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WO1993014108A1 - Selection de molecules de liaison - Google Patents

Selection de molecules de liaison Download PDF

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Publication number
WO1993014108A1
WO1993014108A1 PCT/US1993/000321 US9300321W WO9314108A1 WO 1993014108 A1 WO1993014108 A1 WO 1993014108A1 US 9300321 W US9300321 W US 9300321W WO 9314108 A1 WO9314108 A1 WO 9314108A1
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WO
WIPO (PCT)
Prior art keywords
test
molecule
dna
sequence
molecules
Prior art date
Application number
PCT/US1993/000321
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English (en)
Inventor
Gregory L. Verdine
Original Assignee
President And Fellows Of Harvard College
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Filing date
Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Priority to EP93903482A priority Critical patent/EP0623141A1/fr
Publication of WO1993014108A1 publication Critical patent/WO1993014108A1/fr
Priority to US08/220,272 priority patent/US5783384A/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • the present invention relates to methods of design ⁇ ing and producing a member of a binding pair which spe ⁇ cifically binds to its partner. It further relates to the products resulting from the methods. Such members are referred to herein as specific binding molecules. It particularly relates to designing and synthesizing mole ⁇ cules which specifically bind a desired target, such as a DNA sequence; these molecules are referred to as se- quence-specific DNA binding molecules and are also the subject matter of the present invention.
  • Molecules such as the sequence-specific binding molecules (also referred to herein as specific binding molecules) designed by the present method can be a peptide (D-, L- or a mixture of D- and L-) , a peptidomimetic, a complex carbohydrate or other oligomer of individual units or monomers which binds specifically to its binding partner (e.g., to DNA).
  • the present invention further relates to molecules, particularly sequence-specific DNA molecules, designed and produced by the present method and to uses therefor.
  • Specific binding molecules produced by the present method can be used in any application in which predictable or specific joining of two members of a binding pair is desired.
  • sequence-specific DNA binding molecules produced by the methods described herein are useful as gene regulatory molecules, such as molecules which mimic the tight and specific DNA binding character ⁇ istics of transcription factors, which play important roles in regulation of gene transcription by increasing or decreasing the rate of mRNA synthesis.
  • genes are regulated at the level of transcription by proteins, referred to as transcription factors, which bind promoter DNA.
  • transcription factors which bind promoter DNA.
  • a critical step in gene regulation by transcription factors is binding a factor to its specif- ic, or target, DNA sequences in the promoter.
  • Sequence- specific DNA binding molecules designed and produced by the present method can be used as molecules which mimic the tight and specific DNA binding characteristics of transcription factors and, as a result, exert control over gene expression.
  • Sequence specific DNA binding molecules can be used, for example, to control (enhance or repress) gene expression in vivo and, thus, serve as the basis for development of new therapeutic strategies for treating diseases or conditions in which there is a genetic defect.
  • a sequence-specific DNA binding molecule of the present invention can be used as an artificial or synthetic transcription repressor which is designed to bind a particular promoter and inhibit transcription of the gene under its control.
  • An artifi- cial or synthetic transcription repressor can be used to inhibit expression of a gene whose over-expression is associated with a disease or condition. Genetic diseases showing dominant inheritance, such as Huntington's dis ⁇ ease, are promising candidates for counteraction by transcriptional inhibitors designed and produced by the method of the present invention.
  • the present method of designing and producing a sequence-specific binding molecule is exemplified herein by the method of designing and producing a sequence- specific DNA binding molecule, particularly, a sequence- specific DNA binding peptide.
  • the following steps are carried out:
  • a desired or target molecule (e.g., a desired or target DNA sequence, or molecule) is synthesized or otherwise provided, which contains a first moiety capable of forming a reversible bond with a second moiety.
  • the target DNA sequence is one for which a sequence specific binding molecule, particularly a sequence specific DNA binding peptide, is to be designed and produced.
  • the target DNA sequence is combined with a test-binding mole ⁇ cule, which contains a moiety capable of forming a re ⁇ versible bond with the moiety present on the target sequence, such as the target DNA sequence.
  • the test- binding molecule (also referred to herein as test-mole ⁇ cule) comprises a unit such as an amino acid residue, to be assessed for its ability to bind to the desired DNA sequence.
  • the resulting combination of target DNA se ⁇ quences and test-molecules is maintained under conditions that are appropriate for the formation of a reversible bond between the first moiety (i.e., on the DNA sequence) and the second moiety (i.e., on the test-molecule) and binding of the unit being assessed to a region of the target sequence.
  • DNA sequence-test-binding molecule complexes are formed, or produced.
  • a mixture which contains complexes of the test-molecule bound to the desired target sequence, uncomplexed target molecules and uncomplexed test-molecules.
  • a sequence-specific DNA-binding molecule e.g., a DNA binding peptide
  • the resulting mixture contains complexes, uncomplexed target DNA sequence and uncomplexed test molecules.
  • the identity of the test-molecule present in the complexes, and the order of the units comprising the test-molecule, is determined by the present method by carrying out the above-described process.
  • the process is carried out a sufficient number of times to identify a binding partner, such as a DNA binding protein, of appro ⁇ priate makeup and sufficient length to bind to the target DNA and remain bound to the DNA, and subsequently deter- mining the identity and order of the units (e.g., amino acid residues) in the binding partner produced.
