WO2009077173A2 - Bibliothèques de produits chimiques codés par adn - Google Patents

Bibliothèques de produits chimiques codés par adn Download PDF

Info

Publication number
WO2009077173A2
WO2009077173A2 PCT/EP2008/010770 EP2008010770W WO2009077173A2 WO 2009077173 A2 WO2009077173 A2 WO 2009077173A2 EP 2008010770 W EP2008010770 W EP 2008010770W WO 2009077173 A2 WO2009077173 A2 WO 2009077173A2
Authority
WO
WIPO (PCT)
Prior art keywords
region
complementary
initial
coding
building block
Prior art date
Application number
PCT/EP2008/010770
Other languages
English (en)
Other versions
WO2009077173A3 (fr
Inventor
Dario Neri
Samu Melkko
Luca Mannoci
Fabian Buller
Original Assignee
Philochem Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Philochem Ag filed Critical Philochem Ag
Publication of WO2009077173A2 publication Critical patent/WO2009077173A2/fr
Publication of WO2009077173A3 publication Critical patent/WO2009077173A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis

Definitions

  • the present invention relates to methods of preparing DNA-encoded chemical libraries, libraries prepared by those methods, use of these libraries and particular compounds therein.
  • HTS high-throughput screening
  • DNA-encoded chemical libraries are collections of small organic molecules that are conjugated covalently to DNA tags that serve as identification bar codes.
  • DNA-encoded chemical library the organic molecule, or putative binding moiety, is linked physically to a DNA tag that carries the identity code of the molecule it is attached to.
  • the selection procedure for DNA-encoded chemical libraries comprises incubating the library of encoded molecules with the target protein of choice and, after affinity capture, non-binding library members are separated from binding library members by, for example, coupling the target protein to a solid support and removing the supernatant (that contains the non-binders). The stringency of selection is controlled using suitable washing conditions. After elution of the DNA-tagged binders, the enriched DNA moieties are amplified by polymerase chain reaction (PCR), which allows very low amounts of template DNA to be detected.
  • PCR polymerase chain reaction
  • Subsequent decoding of the enriched DNA uses either DNA sequencing or hybridization to oligonucleotide microarrays, depending on the architecture of the library and its size.
  • the DNA tags not only serve as identification bar codes, but can also guide the synthesis of the displayed molecule.
  • the population of DNA tags of the selected library members would be amplified by PCR and, in theory, utilized for enrichment of the bound molecules by serial hybridization steps to a subset of the library. In principle, the affinity-capture procedure could be repeated, possibly resulting in a further enrichment of the active library members. Finally, the structures of the chemical entities would be decoded by cloning and sequencing the PCR products. The feasibility of the orthogonal, solid-phase synthesis of peptides and oligonucleotides was demonstrated by attaching a test peptide (the pentapeptide leucine enkephalin) and an encoding DNA tag onto controlled-pore glass beads (Nielsen, J., et ai, J.
  • the present inventors have developed a method of preparing DNA-encoded chemical libraries in which the initial coding of each building block of the chemical moiety is carried out by a single stranded oligomer, and where the double stranded DNA moiety in the final library members is completed using a DNA polymerase.
  • a first aspect of the present invention provides a method of preparing a DNA-encoded chemical library comprising members which comprise a chemical moiety, a linking moiety and a DNA moiety, comprising for each member the steps of:
  • the middle building block is coupled to the residue of the initial building block; and (b) the middle coding single strand oligomer comprises a middle coding region and second annealing region, and is coupled by:
  • the final building block is coupled to the residue of the initial building block, or may be additionally or alternatively be coupled to the residue of the middle building block (if present); and (b) the final coding single strand oligomer comprises a final coding region and a second primer region, and is coupled by:
  • a complementary single strand oligomer which comprises a complementary first or second annealing region as appropriate, a complementary final coding region and a complementary second primer region, by interaction between the first or second annealing region and the complementary first or second annealing region, as appropriate; (ii) treating the conjugate formed with a DNA polymerase to elongate the initial coding single strand oligomer to be complementary to the complementary final coding region and the complementary second primer region, and to elongate the complementary coding strand to be complementary to the initial coding single strand oligomer.
