except the last round, resulting in selection of a guanine at nearly all fixed positions.
  • a oligonucleotide group was designed as shown in Table 5, using the oligonucleotide cassette GGGG.
  • N is any of A, G, T or C.
  • the human T-lymphoblastoid CEM cell line was maintained in an exponential growth phase in RPMI 1640 with 10% fetal calf serum, glutamine, and antibiotics. On the day of the assay, the cells were washed and counted by trypan blue exclusion. These cells (CEM-IIIB) were seeded in each well of a 96-well microtiter plate at 5 X 10 3 cells per well. Following the addition of cells to each well, the compounds were added at the indicated concentrations and serial half log dilutions. Infectious HIV-1 IIIB was immediately added to each well at a multiplicity of infection determined to give complete cell killing at 6 days post-infection.
  • the XTT assay measures protection from the HIV-induced cell killing as a result of the addition of test compounds. The supernatant aliquot was utilized to confirm the activities determined in the XTT assay. Reverse transcriptase assays and p24 ELISA were performed to measure the amount of HIV released from the infected cells. Protection from killing results in an increased optical density in the XTT assay and reduced levels of viral reverse transcriptase and p24 core protein.
  • the compound sets are described in Table 6.
  • Table 6 sets forth the IC 50 ( ⁇ M) for four oligonucleotide sets.
  • N is any of A, C, G, or T.
  • Set C sowed 50% inhibition of HIV-induced cytopathic effects at 5 ⁇ M, while the other compound sets were inactive at concentration up to 25 ⁇ M.
  • Confluent monolayer cultures of human dermal fibroblasts were treated with oligonucleotide sets at the indicated concentrations in serum-free fibroblast growth media. After overnight incubation at 37 °C, culture medium containing oligonucleotide was removed, cells were rinsed and human cytomegalovirus was added at a multiplicity of infection of 0.1 pfu/cell. After a 2 hour adsorption at 37 °C, virus was removed and fresh fibroblast growth medium containing oligonucleotide sets at the indicated concentrations was added. Two days after infection, old culture medium was removed and replaced with fresh fibroblast growth medium containing oligonucleotide sets at the indicated concentrations.
  • HCMV antigen expression was quantitated using an enzyme linked immunoassay.
  • Primary reactive antibody in the assay was a monoclonal antibody specific for a late HCMV viral protein. Detection was achieved using biotinylated goat anti-mouse IgG as secondary antibody followed by reaction with streptavidin conjugated B-galactosidase. Color was developed by addition of chlorophenol red B-D-galactopyranoside and absorbance at 575 nanometers measured using an ELISA plate reader. Results are expressed as percent of untreated control and were calculated as follows :
  • a group of 65,536 unique phosphorothioate 8-mers in 4 sets of 16,438 were prepared in accordance with Examples 3 and 6 and screened for activity against the human cytomegalovirus in accordance with Example 14.
  • the results show that compound set B had the greatest activity against cytomegalovirus, causing approximately 20% inhibition at a lOO ⁇ M dose and 90% inhibition at a 200 ⁇ M dose.
  • Sets A, B and D exhibited minimal to no antiviral activity.
  • Vero cells were pretreated overnight with randomer sets by direct addition to the media at 10 ⁇ M and lOO ⁇ M concentrations. After overnight treatment cells were infected with influenza A/PR/8 at a MOI of 0.05. Following infection cells were incubated for 48 hours in the presence of oligonucleotide. After incubation cells were fixed with methanol and air dried. Monolayers were then assayed by ELISA for matrix protein.
  • Primary antibody was a monoclonal antibody specific for matrix protein of influenza A virus (B020 Bioproducts for Science) .
  • Second antibody was goat antimouse IgG conjugated to alkaline phosphatase (BRL, Bethesda, MD) .
  • Substrate was ATTO-PHOS reagent, JBL. Fluorescence was measured using a Millipore Cytofluour 2300 with excitation at 450 nM and emission read at 580 nM.