  • the test-molecule includes one more unit to be assessed than the test-molecule of the previous cycle; the test-molecule in the complex which is formed also has one additional unit than the complex in the previous cycle.
  • a sequence-specific DNA binding molecule is designed and produced.
  • the moiety present on the target DNA and on the target molecule is a thiol group
  • the reversible bond formed between the two moieties is a disulfide bond
  • the test-molecule is a peptide
  • the unit to be assessed is an amino acid residue.
  • a DNA molecule of a desired sequence which contains a thiol group attached at a specific site on the sequence is combined with a synthetic peptide which also contains a thiol group.
  • the peptide has the formula C0 2 H-Cys-Xaa-NH 2 .
  • the DNA molecule and the peptide bind, or associate, via the formation of a reversible disulfide bond, thus, forming a DNA-peptide complex.
  • a mixture of peptides can be used, all of which have the formula C0 2 H-Cys-Xaa-NH 2 and each of which differs in the amino acid residue Xaa (Xaa can be any amino acid residue which lacks an -SH group) .
  • each peptide will have a different association constant for the DNA sequence, and these differences will affect the reversibility, or reducibili- ty, of the disulfide bond.
  • the peptides Under reversing conditions, such as subjecting the formed complexes to a thiol gradient, the peptides are released from the DNA sequence according to their DNA association constants.
  • the strength of the disulfide bond in a disulfide-linked peptide-DNA complex is direct ⁇ ly related to the strength of the peptide-DNA associa- tion. This relationship permits screening of tight- binding peptides from a mixture of peptides. It is reasonable to expect that the peptide that remains complexed to the DNA sequence under conditions using the highest concentration of thiol binds tightest to the DNA. This screening process can be repeated in subsequent cycles with a peptide which has one additional amino acid residue designated Xaa, in each cycle.
  • each Xaa residue can be determined by conven ⁇ tional methods, such as peptide sequencing or UV absorp- tion. The order of the next residue of the peptide, resulting in the tightest binding to the DNA sequence is determined.
  • binding molecules include oligo- meric molecules in which units can be added or removed (e.g., D-, L- r or DL-peptides, peptidomimetic compounds or complex carbohydrates) .
  • Molecules made by the methods of the invention can be used to regulate a wide variety of biological process ⁇ es which depend on the site specific interaction of one molecule with another molecule. For example, processes mediated by the binding of a peptide with a nucleic acid, or of a peptide with a peptide.
  • Binding molecules which bind with a nucleic acid can be used to prevent gene activation by blocking the access of an activating factor to its sequence element, repress transcription by stabi ⁇ lizing duplex DNA or interfering with the transcriptional machinery, or carry out targeted DNA modification by delivering a reagent to a specific sequence.
  • Binding molecules which bind to peptides can be used to mediate or otherwise participate in, various processes such as antibody-antigen interactions, enzyme substrate interac- tions, hormone-receptor interactions, and lymphokine- receptor interactions.
  • the methods of the invention are chemical rather than biological, they can be used to select or discover binding molecules which are not normally synthe- sized by living organisms, such as peptides which include D-amino acids or nonbiogenic polymers (e.g., polymers derived from polyethylene glycol or nonnatural carbohy ⁇ drates) .
  • peptides which include D-amino acids or nonbiogenic polymers (e.g., polymers derived from polyethylene glycol or nonnatural carbohy ⁇ drates) .
  • Figure 1 is a schematic representation of the reac ⁇ tion between a thiol-tethered oligonucleotide and a mixture of -SH-containing peptides.
  • Figure 2 is a graph of a hypothetical reduction- elution profile.
  • Figure 3 shows the components of the CGN4 binding system, including the oligonucleotides GCN4-1 (SEQ ID N0:l); GCN4-2 (SEQ ID N0:2); GCN4-3 (SEQ ID N0:3); GCN4-4 (SEQ ID NO:4) and the GCN4-derived peptide, including the disulfide tether (SEQ ID NO:5).
  • the clear boxed area indicates the location of the tethered disulfide.
  • Figure 4 shows the results of coupling the disul- fide-linked GCN4 peptide (SEQ ID NO:5) with the GCN4 oligonucleotides (SEQ ID NOS:1-4) as analyzed by denatur- ating polyacryla ide gel electrophoresis.
  • X indicates what appears to be peptide-DNA complexes of differing mobility.
  • the present invention relates to methods of design ⁇ ing and producing a member of a binding pair which spe ⁇ cifically binds to its partner as well as to the products resulting from these methods.
  • Such members are referred to herein as specific binding molecules. It particularly relates to methods of designing and synthesizing mole ⁇ cules which specifically bind a desired DNA sequence (i.e., sequence-specific or site-speci ic DNA binding molecules) .
  • Specific binding molecule refers to an enti ⁇ ty, e.g., a molecule, or a portion of a molecule, which binds to a target.
  • a specific binding mole- cule is susceptible to a plurality of successive or serial modifications, e.g., in the case of a polymeric molecule, the addition of monomeric units to the polymer ⁇ ic chain.
  • the binding affinity of a specific binding molecule with the target can be evaluated before and/or after successive modification of the specific binding molecule.
  • a specific binding molecule is capable of reversible attachment to a target, preferably via a tether.
  • Test-binding molecule refers to a specific binding molecule, some or all of the structure of which is evaluated for inclusion in the final structure of a specific binding molecule.