  • a second aspect of the present invention provides a method of preparing a DNA-encoded chemical library comprising a chemical moiety, a linking moiety and a DNA moiety, comprising for each member the steps of: (I) coupling a initial building block to a initial coding single strand oligomer via a linking moiety to form a initial conjugate, the initial coding single strand oligomer comprising a primer region, an initial coding region and an annealing region;
  • the final coding single strand oligomer comprising a final coding region and a second primer region, and is coupled by ligating a terminal double-stranded portion comprising a ligation region that matches the remainder of the restriction region site of the middle conjugate, and a final coding region and second primer region, as well as the complementary regions in the complementary strand.
  • steps (I), (II) if present, and (III) follow a split and pool methodology.
  • This well-known technique in combinatorial chemisty involves carrying out step (I) in an appropriate number of vessels for the number of building blocks, pooling the resulting initial conjugates and then splitting the mixture so obtained into the appropriate number of vessels for the number of building blocks in the subsequent step. This method can be repeated a third time if the method involves three groups of building blocks.
  • a third aspect of the present invention provides a method of preparing a DNA-encoded chemical library comprising members which comprise a chemical moiety, a linking moiety and a DNA moiety, comprising for each member the steps of:
  • the middle coding single strand oligomer comprises a middle coding region and a restriction region, and is coupled by:
  • the coupling take place in either order, wherein: (a) the final building block is coupled to the residue of the initial building block, and additionally or alternatively be coupled to the residue of the middle building block; and (b) the final coding partially double stranded oligomer comprises a final coding region, a second primer region, and a double stranded ligation region that matches the remainder of the restriction region site of the middle conjugate, and is coupled by ligation using an excess of the final coding partially double stranded oligomer.
  • step (I), (II) and (III) follow a split and pool methodology.
  • This well-known technique in combinatorial chemisty involves carrying out step (I) in an appropriate number of vessels for the number of building blocks, pooling the resulting initial conjugates and then splitting the mixture so obtained into the appropriate number of vessels for the number of building blocks in the subsequent steps
  • a fourth aspect of the present invention provides a library synthesised according to the method of either the first, second or third aspects of the invention. This aspect also comprises the particular library described below however made.
  • a fifth aspect of the present invention provides the use of the library of the fourth aspect in a method of screening the library for a library member which shows affinity for a biological target.
  • Figure 1 illustrates the invention where the optional middle step doesn't take place
  • Figure 2 illustrates the invention with the optional middle step.
  • Figure 4 shows the results of the screening of a library of the invention against streptavidin; figure 4a shows the frequency of the library members before selection; figure 4b shows the frequency of the library members after selection on empty beads; figure 4c shows the frequency of the library members after selection against Streptavidin bead, as well as the identity of high frequency library members.
  • Figure 5 shows the results of the screening of a library of the invention against IgG 1 with the identity of high frequency library members.
  • Figure 6 shows the results of use of an IgG binding resin according to the invention.
  • Figure 7 illustrates three embodiments of the first and second aspects of the invention, including gels resulting from the experiments described in Example 7.
  • Each member of the library comprises a chemical moiety, which is made from building blocks, a DNA moiety, which includes codes for the building blocks of the member, and a linking moiety, which links the chemical moiety to the DNA moiety.
  • a library of this invention is a collection of chemical diverse library members.
  • the number of different members in a library represents the complexity of a library and is defined by the number of building blocks in each member of the library, and by number of variants of each building block.
  • the number of different members of any particular library can be determinded by multiplying the number of variants of each building block together. For example, if there are two building blocks, each of which have twenty variants, then the resulting library has 400 members. If, for example, there are three building blocks, each of which has twenty variants, then the resulting library has 8000 members.
  • This size limit is dictated by the level of sensitivity for detecting the presence of the DNA moiety after the screening process. Detection sensitivity is dictated by the threshold of binding between an acceptor molecule to be assayed and a library member.
  • the relative amounts of the individual members within the library can vary from about 0.2 equivalents to about 10 equivalents, where an equivalent represents the average amount of a member within the library. Preferably each member is present in the library in approximately equimolar amounts.
  • a library contains the every possible member based on the mathematical combinations for a given library.
  • Chemical Moiety is formed from the initial, middle (if present) and final building blocks. If there are only two building blocks, initial and final, these may be joined to one another by a chemical reaction (see below) and their residues will be linked by one or more chemical bonds.
  • both middle and final building blocks are linked to the initial building block.
  • both middle and final building blocks are linked to the initial building block, as well as to each other other.
  • each building block for linking to other building blocks will vary depending on the mode of binding desired.
  • the initial building block will also require functionality so as to bind to the linking moiety.
  • reaction conditions used to link the building blocks must be compatible with the DNA moiety of the library members.