  • Example 20
  • a group of 65,536 unique phosphorothioate 8-mers in 4 sets of 16,438 was prepared in accordance with Examples 3 and 6 and was screened for activity against the Influenza A virus as described in Example 16.
  • the results show that sets C and D had the greatest antiviral activities, set C exhibited approximately 50% inhibition and set D exhibited approximately 35% inhibition of viral activity.
  • a and B exhibited minimal activity.
  • Data are the arithmetic mean and standard error of triplicate data points of a single experiment.
  • a phosphorothioate oligonucleotide group comprising 20 sets having the sequence N N N N X N N N where N is any of adenine, guanine, cytosine or thymidine and X is one of adenine, guanine, cytosine or thymidine is prepared in accordance with Examples 3 and 6.
  • the sets are set forth in Table 7.
  • An ELISA is performed to determine the set which is most effective to induce interferon.
  • the nucleotide in the most effective set is fixed and sets having the fifth position fixed and the fourth position one of adenine, guanine, cytosine or thymidine is prepared.
  • An ELISA is performed to determine the set which is most effective to induce interferon. The steps are repeated until all of the positions are determined.
  • the HIV TAR element is a structured RNA found on the 5 ' -end of all HIV transcripts. A gel shift has been used to analyze the binding of four oligonucleotide sets to the HIV TAR element.
  • the target RNA has a three base bulge that is required for binding of the transcriptional activation protein tat.
  • the oligonucleotides set forth in Table 8 were prepared in accordance with Examples 5 and 6, each containing a pyrene analog (indicated by A * ) .
  • the assay uses a 15pM concentration of the radioactively labeled target and an 0.1, 1, 10, and 100 ⁇ M concentrations of each set. Binding of molecules from the set to the target results in a slower mobility complex.
  • Set 3 binds best to TAR wherein 100 ⁇ M of the oligonucleotide set caused a shift of approximately 50% of the target.
  • 100 ⁇ m of the oligonucleotide set 2 caused a shift of approximately 25% of the target .
  • Sets 1 and 4 caused minimal shift of the target.
  • the sixth position will be fixed as a G and another position unrandomized in the second round of synthesis and assays.
  • Binding to double stranded DNA or RNA is possible by formation of a three stranded complex with the incoming third strand binding in the major groove of the duplex RNA or DNA.
  • the molecular nature of the interaction between the oligomer and target need not be known in order to practice the technique. Thus, it is possible that novel interactions between oligomers and DNA or RNA will be responsible for binding.
  • One of the limitations in the design of triple strand interactions is the need to have a long stretch of homopurines as a target.
  • the 3' (right) side of the gag-pol stem loop is homopurine except for a pair of cytosines near the bottom of the stem.
  • RNA oligonucleotide sets was designed to bind to the purine- rich strand of the gag-pol stem-loop by Hoogstein base pairing and prepared in accordance with Examples 2 and 6.
  • the sequence was randomized to provide the sequences set forth in Table 9. Binding to the gag-pol stemloop was measured by gel shift analysis as previously described in Example 8 with the following modifications: the radiolabeled gag-pol RNA was incubated with the oligonucleotide in lOOmM NaCl, 25 mM TRIS acetate pH5, 2mM Mg Cl 2 , ImM spermidine. The gel was a 15% acrylamide with 50 mM NaCl 2mM MgCl 2 added to the running buffer.
  • CCCUUCCCCUC (SEQ ID NO: 12) had the greatest affinity for the target in the ninth round with a K D of 1. Thus, a triple strand-binding sequence can be optimized.
  • a radiolabeled oligonucleotide group was prepared having the sequence NNGGGGNX wherein N is any of A, G, T or C and X is one or A, G, T or C as described in Examples 3, 6 and 7.
  • the group was screened for binding to the HIV tat protein, which is a transcription factor produced by the virus as described in Example 24. Binding activity was observed.