  • the specific binding molecule e.g., a final full length peptide, which is the product of the entire process, can be referred to as a final or finished spe ⁇ cific binding molecule.
  • Target refers to an entity with which a specific binding molecule binds. Methods of the invention optimize binding affinity between a target and a specific binding molecule.
  • a target can be a molecule, a portion of a molecule, or an aggregate of molecules.
  • a target and a specific binding molecule can be separate molecules, or they may be different moieties on one molecule.
  • a target includes a target site.
  • a target is capable of reversible attachment to a binding molecule via a tether.
  • targets include: nucleic acids (e.g., RNA or DNA, double stranded DNA, single stranded DNA, or supercoiled DNA) , peptides or proteins (e.g., enzymes, receptors or antibodies), carbohydrates, and other molecular structures, such as nucleic acid- protein complexes, chromatin or ribosomes, lipid-bilayer containing structures, such as membranes, or structures derived from membranes, such as vesicles.
  • nucleic acids e.g., RNA or DNA, double stranded DNA, single stranded DNA, or supercoiled DNA
  • peptides or proteins e.g., enzymes, receptors or antibodies
  • carbohydrates e.g., lipid-bilayer containing structures, such as membranes, or structures derived from membranes, such as vesicles.
  • Target site or specific site refers to a site on a target to which a specific binding mole ⁇ cule binds.
  • Methods of the invention optimize binding affinity between a specific binding molecule and a target site on a target.
  • a target site will usually include a specific sequence of monomeric subunits or a three dimensional structure.
  • the actual structure (e.g., the chemical structure, or three dimensional structure) of the target site need only be known with enough particularity to allow formation of a reversible bond to the target.
  • the molecular interactions between a binding molecule and a target site are noncovalent and have energies of less than 25 kcal/mol at 25°C. These molecu ⁇ lar interactions include hydrogen bonds, Van de Waals interactions and electrostatic interactions.
  • Aggregate of molecules refers to two or more molecules which are connected by covalent or noncovalent interactions.
  • Tether refers to a structure which includes a moiety capable of forming a reversible bond with another moiety (e.g., a moiety on another tether) and (optionally) a spacer element. Alkane chains are suitable spacer moieties.
  • Reversible bond refers to a bond linking a binding molecule and a target (i.e., a binding pair) which is thermodynamically stable but capable of being broken by a reversing agent which is a physical or chemical agent capable of breaking the bond. For any given bond an appropriate reversing agent can be readily chosen based on the chemical nature of the bond.
  • a reversing agent for a disulfide bond is a reducing agent such as thiol.
  • the reversible bond is between a tether on a specific binding molecule and a tether on a target, a bond between tether on a specific binding molecule and a target, a bond between a specific binding molecule and a tether on a target, or a bond directly between a target and a specific binding mole- cule.
  • thermodynamically stable is meant a bond whose strength is greater than 10, preferably greater than 20, more preferably greater than 50, even more preferable greater than 65, but preferably less than 100 Kcal/mol at 25°C.
  • Suitable examples of reversible bonds include: R,- S-S-R-,, R.-S-Cd-S-R-,, and R ⁇ S-Hg-S-R., wherein R 1 includes a binding molecule or entity and R 2 includes a target and the reversible bond is within the underlined area.
  • bonds in which a metal e.g., Fe 3+ , Co 2+ , Ni 2* , Cu 2+ , Zn 2+ , Cd 2+ , or Hg 2+
  • a multidentate ligand i.e., a ligand having two (or more) moieties with which to complex an atom or group, prefera ⁇ bly a metal atom
  • a moiety on the binding molecule can be, e.g., S, N, or an imidaz- ole group
  • a multidentate ligand on a target wherein a moiety on the target can be S, N, or an imidaz- ole group.
  • multidentate ligands follow: SH
  • R can be either a binding molecule or a target.
  • multidentate ligands and monodentate ligands i.e., a ligand having one moiety with which to complex a metal or other atom or group
  • a binding molecule having a multidentate ligand and a target having a multidentate ligand a binding molecule having a multidentate ligand and a target having a multidentate ligand and a target having a monodentate ligand, or a binding molecule having a monodentate ligand and a target having a multidentate ligand can be used.
  • Methods of the invention can be used to design specific binding molecules which bind to a target site (i.e., a specific sequence) on a target molecule. These methods include an iterative process comprising successive ⁇ sive cycles of: (1) modifying a test-binding molecule (also referred to as a test-molecule) ; and (2) evaluating the affinity of the modified test-binding molecule for a target site on the target molecule.
  • the evaluation includes evaluating the relative affinity of a test- binding molecule for a target site as compared with other test-binding molecules in a pool, or mixture of test- binding molecules.
  • the affinity of the test-binding molecule for the target can be determined by forming a reversible bond between the test-binding molecule and the target.
  • the susceptibility of the reversible bond to reversal is related to the affinity of the test-binding molecule for the target site on the target.
  • a number of species of test-binding mole ⁇ cules, representing alternative modifications of a test- binding molecule i.e., modifications of the initial test-binding molecule or a test-binding molecule from the previous cycle of the method
  • the structure of the species (at each cycle) which gives the optimum results is chosen to supply an element of the structure of the final specific binding molecule.
  • a moiety capable of forming a reversible bond with a moiety on the test-binding molecule is attached to target DNA mole ⁇ cules.
  • a sulfhydryl group is tethered by an alkane chain to a site such as a site in a major or minor groove in a DNA molecule.