  • the type of building blocks that are suitable for use in the present invention will be limited by suitable linking chemistry.
  • a subsequent building block may be a carboxylic acid (or activated version thereof, e.g. ester, acid anhydride, acid halide).
  • carboxylic acid or activated version thereof, e.g. ester, acid anhydride, acid halide.
  • Appropriate conditions are shown in scheme 1 , where the method is illustrated with respect to an amino acid residue:
  • the first building block comprises a protected amine group
  • this is deprotected before being coupled with a carboxylic acid or activated form thereof.
  • first building block comprises a double bond conjugated to an electron withdrawing group
  • a subsequent building block being a thiol, alcohol, primary amine or enolate may be added, as shown in scheme 2:
  • Click chemistry may be used to join an azide containing moiety to a alkyne containing moiety, as shown in scheme 3:
  • Ether-bond formation could be used to join a moiety comprising a hydroxy group and a moiety comprising a leaving group, such as halogen, OTf, ONf or OTs, as shown in scheme 4:
  • Reductive amination could be used to link a moiety comprising a ketone group with a moiety comprising a primary amine group, as shown in scheme 5:
  • the chemical moiety is formed from an initial and final building block, where the initial building block is an amino acid, and the final building block is a carboxylic acid.
  • the collection of 20 initial building blocks is set out in Table 1:
  • the present invention provides a DNA-encoded chemical library comprising members which comprise a chemical moiety, a linking moiety and a DNA moiety, wherein the chemical moiety is the reaction product of an initial building block and a final building block, the initial building blocks being selected from table 1 and the final building blocks being selected from table 2 where the library comprises members representing the reaction product of at least three initial building blocks and at least 5 final building blocks.
  • the library may comprise members representing the reaction product of at least five, ten fifteen or twenty initial building blocks and at least 10, 50, 100 or 200 final building blocks.
  • the library comprises members representing every possible reaction product of the initial and final building blocks, in some embodiments, the library comprises members representing at least 75, 90 or 95% of the possible reaction products.
  • the linker moiety can be any moiety that performs the function of operatively linking the chemical moiety to the DNA moiety.
  • the linker moiety can vary in structure and length, and provide at least two features: (1) operative linkage to the chemical moiety and (2) operative linkage to the DNA moiety.
  • any of a variety of chemistries may be utilized to effect the indicated operative linkages to both the chemical and DNA moieties, the nature of the linkage is not considered an essential feature of this invention.
  • the size of the linker moiety in terms of the length between the chemical and DNA moieties can vary widely, but for the purposes of the invention, need not exceed a length sufficient to provide the linkage functions indicated. Thus, a chain length of from at least one to about 20 atoms is preferred.
  • Typical linkers may be amino modifiers (such as 3'-Amino-Modifier C3, 3'-Amino-Modifier C6 dC, 3'-Amino-Modifier C6 dT, 3'-Amino-Modifier C7, 3'-PT-Amino-Modifier C3, 3'-PT-Amino- Modifier C6, 5'-Amino-dT-CE, 5'-Amino-Modifier 5, 5'-Amino-Modifier C12, 5'-Amino-Modifier C3, 5'-Amino-Modifier C6, Amino-Modifier C2 dT, Amino-Modifier C6 dA, Amino-Modifier C6 dC, Amino-Modifier C6 dG, Amino-Modifier C6 dT, Amino-Modifier C6-U, 5'-Amino Modifier C12), thiol modifiers (e.g.
  • 3'-Thiol-Modifier C3 S-S 1 5'-Thiol-Modifier C6, 3'-C6-Thiuol-Modifier S-S, 3'- C6-Thiol-Modifier, 5'-C6-Thiol-Modifier S-S, 5'-C6-Thiol-Modifier), carboxy modifiers (e.g. 3 1 - Carboxylate Photolabile C6, 5'-Carboxy-Modifier C10, Carboxy-dT), or aldehyde modifiers (e.g. 5'-Aldehyde-Modifier C2). These are readily commercially available
  • the linker moiety is a 5'-amino modifier C12.
  • the DNA moeity is double-stranded and comprises in the first aspect of the invention a first primer region, an initial coding region, a first annealing region, a final coding region and a second primer region, as well as optionally a middle coding region and second annealing region between the first annealing region and the final coding region.
  • the DNA moiety comprises a first primer region, an initial coding region, an annealing region, a middle coding region, a restriction/ligation region, a final coding region and a second primer region.
  • the first and second primer regions provide a means to produce a PCR-amplified duplex DNA fragment derived from the library member using PCR.