  • Receptor and radiolabeled ligand were supplied in a kit obtained from DuPont/NEN. Assays were performed according to the manufacturer's instructions. A random 2 ' -O- methyl group was prepared in accordance with Examples 4 and 6 to provide four sets having the sequences GCGNNNANNNNNNCGC (SEQ ID NO: 14); GCGNNNGNNNNNNCGC (SEQ ID NO:15) ; GCGNNNCNNNNNNCGC (SEQ ID NO:16) ; GCGNNNUNNNNNNCGC (SEQ ID NO: 17) where N is any of A, G, C or U.
  • Each set was diluted to 100 ⁇ M in an assay buffer provided in the kit, then incubated with the receptor and ligand as per the manufacturer's protocol. Following the incubation, ligand- bound receptor was separated from unbound by vacuum filtration through glass filters. The bound ligand was then eluted from the filter in scintillation fluid and counted in a scintillation counter. Receptor and ligand were incubated with an excess of unlabeled ligand in order to establish the level of non-specific binding (NSB) to the filters and with no oligonucleotide set (zero) to establish the level of complete binding.
  • NBS non-specific binding
  • Random 2'-0-Methyl Oligonucleotide Binding to Leukotriene B4 Receptor and radiolabeled ligand were supplied in a kit obtained from DuPont/NEN. Assays were performed according to the manufacturer's instructions. A random 2 ' -0- methyl group was prepared in accordance with Examples 4 and 6 to provide four sets having the sequences GCGNNNANNNNNNCGC (SEQ ID NO: 14) ; GCGNNNGNNNNNNCGC (SEQ ID NO:15) ;
  • GCGNNNCNNNNNNCGC SEQ ID NO:16
  • GCGNNNUNNNNNNCGC SEQ ID NO: 17 where N is any of A, G, C or U.
  • Each set was diluted to 100 ⁇ M in an assay buffer provided in the kit, then incubated with the receptor and ligand as per the manufacturer's protocol. Following the incubation, ligand- bound receptor was separated from unbound by vacuum filtration through glass filters. The bound ligand was then eluted from the filter in scintillation fluid and counted in a scintillation counter.
  • a phosphorothioate oligonucleotide group was prepared in accordance with Examples 3 and 6.
  • a 2'-0-methyl oligonucleotide group was prepared in accordance with
  • each group of random oligonucleotides is 5' end labeled to high specific activity with [ ⁇ - 32 P] ATP and T4 polynucleotide kinase.
  • poly dl*dc is added as indicated as a non-specific competitor.
  • Polypeptides may be used in the practice of this invention. Monomer amino acids are easily oligomerized into peptides using the appropriate precursor chemicals and instruments available to those skilled in the art, such as those that can be purchased from Applied Biosystems.
  • the first round of synthesis is as follows:
  • A is defined as an acidic amino acid
  • B is defined as a basic amino acid
  • W is defined as a neutral amino acid
  • L is defined as a lipophilic amino acid
  • X is defined as any amino acid from the above identified group.
  • 0.2 ⁇ M of a target oligonucleotide having the sequence 3'dBAB AGA CGT CTT GCG 5' (SEQ ID NO: 18) wherein B is biotin was incubated for 30 minutes at room temperature with lO ⁇ M of a radiolabeled 2'-0-methyl oligonucleotide group prepared in accordance with Examples 4, 6 and 7 having the sequence NNN NCN CNN wherein N is any of adenine, cytosine, thymidine or guanine, and O.l ⁇ M of a radioactively labeled oligonucleotide complementary to the target (dTCTGCAGAACGC; SEQ ID NO: 19) .
  • the target oligonucleotide and any bound radioactively labeled oligonucleotide was captured on streptavidin-coated magnasphere beads (Promega) , the beads were washed, and supernatant removed.
  • the captured radioactively labeled oligonucleotide was removed from the beads and run on a polyacrylamide gel .