  • the DNA- [C] n -SH is then attached to an immobilizing matrix.
  • the DNA-[C] n -SH molecules are then complexed, via a disulfide bond, to a mixture of synthetic peptides and placed in a chromatography column as shown in Figure 1.
  • X in Figure 1 represents the number of species of peptides in a mixture of peptides.
  • the curved line connecting the peptide to the DNA target represents the tether.
  • the vertical arrows between the peptide and the DNA target represent the specific binding molecule/target site interaction, which, preferably, is the interaction the method optimizes.
  • the synthetic peptides are all of the formula C0 2 H- Cys-Xaa-NH 2 (where Xaa equals any amino acid residue which lacks an -SH group) .
  • N or C terminal can be modified, or blocked, as in the structure HN 2 C0 2 -Cys-Xaa-NHC0 2 CH 3 , to prevent unwanted interaction between the specific binding molecule and the target.
  • Amino acids may be added at either end of the molecule.
  • the mixture of synthetic peptides includes a variety of species (i.e., a plurality of peptides of different sequences) with differences in sequences arising from various candidate residues occupying the second (Xaa) position in different peptides.
  • the candidate residues may be any moiety which lacks an -SH group and which can be incorporated into the peptide chain, including, for example, D- or L-amino acids, naturally occurring or non- naturally occurring amino acids, or - , ⁇ - r or ⁇ amino acids.
  • the test-binding molecule will have different bind ⁇ ing affinities for the target DNA sequence, and these differences will affect the reducibility of the disulfide bond between the peptide and the DNA molecule with which it is complexed.
  • passage of a thiol gradient through the peptide-DNA column results in the release of the peptides according to the susceptibility of the binding molecule-target disulfide bond to reduc- tion (i.e., reversal).
  • reduc- tion i.e., reversal
  • Figure 2 shows a hypothetical elution profile.
  • the concentration of thiol is represented by a dashed line and the elution profile by a solid line.
  • the peak la ⁇ beled A represents the species with the highest binding affinity for the target.
  • C0 2 H-Cys-XAA-Xaa-NH 2 where XAA is the optimum second position residue and Xaa is defined as above, is cycled through the process to determine the optimum residue for the third position in the binding peptide.
  • Subsequent cycles extend the sequence of the binding peptide to the desired length.
  • the desired length can be a predetermined number of amino acid resi ⁇ dues, or can be a length at which the binding molecule exhibits useful or optimum binding affinity and/or se ⁇ quence specificity.
  • the site at which the reversible bond or tether is placed should be chosen so as to allow a specific binding molecule coupled to the target unhindered access to the target site on the target.
  • Stearic hindrance imposed by the location or structure of the bond or tether(s) can interfere with the correlation between bond reversibility and binding molecule-target site affinity.
  • the inclusion of a spacer element can reduce stearic hindrance.
  • an alkane of appropriate length can be used to provide both flexibility and sufficient separation be ⁇ tween the binding molecule and the target site.
  • nucleic acid When a nucleic acid is the target molecule a nucleic acid of any strandedness and of any topology can be used in methods of the invention.
  • the tether In the case of double stranded DNA, the tether can be located in a major or minor groove close to the target sequence, but not so close as to result in stearic hindrance to binding from strain on the bond between the binding peptide and the targe .
  • the reversible bond or tether can be located such that either binding molecule-target interactions or binding molecule-solution interactions are favored.
  • the reversible bond or tether can be placed at or near a terminus of the mole ⁇ cule to favor binding molecule-solution interactions, or in the central areas (away from the termini) , to favor binding molecule-target interactions.
  • a tether can be attached to DNA, or the reversible bond formed, on a base at any exocyclic amine or any vinyl carbon, such as the 5 or 6 position of pyrimidines, 8 or 2 positions of purines, at the ultimate 5' or 3' carbons, at the sugar phosphate backbone, or at internucleotide phosphorus atoms.
  • the binding molecule is conjugated to, or associated with, the target by a reversible bond.
  • the reversible bond is between a tether on the target and a tether on the specific binding molecule.
  • the tether on the binding molecule can be the same as the tether used on the target. Alterna ⁇ tively, different tethers can be used on each. In other embodiments only one tether is used, and in some embodi ⁇ ments the reversible bond is formed directly between the binding molecule and the target.
  • the tethers and the reversible bond should have the following characteristics.
  • a tether should be capable of attachment to the target without substantial alteration of the three dimensional structure of the target.
  • the reversible bond or tether-bearing-target should remain similar enough in conformation to the in vivo target so that the binding molecules generated will recognize and bind to the in vivo target with a useful affinity and site specificity.
  • the reversible bond formed between the target and the binding molecule should reversibly couple, by a covalent or ionic bond, the target to the binding molecule.
  • the susceptibility to reversal, or breakage, of the reversible bond formed between the target and the binding molecule should vary with the affinity of the binding molecule for the target site on the target.
  • the tether or tethers should be of appropriate length and flexibility such that the binding molecule has free access to the target site, and under the conditions used in methods of the invention, the reversible bond and/or tethers should be substantially unreactive with other sites on the binding molecule or target molecule.
  • Thiol groups are suitable moieties for forming a reversible bond.
  • a reversible bond e.g., a disulfide or metal-bridged disulfide bond, formed between -SH groups can be broken by contacting the bond with a reducing agent.