  • the typical length of primer regions is between 16 and 28 nucleotides (in single stranded format), or base pairs (in double stranded format).
  • double stranded format one of the two DNA strands of the primer region can form a sequence specific dimer with a PCR primer at an appropriate hybridization temperature.
  • Typical hibridization temperatures for the sequence specific hybridization of PCR primers to PCR primer regions is between 40 and 70 0 C.
  • PCR primers can be longer than the hybridization region complementary to the primer region (at their respective 5' ends), e.g.
  • the sequences that are not underlined, are complementary to one of the 2 strands of the PCR primer regions on the double stranded library members.
  • the coding region may be longer than necessary. The benfit of employing coding regions that are longer than necessary is that they provide the opportunity to differentiate codes by more than just a single nucleotide difference, which gives more confidence in the decoding process.
  • the initial building block (20 compounds) was encoded by 6 nucleotides
  • the second building block (200 compounds) was encoded by 8 nucleotides.
  • the annealing regions need to be of a sufficient length to allow for good recognition and binding between the oligonulceotides.
  • an annealing region will contain at least 10 nucleotides, and more preferably at least 18 nucleotides. Normally, the annealing region will contain no more than 28 nucleotides. If more than one annealing region is present, they should have different sequences. In the examples below, the annealing region contains 18 nucleotides.
  • typical hybridization temperatures for the sequence specific annealing of two annealing regions are between 40 0 C and 70 0 C.
  • the restriction region used in the second aspect of the invention provides a site for restriction with a non-palindromic restriction enzyme that leaves a non-blunt (cohesive) end. This non- blunt end is matched by the ligation region used to couple the final coding region and second primer.
  • the restriction region will contain preferably 6 nucleotides and normally no more then 8 nucleotides.
  • the non-palindromic restriction site is required to prevent any non-specific ligation on the final coding step.
  • the non-palindromic restriction enzyme may be, for example, BssSI (5'-CAC GAG-3'). BmgBI (5'-CAC GTC-3'). BbvCI (5'-CCT TCA GC-3'), BseYI (5'-CCC AGC-3"> or BsrBI (5'-CCG_CTC-3').
  • the enzyme restriction sequences are underlined.
  • restriction region contains 6 nucleotides and is specific for BssSI enzyme (5'-CAC GAG-3').
  • the restriction region contains 6 nucleotides and is specific for BssSI enzyme (5'-CAC GAG-3').
  • the chemical modifier used in the first and second aspects of the invention may be a biotin moiety, or an imminobiotin moiety or any suitable chemical moiety that allows a selective capture by a macromolecular entity immobilized on a solid support, preferably high density coated streptavidin sepharose resin.
  • the chemical modifier is either a 3'-Biotin-C6-modification or a 5'- Biotin-C6-modification.
  • the modification has the structure:
  • the restriction region used in the third aspect of the invention provides a site for restriction with a restriction enzyme that leaves a non-blunt (cohesive) end. This non-blunt end is matched by the ligation region used to couple the final coding region and second primer.
  • the restriction region will contain preferably 6 nucleotides and normally no more then 8 nucleotides.
  • the restriction site is not required to be non-palindromic, as in the third aspect of the invention an excess of the final coding partially double stranded oligomers are used in the ligation.
  • the restriction enzyme may be, for example, BamHI (5'-GGA TCC-3'). or EcoRI (5'-GAA TTC-3'). The enzyme restriction sequences are underlined.
  • restriction region contains 6 nucleotides and is specific for BamHI enzyme (5'-GGA TCC-3').
  • the restriction region contains 6 nucleotides and is specific for BamHI enzyme (5'-GGA TCC-3').
  • the initial coding strand oligomer may be derivatised by a linking moiety using the appropriate conditions, as are well known in the art.
  • the initial coding strand oligomer may be puchased with the linking moiety already attached.
  • the initial building block is reacted with the reactive terminus of the linking moiety under conditions which do not affect the conjugated oligomer.
  • Different initial building blocks are conjugated to different oligonucleotides that bear coding regions specific for the initial building block they are coupled to.
  • the building blocks may be coupled to each other using, for example, the chemistries set out above. As discussed, the reaction conditions used must be suitable to be used in the presence of single or double stranded DNA.
  • the annealing of the various oligonucleotides described above occurs by virtue of hybridization mediated by the complementary sequences of the annealing and complementary annealing regions. Incubating the mixture at an appropriate hybridization temperature (typically between 40 and 70 0 C) facilitates sequence specific annealing.