  • a sample gel indicates that a "winner" can be separated from an excess of random sequence oligonucleotides. The procedure was repeated. In lane 1 was run a 1:10 dilution of the original solution prior to capture. Lane 2 is the supernatant diluted 1:10. Lane 3 is the bound material from the first round.
  • Lanes 4 and 5 are the supernatant (1:10 dilution) and bound material from the second round, respectively.
  • the second round results in a "winner” band of greater purity.
  • Lanes 6 and 7 are the supernatant (1:10 dilution) and bound material from the third round, respectively.
  • the supernatant does not contain any radiolabeled oligonucleotides.
  • the third round results in a "winner" band with little to no non-specific oligonucleotide.
  • a group of oligonucleotides having the sequence NNNNNNNN wherein N is any one of adenine, guanine, thymidine or cytosine is prepared in accordance with Examples 3 and 6.
  • the group is labeled ' using [ ⁇ - 32 P] ATP and T4 polynucleotide kinase.
  • oligonucleotide analog group comprising four sets of oligonucleotides eight positions in length is prepared in accordance with Examples 3 and 6 and each set is tested for binding against the nitrocellulose-bound proteins identified in accordance with Example 27.
  • the set having the highest affinity for each protein, as indicated by counts per well is the "winner set" for each protein. Results of the first round are as set forth in Table 13.
  • Oligonucleotides An oligonucleotide analog group comprising four sets of oligonucleotides eight positions in length is prepared in accordance with Examples 3 and 6 wherein each of the sets has a different one of adenine, guanine, thymidine and cytosine in the 5th position, and the rest of the positions are randomized to provide the group: NNNNANNN, NNNNGNNN, NNNNTNNN, and NNNNCNNN. Each set is subfractionated by charge with an anion exchange column. Each subfraction is tested for affinity for the target protein by gel shift assay. The subfraction from the set having an adenine in the 5th position has the highest binding affinity.
  • the 5th position is fixed to contain an adenine in the 5th position, and each set has a different nucleotide in the fourth position to provide the group NNNAANNN, NNNTANNN, NNNGANNN, and NNNCANNN.
  • the sets are again subfractionated by charge with an anion exchange column and the subfractions are tested for affinity for the target protein by gel shift assay. The steps are repeated until each position is determined.
  • the complementary oligonucleotides were then hybridized to create the double stranded NF-kB binding site, which was used as a positive control in the assay.
  • Each pool of the library was incubated in triplicate at a concentration of 1 ⁇ M with 100 ⁇ l of the extract for 20 minutes at room temperature.
  • the native double stranded NF-kB binding site was incubated at 100 nM with the same amount of extract.
  • each mixture was added to streptavidin coated microtiter plates (Elkay Lab Systems) and incubated 20 minutes. Unbound molecules were washed away with PBS. 10 ⁇ l of PBS plus 5% fetal calf serum was added to each well for 20 minutes at room temperature followed by aspiration and addition of 100 ⁇ l of 1:500 tat antisera in PBS plus 1% FCS for 2 hours at room temperature.
  • HIV mRNAs contain a stable hairpin structure known as TAR on the extreme 5' end of the message. This region has been shown to specifically bind an HIV regulatory protein known as tat. The mechanisms mediating increased gene expression have been intensely studied and may involve increased transcriptional efficiency. Cullen, et al . , Cell 1990, 63 , 655.
  • a truncated version (residues 16-45; ⁇ TAR) of the TAR hairpin previously shown to bind tat was synthesized using 2'-0-methyl oligonucleotides as described in Example 4 and was biotin conjugated on the 3' end ( ⁇ TAR-B) as described in Example 9.
  • the pools of the library at 0, 1, 10, or 100 ⁇ M were incubated with 100 ⁇ l of purified recombinant tat transcription factor (Repligen) at a concentration of 20 pg/ ⁇ l for 15' . This and all other steps were carried out at room temperature.