  • the reversible bond can be reversed with a ligand which competes with the metal atom for its position in the bridge.
  • the binding molecule is a peptide
  • the amino acid residue, cysteine is a convenient source of an -SH group for use as the binding molecule tether.
  • Alkane chains are suitable spacer moieties.
  • the reversible bond between the binding molecule and the target is disrupted with a reversing agent
  • immobilize the target molecule before exposure to the reversing agent This can be done by attaching, or linking the target to a matrix, such as a resin. Methods for attaching molecules to resins are known to those skilled in the art.
  • Test-binding molecules i.e., putative or candidate binding molecules
  • GCN4 a derivative of the DNA binding protein, GCN4, (O'Shea, E. K. , et al. , Science 243:538-542 (1989); Talanian, R. V., et al. , Science 249:769-771 (March 1990); Talanian, R. V., et al. , Biochem. 31:6871-6875 (1992)) was synthe- sized.
  • the GCN4-derived peptide is a monomer, comprised of 24 amino acid residues (SEQ ID NO:5).
  • the peptide was reduced, also as described in the Example, and, using the reaction conditions described in the Example, formation of the disulfide bond between the CGN4-derived peptide and the four DNA oligonucleotides was carried out. After incubation of the coupling reac ⁇ tion mixture, aliquots were taken and analyzed on poly- acrylamide gels under denaturing or native conditions.
  • Figure 3 shows the results of the analysis of aliquots from the four reaction mixtures containing the CGN4-derived peptide and the modified DNA sequences, on a denaturing gel. In all four reaction mixtures, a disul- fide-linked GCN4 peptide-DNA complex was formed, as indicated by the arrows denoting uncomplexed DNA and peptide-DNA complexes.
  • the structures of the disulfide-linked GCN4-DNA complexes were also analyzed to determine whether the peptides associated with the DNA oligonucleotides in a way that mimics their natural counterparts, or at least to discern that the peptide is bound in a sequence-spe ⁇ cific manner.
  • Preliminary data using DNA footprinting techniques indicate that three out of the four modified DNA oligonucleotides bound the GCN4-derived peptide in the anticipated region. That is, the data is strongly suggestive that the peptide bound to three DNA sequences in a site-specific manner.
  • binding of peptides to thiol- tethered DNA via formation of a disulfide bond can be performed as follows. Peptides can be bound quantita ⁇ tively to a thiol-tethered DNA molecule that is bound to a polymer resin, by formation of a disulfide bond between the DNA and the peptides. In these experiments, the object is to bind approximately 100% of the peptides to the resin-bound DNA, hence, an excess (2-10-fold mole excess based on the thiol-containing DNA strand) of resin-bound DNA, relative to moles of thiol groups (or disulfide groups) on the peptides is used.
  • the resin-bound DNA is prepared in the reduced state by treatment with common disulfide-reducing agents (alkanethiols or borohydride compounds) .
  • This incubation can be done in a batch mode or by passage of reagents through a column containing the resin-bound DNA.
  • the excess reducing agents can be removed by filtration (batch mode) or elution (column mode) .
  • Charging of the peptides onto the resin can either be done in batch mode or column mode.
  • the thiol group of the peptides will first be activated by conversion to the corresponding 2-thiopyridyl or 5- thio-2-nitrobenzoyl disulfide, using standard methods.
  • the activated peptides, in deaerated buffer, pH 7-9 (for example 50 mM Tris, pH 8.0) will be incubated with the reduced DNA-bound resin either with shaking or stirring (batch mode) or with recirculation (column mode) .
  • the resin-bound DNA can be prepared as the 2- thiopyridyl or 5-thio-2-nitrobenzoyl disulfide, and the reduced peptides bound as described above.
  • the binding reactions can be quantified by UV mea ⁇ surements, monitoring release of the pyridine-2-thione or 5-thio-2-nitrobenzoate chromophores.
  • the amount of peptides bound to the resin or free in solution can be quantified by a routine ninhydrin test.
  • the presence of free thiol groups on any material at any stage of the experiments can be monitored by alkylation with 14 C-iodoacetamide.
  • Binding can be optimized by examination of % pep- tides bound versus method of activation (DNA-disulfide or peptide-disulfide) , activating agent (2-thiopyridyl or 5- thio-2-nitrobenzoyl) , binding mode (batch or column) , time of incubation, temperature, and structure of the thiol-containing tether in the DNA.
  • activating agent (2-thiopyridyl or 5- thio-2-nitrobenzoyl
  • binding mode (batch or column)
  • time of incubation temperature
  • structure of the thiol-containing tether in the DNA In another embodiment, equilibrium binding of peptides to thiol-tethered DNA via formation of a disul ⁇ fide bond can be performed.
  • Peptides can be bound under equilibrium conditions to a thiol-tethered DNA molecule that is bound to a polymer resin, by formation of a disulfide bond between the DNA and the peptides.
  • the disulfide bond between the DNA and peptides can be formed under freely reversible conditions, so the noncovalent interaction of the peptide with DNA will cooperate with the covalent interaction (i.e., disulfide bond formation) to -establish a stable complex.
  • the thiol-tethered DNA is mixed with a stoichiomet- ric amount of the peptides in a deaerated redox buffer.
  • the redox buffer can be the same as the redox eluent described above.