  • DNA polymerase By addition of desoxynucleotides (dNTPs) and a polymerase (e.g. Klenow polymerase) in a suitable buffer at the appropriate point in the method, and incubation at a suitable temperature (e.g. 37°C for Kenow polymerase), the DNA strands are elongated, forming a double stranded DNA segment.
  • dNTPs desoxynucleotides
  • Klenow polymerase e.g. Klenow polymerase
  • the double stranded DNA with suitable chemical modification is incubated with denaturing buffer (e.g. urea 4M aqueous) and eventually heated at 94°C.
  • denaturing buffer e.g. urea 4M aqueous
  • suitable macromolecular capturing agent immobilized on a solid support (e.g. high density coated streptavidin sepharose resin)
  • the sample is incubated at an appropriate temperature, the supernatant separated by filtration and the desired single strand DNA isolated in the suitable buffer.
  • Conjugates that are part of the second aspect of the invention containing a restriction site are treated with a restriction enzyme (e.g. BssSI) in a suitable buffer, and then incubated at the appropriate temperature for the enzyme (e.g. 37°C for BssSI). Two restriction fragments are formed. On the one hand, a DNA fragment with the putative binding moiety conjugated to a DNA restriction fragments containing the coding tags, and on the other hand, a DNA restriction fragment with a chemical modifier, suitable for affinity capture. Subsequent addition of a suitable macromolecular ligand immobilized on a solid support (e.g. high density coated streptavidin sepharose resin), allows the separation by filtration of both restriction fragments.
  • a restriction enzyme e.g. BssSI
  • a suitable buffer e.g. high density coated streptavidin sepharose resin
  • Conjugates that are part of the third aspect of the invention containing a restriction site are treated with a restriction enzyme (e.g. BamHI) in a suitable buffer, and then incubated at the appropriate temperature for the enzyme (e.g. 37°C for BamHI). Two restriction fragments are formed. On the one hand, a DNA fragment with the putative binding moiety conjugated to a DNA restriction fragments containing the coding tags, and on the other hand, a DNA restriction fragment. This DNA restriction fragment can be removed by chromatographic means.
  • a restriction enzyme e.g. BamHI
  • Ligation may be carried out by mixing of two sets of pre-hybridized suitable pairing oligonucleotides with cohesive ends, and a ligase enzyme (e.g. T4 ligase) in a suitable buffer, and incubation at appropriate temperature (e.g. 25°C for T4 ligase).
  • a ligase enzyme e.g. T4 ligase
  • an excess of oligonucleotides is used in the ligation step.
  • the libraries of this invention provide a repertoire of chemical diversity such that each chemical moiety is linked to a DNA moiety that facilitates identification of the chemical moiety.
  • the invention therefore also contemplates a method for identifying a chemical moiety that participates in a preselected binding interaction between the chemical moiety and a biological macromolecule.
  • the chemical moiety to be identified is represented by one of the members of a library of this invention, and the method comprises the following steps:
  • a library according to the present invention is admixed with a preselected biological macromolecule under binding conditions (i.e., a binding reaction admixture) for a time period sufficient for the biological macromolecule to interact with at least one member of the library and form a binding reaction complex;
  • binding conditions i.e., a binding reaction admixture
  • binding reaction complex is then isolated from the library admixture to form an isolated complex
  • a typical biological macromolecule exhibiting a preselected binding interaction can be any of a variety of molecules (e.g. proteins) that bind selectively to another molecule, including antibodies to antigens, lectins to oligosaccharides, receptors to ligands, enzymes to substrates and the like mediators of molecular interactions. Therefore, a preselected binding interaction is defined by the selection of the biological macromolecule with which a library member is to bind.
  • the admixture of a library of the invention with a biological macromolecule can be in the form of a heterogeneous or homogeneous admixture.
  • the members of the library can be in the solid phase with the biological macromolecule present in the liquid phase.
  • the biologically active molecule can be in the solid phase with the members of the library present in the liquid phase.
  • both the library members and the biological macromolecule can be in the liquid phase.
  • Binding conditions are those conditions compatible with the known natural binding function of the biological macromolecule. Those compatible conditions are buffer, pH and temperature conditions that maintain the biological activity of the biological macromolecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction. Typically, those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the biological macromolecule of interest.
  • the preferred binding conditions would be conditions suitable for the antibody to immunoreact with its immunogen, or a known immunoreacting antigen.
  • the binding conditions would be those compatible with measuring receptor- ligand interactions.