  • the competitor, ⁇ TAR-B was then added at a concentration of lOOnM. Following a 20' incubation, the mixture was bound to streptavidin-coated microtiter plates as in Example 33. After the non-bound molecules were washed away with PBS, 100 ⁇ l of 1:500 tat antisera was added to each well for 2 hours. Protein A/G-alkaline phosphatase was bound to the tat antibodies followed by the addition of the PNPP substrate and quantitation of A 405 as detailed in Example 33.
  • Repligen purified recombinant tat transcription factor
  • Example 33 Each pool of the phosphorothioate combinatorial library described in Example 33 was incubated at a concentration of lOOnM with tat protein as described in Example 34. Following incubation of the protein with the library pools, the complexes were captured on streptavidin plates and binding quantitated as in Example 33. The results are shown in Figure 4.
  • Myc is nuclear protein which functions in cell proliferation, differentiation and neoplastic disease, Nissen, et al . , Cancer Research 1986, 46, 6217. It has been shown to bind DNA in a sequence specific manner. Blackwell, et al., Science 1990, 250, 1149.
  • Example 34 Each pool of the library was incubated in triplicate at a concentration of 50 ⁇ M with the HL-60 extract described above.
  • An antibody directed to the leucine zipper region of the myc protein (Santa Cruz Biotechnology) was added at a 1:1000 dilution, myc bound to biotinylated c-myc transcription factor was quantitated as described in Example 34.
  • the results are shown in Figure 5.
  • the results, shown in Figure 6, demonstrate that the pool fixed at bilm A has the greatest ability to disrupt c-myc association to its natural DNA binding site.
  • Example 34 Each pool of the library was incubated at either 20 or 100 ⁇ M with 100 ⁇ l of the extract. This was followed by the addition of the NF-kB binding site competitor. Following the wash antibody to rel was added as in Example 33. The amount of rel bound was quantitated as in Example 34.
  • the pool fixed at nonyl C has the least affinity for the rel .
  • a second combinatorial library was synthesized based upon the first round winner.
  • the pools of the library were tested for inhibition of rel binding as described above.
  • the results, as shown in Figure 8, indicate that the pool fixed at nonyl C gave the greatest amount of inhibition.
  • c-jun is a major component of the AP-1 binding site which was originally shown to regulate TPA induced expression of responsive genes through the TPA response element (TRE) .
  • the June protein forms homo- or heterodimers which bind the TRE, but the Fos protein is only active as a heterodimer with any of the Jun family of proteins .
  • Fos/Jun heterodimers have a much higher affinity for the TRE than Jun homodimers ⁇ 3644 ⁇ . Both the fos and the jun cDNA have been cloned downstream of the Sp6 promoter.
  • MOLECULE TYPE RNA (genomic)
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  • MOLECULE TYPE DNA (genomic)
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  • MOLECULE TYPE DNA (genomic)
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  • MOLECULE TYPE DNA (genomic)
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Abstract

L'invention concerne des procédés utiles pour l'identification d'oligomères, qui sont spécifiques vis-à-vis d'un facteur de transcription ou d'une autre molécule cible dans un ensemble de sous-unités assemblées essentiellement d'une manière aléatoire. Les procédés décrits font appel à des synthèses répétées de jeux d'oligomères qui vont en se simplifiant, associées à de nouvelles procédures de sélection pour identifier les oligomères ayant l'activité la plus élevée. Le fait de ne pas avoir à utiliser des enzymes permet d'appliquer ces procédures à n'importe quelle molécule se prêtant à une oligomérisation contrôlée.