  • the most important components are the reduced and oxidized forms of a thiol reducing agent, such as 2-thiopyridine, 5-thio-2-nitrobenzoate, dithiothreitol, 2-mercaptoethanol, and N,N'-dimethy1- N,N'-bis(mercaptoacetyl)hydrazine (DMH) .
  • the reactants are allowed sufficient time to reach equilibrium.
  • DNA-bound peptides are then eluted by incubation of the resin under strongly reducing conditions (such as 100 mM dithiothre- itol) .
  • strongly reducing conditions such as 100 mM dithiothre- itol
  • parallel incubations should be set up and analyzed separately.
  • the following conditions can be varied to optimize the system: chemical structure of redox eluent, concen ⁇ tration of redox eluent, temperature, flow rate, buffer conditions (pH, ionic strength, addition of organic co- solvents such as trifluoroethanol) .
  • Peptides can be quantified by amino acid analysis and sequenced by automated phenylthiohydantoin methods.
  • Binding Molecule-Target Site Binding Affinity The affinity of a specific binding molecule for the target site on a target can be determined by evaluating the ease with which a reversible bond between the binding molecule and the target can be reversed. These determi ⁇ nations can be made by immobilizing the binding molecule- target complex, such as on a matrix or a resin, and passing a gradient of a reversing agent (an agent which reverses, that is, breaks, or disrupts, the reversible bond and thus releases the binding molecule from the tar ⁇ get site) over the immobilized complexes.
  • a reversing agent an agent which reverses, that is, breaks, or disrupts, the reversible bond and thus releases the binding molecule from the tar ⁇ get site
  • test-binding molecules In most embodiments of the methods described herein, several species (also refrred to herein as a plurality) of test-binding molecules will be screened simultaneously to determine which test-molecule possesses the optimum binding properties.
  • the elution profile allows determi- nation and comparison of the binding affinities of vari ⁇ ous species of test-binding molecule and selection of the species which represents the optimum or desired structure for the final specific binding molecule.
  • the resin bound peptide-DNA complexes are placed in a chromatogra- phy column.
  • a gradient of a reducing agent e.g., a thiol reagent, is applied to the column. This results in the release of peptides according to their DNA associa ⁇ tion constants, producing a reductive elution profile. The peptide that elutes last has the highest affinity for the target DNA. This chemical screening process thus provides the optimal residue at the tested position.
  • Elution of peptides coupled to a target by a disul ⁇ fide bond can be performed, either in batch or column mode, as follows. Column mode allows more precise con ⁇ trol over the elution conditions, since the column can be attached to a commercially available gradient elution system, such as the Fast Protein Liquid Chromatograph
  • FPLC FPLC
  • Pharmacia Pharmacia
  • Batch mode operation may be necessary if the conditions required for elution (e.g., high temperatures, long elution times) are incompatible or inconvenient with FPLC.
  • a redox gradient is passed through the column, causing peptides to be released depending on their redox potential.
  • the redox gradient consists of mixtures of a thiol or dithiol compound and its corresponding disulfide. In the beginning of the gradient, the redox eluent contains 100% of the disulfide form, and at the end of the gradi ⁇ ent, 100% of the thiol (or dithiol) form.
  • Typical redox eluents consist of the thiol and disulfide forms of 2- thiopyridine, 5-thio-2-nitrobenzoate, dithiothreitol, 2- mercaptoethanol, and the N,N'-dimethyl-N,N'—bis(mercapto- acetyl)hydrazine (DMH) reagent recently reported by Whitesides fJ. Org. Chem. 56:2332-2337 (1991)).
  • DMH N,N'-dimethyl-N,N'—bis(mercapto- acetyl)hydrazine
  • the latter may be preferable because of its exceptionally fast kinetics of disulfide reduction. Elution of peptides from the column is monitored by on-line UV detection at 214 n and post-column derivati- zation with ninhydrin.
  • Peptides are quantified by amino acid analysis and sequenced by automated phenylthiohydan- toin methods.
  • the following conditions can be varied to optimize elution for speed, ease, or resolution: chemical struc ⁇ ture of redox eluent, concentration of redox eluent, slope of gradient, shape of gradient (linear, step, exponential) , temperature, flow rate, buffer conditions (pH, ionic strength, addition of organic co-solvents such as trifluoroethanol) .
  • the resin containing DNA-bound peptides is incubated in an Eppendorf tube with deoxygen- ated buffer containing the redox eluent.
  • Redox eluents, quantification and identification of peptides are the same as described above for the column mode.
  • the follow ⁇ ing conditions can be varied to optimize elution: chemi ⁇ cal structure of redox eluent, concentration of redox eluent, number and spacing of stepwise elutions, elution time, temperature, buffer conditions (pH, ionic strength, addition of organic co-solvents such as trifluoroetha ⁇ nol) .
  • a second modification can be performed on the test- binding molecule (e.g., the addition of a subsequent residue to a polymeric binding molecule) and the process of evaluating the binding affinity of the newly modified test-binding molecule repeated. This cycle may be re ⁇ peated a number of times.
  • test-binding molecules representing a number of different modifications
  • a number of species i.e., a plurality
  • test-binding molecules representing a number of different modifications
  • a set of tripeptide ⁇ of the formula C0 2 H-Cys-XAA-Xaa-NH 2 (where XAA is the optimum second position amino acid and Xaa represents any amino acid which lacks an -SH group) , is synthesized.
  • Each peptide of the set differs at Xaa.