  • a time period sufficient for the admixture to form a binding reaction complex is typically that length of time required for the biological macromolecule to interact with its normal binding partner under conditions compatible with interaction. Although the time periods can vary depending on the molecule, admixing times are typically for at least a few minutes, and usually not longer than several hours, although nothing is to preclude using longer admixing times for a binding reaction complex to form.
  • a binding reaction complex is a stable product of the interaction between a biological macromolecule and a bifunctional molecule of this invention.
  • the product is referred to as a stable product in that the interaction is maintained over sufficient time that the complex can be isolated from the rest of the members of the library without the complex becoming significantly disassociated.
  • a binding reaction complex is isolated from the binding reaction admixture by any separation means that is selective for the complex, thereby isolating that library member, or members, which hase/have bound to the biological macromolecule.
  • separation means There are a variety of separation means, depending on the status of the biological macromolecule.
  • the biological macromolecule can be provided in admixture in the form of a solid phase reagent, i.e., affixed to a solid support, and thus can readily be separated from the liquid phase, thereby removing the majority of library members. Separation of the solid phase from the binding reaction admixture can optionally be accompanied by washes of the solid support to rinse library members having lower binding affinities off of the solid support.
  • a secondary binding means specific for the biological macromolecule can be utilized to bind the molecule and provide for its separation from the binding reaction admixture.
  • an immobilized antibody immunospecific for the biological macromolecule can be provided as a solid phase-affixed antibody to the binding reaction admixture after the binding reaction complex is formed.
  • the immobilized antibody immunoreacts with the biological macromolecule present in the binding reaction admixture to form an antibody-biological macromolecule immunoreaction complex. Thereafter, by separation of the solid phase from the binding reaction admixture, the immunoreaction complex, and therefor any binding reaction complex, is separated from the admixture to form isolated library member.
  • a binding means can be operatively linked to the biological macromolecule to facilitate its retrieval from the binding reaction admixture.
  • Exemplary binding means are one of the following high affinity pairs: biotin-avidin, protein A-Fc receptor, ferritin-magnetic beads, and the like.
  • the biological macromolecule is operatively linked (conjugated) to biotin, protein A, ferritin and the like binding means, and the binding reaction complex is isolated by the use of the corresponding binding partner in the solid phase, e.g., solid-phase avidin, solid-phase Fc receptor, solid phase magnetic beads and the like.
  • solid supports on which to operatively link proteinaceous molecules is generally well known in the art.
  • Useful solid support matrices are well known in the art and include cross- linked dextran such as that available under the tradename SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N. J.); agarose, borosilicate, polystyrene or latex beads about 1 micron to about 5 millimeters in diameter, polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose or nylon-based webs such as sheets, strips, paddles, plates microtiter plate wells and the like insoluble matrices.
  • PCR polymerase chain reaction
  • a preferred method for decoding is the use of high throughput sequencing methods, such as the 454-Roche Genome Sequencer system.
  • PCR products For sequencing with the 454-Roche Genome Sequencer system, PCR products have to contain suitable adaptor sequences at their extremities (calles adaptor sequence A and B), which can be either added after a PCR reaction by ligation, or they can be incorporated in the PCR reactions, if the PCR primers contain on their 5'-ends sequences corresponding to an adaptor region.
  • PCR primers DEL_P1_A (5'-GCC TCC CTC GCG CCA TCA GGG AGC TTG TGA ATT CTG G-3 1 ) and DEL_P2_B (5'-GCC TTG CCA GCC CGC TCA GGT AGT CGG ATC CGA CCA C-3 1 ) were used.
  • the sequences that are not underlined, are complementary to one of the 2 strands of the PCR primer regions in the compounds.
  • the underlined sequence of DEL_P1_A corresponds to the sequence of adaptor region A
  • the underlined sequence of DEL-P2_B corresponds to adaptor sequence B.
  • the next step of a particular sequencing process is the annealing of PCR amplicons on DNA Capture Beads, emulsification of beads and PCR reagents in water-in-oil microreactors, and clonal emPCR amplification inside these microreactors.
  • the Capture beads After breaking of the emulsion, the Capture beads are mixed with Enzyme Beads, and loaded on a PicoTiterPlate. Pyrosequencing allows the recording of individual sequences for each DNA species displayed at Capture Beads, trapped in the wells of PicoTiterPlates. This allows the parallel sequencing of a vast amount (typically more than 100,000 per PicoTiterPlate) of individual DNA species at a time. With further improvement of the sequencing technology, it will be possible to sequence more than 1 ,000,000 individual DNA species at a time.