PCT/US1994/002166 1993-03-16 1994-03-01 Tests combinatoires pour la determination de facteurs de transcription et d'autres biomolecules par immunoabsorption d'oligomeres WO1994021825A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017955A2 (fr) * 1994-12-05 1996-06-13 Chiron Corporation Conception de sonde discontinue utilisant la cartographie hybritope
WO1997027330A1 (fr) * 1996-01-24 1997-07-31 Yale University Procede de selection multiple pour identifier des sites de liaison avec une proteine pour des proteines se liant a l'adn
WO1999013338A1 (fr) * 1997-09-12 1999-03-18 Genelabs Technologies, Inc. Procedes et compositions ayant trait a une proteine de fixation d'arn bicatenaire/arn bicatenaire
US6066452A (en) * 1997-08-06 2000-05-23 Yale University Multiplex selection technique for identifying protein-binding sites and DNA-binding proteins
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
JP2017504043A (ja) * 2014-01-22 2017-02-02 ユニバーシティー・オブ・ブレシアＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｂｒｅｓｃｉａ パーキンソン病を診断するｉｎ ｖｉｔｒｏの方法

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Title
ANALYTICAL BIOCHEMISTRY, Vol. 169, issued 1988, MATTHEWS et al., "Analytical Strategies for the Use of DNA Probes", pages 1-25. *
CELL, Vol. 44, issued 14 March 1986, GILMORE et al., "Different Localization of the Product of the V-Rel Oncogene in Chicken Fibroblasts and Spleen Cells Correlates with Transformation by REV-T", pages 791-800. *
NUCLEIC ACIDS RESEARCH, Vol. 19, No. 12, issued 1991, VICKERS et al., "Inhibition of HIV-LTR Gene Expression by Oligonucleotides Targeted to the TAR Element", pages 3359-3368. *
NUCLEIC ACIDS RESEARCH, Vol. 19, No. 13, issued 1991, NILLER et al., "Formation of Several Specific Nucleoprotein Complexes on the Human Cytomegalovirus Immediate Early Enhancer", pages 3715-3721. *
NUCLEIC ACIDS RESERACH, Vol. 18, No. 11, issued 1990, THIESSEN et al., "Target Detection Assay (TDA): A Versatile Procedure to Determine DNA Binding Sites as Demonstrated on Sp1 Protein", pages 3203-3209. *
SCIENCE, Vol. 250, issued 1990, BLACKWELL et al., "Differences and Similarities in DNA-Binding Preference of MyoD and E2A Protein Complexes Revealed by Binding Site Selection", pages 1104-1110. *
THE EMBO JOURNAL, Vol. 8, No. 9, issued 1989, KONIG et al., "Autoregulation of Fos: the Dyad Symmetry Element as the Major Target of Repression", pages 2559-2566. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996017955A2 (fr) * 1994-12-05 1996-06-13 Chiron Corporation Conception de sonde discontinue utilisant la cartographie hybritope
WO1996017955A3 (fr) * 1994-12-05 1996-08-29 Chiron Corp Conception de sonde discontinue utilisant la cartographie hybritope
US5736327A (en) * 1994-12-05 1998-04-07 Chiron Corporation Discontinuous probe design using hybritope mapping
US5747248A (en) * 1994-12-05 1998-05-05 Chiron Corporation Discontinuous probe design using hybritope mapping
WO1997027330A1 (fr) * 1996-01-24 1997-07-31 Yale University Procede de selection multiple pour identifier des sites de liaison avec une proteine pour des proteines se liant a l'adn
US5861246A (en) * 1996-01-24 1999-01-19 Yale University Multiple selection process for binding sites of DNA-binding proteins
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
US6066452A (en) * 1997-08-06 2000-05-23 Yale University Multiplex selection technique for identifying protein-binding sites and DNA-binding proteins
WO1999013338A1 (fr) * 1997-09-12 1999-03-18 Genelabs Technologies, Inc. Procedes et compositions ayant trait a une proteine de fixation d'arn bicatenaire/arn bicatenaire
JP2017504043A (ja) * 2014-01-22 2017-02-02 ユニバーシティー・オブ・ブレシアＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｂｒｅｓｃｉａ パーキンソン病を診断するｉｎ ｖｉｔｒｏの方法

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