  • the elution and determination of binding affinity is repeated with the tripeptide to yield the optimum amino acid residue at the third position. The process is repeated until the de ⁇ sired length is reached.
  • modifications can be performed on the binding molecule. These modifications may be in the form of a second round of selected optimizations of a different binding molecule characteristic. For example, after an initial determination of the optimum primary sequence of a peptide, a second iterative selection can be applied to determine an optimum level of glycosylation, the effect of cofactors, the effect of homo- or heterodimerization. or the effect of inter- or intra-chain cross linking. These, or other modifications may be tested for their effect on binding by non-iterative methods as well.
  • a second iterative selection can be per- formed to select a second specific binding molecule to form a heterodimer with the binding molecule selected in the first iterative cycle.
  • These two specific binding molecules may be cross-linked by conventional methods. Modifications such as the formation of homo- or heterodimers, may require alteration of a selected bind ⁇ ing molecule. For example, new peptides may be constructed to optimize the spacing of binding units relative to each other and the center of target sites .in the DNA, or to allow the introduction of specifically desired residues. Molecular modeling can be used to facilitate the choice of modifications.
  • dimerized peptides can be tested by meth ⁇ ods known to those skilled in the art (e.g., by competi ⁇ tion electrophoretic mobility shift assays, PCR-based target detection assay, or chemical or enzymatic footprinting) .
  • the X-ray crystal structures of the bacteriophage repressor (Jordan et al.. Science 242:893 (1988)) and the murine Zif268 protein (Pavletich et al. , Science 252:809 (1991)) bound to their respective DNA sites are deposited in the Brookhaven Protein Data Bank.
  • These can also be retrieved and molecular modeling methods used to trim the structures down to a peptide-bound DNA core structure, as was done with GCN4.
  • Disulfide tethers can be designed to link the resulting peptides to DNA, bearing in mind that the connector should be as short as possible without generat ⁇ ing strain.
  • the ⁇ repressor and Zif268 systems are favorable for optimization because they represent respec ⁇ tively, examples of extended and ⁇ -helical peptides that bind DNA as isolated units and for which high-resolution structures in the DNA-bound form are available.
  • DNA-binding peptides designed on the basis of X-ray structures can be synthesized by standard methodology.
  • Thiol-tethered oligonucleotides designed similarly (“wild-type” oligonucleotides) can be synthesized by methods and linked to a resin, as described above.
  • the peptides can be tethered to DNA both in solution (for use in high-resolution structural studies) and on a solid matrix (for reductive elution studies) .
  • the conditions for forming and releasing the peptide-DNA reversible bond can be optimized using these molecules, as described in the Example.
  • the structures of the DNA-tethered peptide systems constructed in the previous state can be evaluated to discern whether the peptides are associated with DNA in a way that mimics their natural counterparts, or at least in a way that is discernibly sequence-specific.
  • 1 H-NMR, 15 N-NMR, chemical footprinting, and circular dichroism spectroscopy can be used to evaluate these molecules.
  • Wild-type and mutant peptide-DNA systems, assembled on a solid matrix in a column can be subjected to reduc ⁇ tive elution by a thiol gradient. Parameters affecting elution, such as reducing agent, temperature, pH and slope of the gradient, can be optimized. For example, this approach can be used to find conditions in which wild-type ⁇ and Zif268 peptides are strongly retained (elute late in the gradient) while peptide from mutant systems are not strongly retained (elute early) .
  • the wild-type peptides can be elongated by one peptide unit, using a mixture of any amino acids that lack an -SH group.
  • This 19 peptide mixture can then be coupled to the solid matrix, loaded into a column, and eluted reductively.
  • the late-eluting peptides will be sequenced (e.g., by fast atom bombardment mass spectrometry and/or phenylthiohydantoin degradation) . This synthesis and screening process can be repeated iteratively until either the efficiency of synthesis or resolution of the column procedure falls off.
  • Elongated peptides that are obtained by iterative selection should bind selectively to longer target DNA sequences than the starting peptides.
  • the interaction of these peptides with DNA can be studied by the same meth ⁇ ods as described above for the starting peptides.
  • the three dimensional molecule can serve as a guide in choosing the modifications. This can allow the optimization of residues on the same face or side of a structure. For example, in the case of a binding mole- cule which is a helical molecule, it may be desirable to add subunits in groups of n, where n is the number of subunits involved in one full turn of the helix.
  • the desired three-dimensional structure of the binding molecule can also influence choice of modifica ⁇ tion in other ways.
  • residues which promote the formation of a helical structure such as 2-aminoisobutyric acid or ⁇ -methyl amino acids, can be added.
  • pro-gly could be added to a sequence to interrupt a helical structure.
  • a pro-gly series can be added to a peptide sequence to introduce a fold in a / 5-sheet or ⁇ -ribbon structure.
  • Peptide-on-phage libraries can be used to * supply the binding entities in methods of the invention.
  • a fully degenerate phage library could include all peptide test-binding entities to be tested in one batch.
  • the peptides could be coupled to the target and eluted as a batch.
  • oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer Model 381A using conventional and modified phosphoramidites according to the "convert- ible nucleoside approach" described in MacMillan, A. M. and Verdine, G. L. , J. Org. Chem. 55_:5931 (1990) and Ferentz, A. E. , and Verdine, G. L. , J. Am. Chem. Soc. 113:4000-4002 (1991) .