  • a first further aspect of the present invention provides a compound selected from 02-40, 16-40 02-69 and 18-69. These compounds may used as affintity ligands in an affinity chromatography resin for IgG.
  • a second further aspect of the present invention provides an affinity chromatography resin for IgG comprising as the affinity ligand a moiety comprising a pair of constituent moieties being:
  • the constituent moieties are linked together and to the sold support by a chemical linking group.
  • the chemical linking group may be of the formula:
  • the affinity ligand is selected from 02-40, 16-40, 02-69 and 18-69, and more preferably 02-40 and 16-40.
  • a third further aspect of the invention relates to the use of the affinity chromatography resin of the second further aspect for the purification of IgG.
  • the affinity chromatograpy resin used to immobilize the IgG binding moieties may comprise an agarose carbohydrate polymer, for example, Sepharose Fast Flow.
  • a highly cross-linked matrix makes the support more rigid which in turn improves the pressure/flow characteristic.
  • the particle size needs to be no higher than 180 ⁇ m and preferebly spherical in the range 45-165 ⁇ m.
  • the swelling factor is preferably higher then 3 drained medium/g.
  • the resin needs a wide range of pH and chemical stability to prevent any side reactivity during the washing and elution in various conditions.
  • the resin support is Sepharose 4 Fast Flow resin (GE Healthcare) with a particle size 45-165 ⁇ m, swelling factor 4-5 drained medium/g and a pH stability range between 3-11.
  • a DNA-encoded chemical library was constructed consisting of 20 x 200 modules, joined together by the formation of an amide bond. Initially, 20 Fmoc-protected aminoacids were chemically coupled to 20 individual amino-tagged oligonucleotides. Following deprotection and HPLC purification, the 20 resulting DNA-encoded primary amines were coupled to 200 carboxylic acids, in order to generate a library of 4000 members. In order to ensure that each library member contained a different DNA code, a split-and-pool strategy was chosen, which minimizes the number of oligonucleotides needed for library construction.
  • the 20 Fmoc- protected aminoacids covalently linked to individual single-stranded oligonucleotides were mixed and aliquoted in 200 reaction vessels, prior to the coupling step with the 200 different carboxylic acids (one per well).
  • the identity of the carboxylic acids used for the coupling reactions was encoded by performing an annealing step with individual oligonucleotides, partially complementary to the first oligo carrying the chemical modification.
  • a successive Klenow polymerization step yielded double stranded DNA fragments, each of which contained two identification codes (one corresponding to the initial 20 compounds and one corresponding to the 200 carboxylic acids).
  • the 200 compound mixtures were then purified on an anion exchange cartridge and pooled.
  • Table 1 lists the amino acids and the oligonucleotide code used to identify them:
  • DMSO-dissolved carboxylic acid 3OmM
  • N- hydroxysulfosuccinimide Fluka
  • N-ethyl-N'-(3-dimethylaminopropyl)- carbodiimide Fluka
  • triethylaminehydrochloride pH9.0 water solution, 8OmM
  • DNA-oligonucleotide sub-library pool 1.5 ⁇ M.
  • Test coupling reactions were also performed with the reaction conditions described above; using model 42mer 5'-fmoc-deprotected aminoacids-oligonucleotide conjugate and model carboxylic acids.
  • the reactions were analysed by HPLC and the masses of the reacted oligonucleotides detected by LC-ESI-MS. Typical HPLC coupling yields on this step were >58% with purity >46%.
  • Table 2 lists the carboxylic acids and the oligonucleotide code used to identify them:
  • D-desthiobiotin oligonucleotide-conjugate (positive control)
  • the D-desthiobiotin-oligonucleotide-conjugate was synthesized and unambiguously encoded as described above.
  • Prior Polymerase Klenow reaction HPLC purification was performed and LC- ESI-MS showed the expected D-desthiobiotin oligonucleotide conjugated mass.
  • Example 1 The library of Example 1 , 30OnM, was diluted 1 :30 in PBS, spiked with D-desthiobiotin oligonucleotide-conjugate (final concentration 1OpM) and 100 ⁇ L was added to 50 ⁇ l of streptavidin-sepharose slurry (GE Healthcare, cat.no 17-5113-01) or of sepharose slurry without streptavidin preincubated with PBS, 0.3mg/mL herring sperm DNA (Sigma). After incubation for 1 hour at 25°C the mixture was transferred to a SpinX column (Corning Costar Incorporated), the supernatant removed, and the resin washed 4x400 ⁇ L of PBS. At last the resin was resuspended in 100 ⁇ l_ of water.