  • the lyophilized GCN4-derived peptide was dissolved in 0.1 ml of lxTE8 (Tris-EDTA buffer, pH 8) and peptide concentration determined by UV spectroscopy (210 and 220 nm) was 3 mM.
  • the peptide was reduced by the addition of 1 microliter of 1:10 dilution of 2-mercaptoethanol stock (14.4M, obtained from Bio-Rad Laboratories) and incubated at 50° for 30 minutes.
  • the reaction mixture was subse- quentlyophilized in the speedvac concentrator (Savant) to evaporate 2-mercaptoethanol and the dry pellet was dissolved in 0.1 ml of 10xTE8.

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Abstract

L'invention concerne des procédés de conception et de production de protéines de liaison d'ADN spécifique à une séquence, des procédés de détermination de l'affinité d'une molécule de liaison spécifique pour une cible ainsi que des produits obtenus par ces procédés. Les procédés comprennent la formation d'une liaison réversible entre une molécule de liaison spécifique et la cible ainsi que la détermination de la susceptibilité à l'inversion de la liaison réversible comme une mesure de l'affinité de la molécule de liaison pour la cible.
PCT/US1993/000321 1992-01-13 1993-01-13 Selection de molecules de liaison WO1993014108A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6015709A (en) * 1997-08-26 2000-01-18 Ariad Pharmaceuticals, Inc. Transcriptional activators, and compositions and uses related thereto
US6818394B1 (en) 1996-11-06 2004-11-16 Sequenom, Inc. High density immobilization of nucleic acids
US7232688B2 (en) 1997-01-23 2007-06-19 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
US8999266B2 (en) 2000-10-30 2015-04-07 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
US9068953B2 (en) 2007-09-17 2015-06-30 Agena Bioscience, Inc. Integrated robotic sample transfer device
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4182654A (en) * 1974-09-18 1980-01-08 Pierce Chemical Company Production of polypeptides using polynucleotides
WO1989010931A1 (fr) * 1988-05-02 1989-11-16 The Regents Of The University Of California Procede general de production et de selection de peptides ayant des proprietes specifiques
WO1991019813A1 (fr) * 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4182654A (en) * 1974-09-18 1980-01-08 Pierce Chemical Company Production of polypeptides using polynucleotides
WO1989010931A1 (fr) * 1988-05-02 1989-11-16 The Regents Of The University Of California Procede general de production et de selection de peptides ayant des proprietes specifiques
WO1991019813A1 (fr) * 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
CONNOLLY ET AL., NUC. ACID RES., vol. 13, 1985, pages 4485
FERENTZ ET AL., J. AM. CHEM. SOC., vol. 113, 1991, pages 4000 - 4002
FERENTZ, A. E.; VERDINE, G. L., J. AM. CHEM. SOC., vol. 113, 1991, pages 4000 - 4002
FERENTZ, A. E.; VERDINE, G. L., J. AM. CHEM. SOC., vol. 113, pages 4000 - 4002
FIDANZA ET AL., J. AM. CHEM. SOC., vol. 111, 1989, pages 9117 - 9119
GALAS, D. J.; SCHMITZ, A., NUCLEIC ACID RES., vol. 5, 1978, pages 3157 - 3170
J. ORCT. CHEM., vol. 56, 1991, pages 2332 - 2337
JORDAN ET AL., SCIENCE, vol. 242, 1988, pages 893
LETSINGER ET AL., J. AM. CHEM. SOC., vol. 103, 1981, pages 7394 - 7396
MACMILLAN ET AL., J. ORQ. CHEM., vol. 55, 1990, pages 5931 - 5933
MACMILLAN ET AL., TETRAHEDRON, vol. 47, 1991, pages 2603 - 2616
MACMILLAN, A. M.; VERDINE, G. L., J. ORG. CHEM., vol. 55, 1990, pages 5931
O'SHEA, E. K. ET AL., SCIENCE, vol. 243, 1989, pages 538 - 542
PAVLETICH ET AL., SCIENCE, vol. 252, 1991, pages 809
TALANIAN, R. V. ET AL., BIOCHEM., vol. 31, 1992, pages 6871 - 6875
TALANIAN, R. V. ET AL., SCIENCE, vol. 249, August 1990 (1990-08-01), pages 769 - 771
ZUCKERMAN ET AL., NUC. ACID RES., vol. 15, 1987, pages 5305

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6818394B1 (en) 1996-11-06 2004-11-16 Sequenom, Inc. High density immobilization of nucleic acids
US7232688B2 (en) 1997-01-23 2007-06-19 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
US6015709A (en) * 1997-08-26 2000-01-18 Ariad Pharmaceuticals, Inc. Transcriptional activators, and compositions and uses related thereto
US8999266B2 (en) 2000-10-30 2015-04-07 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
US9669376B2 (en) 2000-10-30 2017-06-06 Agena Bioscience, Inc. Method and apparatus for delivery of submicroliter volumes onto a substrate
US9068953B2 (en) 2007-09-17 2015-06-30 Agena Bioscience, Inc. Integrated robotic sample transfer device
US11981703B2 (en) 2016-08-17 2024-05-14 Sirius Therapeutics, Inc. Polynucleotide constructs
US11597744B2 (en) 2017-06-30 2023-03-07 Sirius Therapeutics, Inc. Chiral phosphoramidite auxiliaries and methods of their use

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