  • D-desthiobiotin oligonucleotide-conjugate final concentration 1OpM
  • streptavidin-sepharose slurry GE Healthcare, cat.no 17-5113
  • the codes of the oligonucleotide-compound conjugates were amplified before and after selection by PCR (50 ⁇ L, 25 cycles of 1min at 94 0 C, 1 min at 55 0 C, 40s at 72 0 C) either of 5 ⁇ l_ of the resuspended resin or of 5 ⁇ L of 10OfM DEL4000 using primers DEL_P1_A (5 1 - GCCTCCCTCGCGCCATCAGGGAGCTTGTGAATTCTGG-S') and DEL_P2_B (5 1 - GCCTTGCCAGCCCGCTCAGGTAGTCGGATCCGACCAC-S') that additionally contain at one extremity a specific domain (19 nucleotide) required for high-throughput sequencing.
  • the PCR product was purified on ion-exchange cartridge (Qiagen, 28104). Subsequent high-throughput sequencing was performed on a 454 Life Sciences-Roche GS FLX Sequencer platform.
  • Example 3 Resynthesis of binding molecules to assess dissociation constants In order to assess whether the extension of the pharmacophore 49 and 78 moieties within the 4,000-membered chemical library (see Figure 4) would contribute to an increased affinity towards streptavidin, the dissociation constants of the most relevant compounds were measured, following conjugation to fluorescein.
  • Fluorescein-compound conjugates (50OnM) were incubated with increasing amounts of streptavidin (BIOSPA, cat.no S002-6) in PBS, 5% DMSO, for 1 hour at 25°C.
  • the library of example 1 (total oligonucleotide conjugate concentration 30OnM) was diluted 1:30 in PBS. 100 ⁇ l_ of the library was either added to 50 ⁇ l IgG-sepharose slurry or to sepharose slurry without streptavidin. Both resins were preincubated with PBS, 0.3mg/ml_ herring sperm DNA (Sigma). After incubation for 1 hour at 25°C the mixture was transferred to a SpinX column (Corning Costar Incorporated), the supernatant was removed, and the resin washed 4x with 400 ⁇ l_ PBS. After washing, the resin was resuspended in 100 ⁇ l_ water. Identification of binding molecules
  • the codes of the oligonucleotide-compound conjugates were amplified by PCR (50 ⁇ L, 25 cycles of 1 minute at 94°C, 1 minute at 55°C, 40 seconds at 72°C) with either 5 ⁇ l_ of 10OfM DEL4000 library before selection as template, or 5 ⁇ L of each resuspended resin after selection as template.
  • the PCR primers DEL_P1_A (5'-GCC TCC CTC GCG CCA TCA GGG AGC TTG TGA ATT CTG G-3') and DEL_P2_B (5'-GCC TTG CCA GCC CGC TCA GGT AGT CGG ATC CGA CCA C-3') additionally contain at one extremity a 19 nucleotide domain (underlined) required for high-throughput sequencing with the 454 Genome Sequencer system.
  • the PCR products were purified on ion-exchange cartridges (Qiagen, 28104). Subsequent high- throughput sequencing was performed on a 454 Life Sciences-Roche GS Fix Sequencer platform.
  • the decoding of the polyclonal IgG selection showed a preferential enrichment of certain classes of structurally-related compounds. Typically an enrichment of the derivatives of compound 40 as well as of the pharmacophore 69 was observed. Additionally the derivatives of the bromide 02 and of thiophene 16 revealed an exceptional enrichement in combination both with the moiety 40 and the compound 69.
  • O-bis-(aminoethyl)ethylene glycol trityl resin (Novabiochem, cat.no 01-64-0235) was suspended in a mixture of the appropriate fmoc- protected amino acid (100 ⁇ mol , 1mL), HBTU (Aldrich, 200 ⁇ mol , 1mL), and DIEA (Fluka, 400 ⁇ mol , 0.5mL) in dry DMF. After overnight incubation at 25°C, the resin was washed 6x with 2mL dry DMF and the fmoc moiety was removed by addition of 1 mL piperidine (50% in dry DMF) for 1 hour at 25°C.
  • Fluorescein-compound conjugates 500 nM were incubated with increasing amounts of polyclonal human IgG (Sigma-Aldrich-Fluka, Buchs, Switzerland) in PBS, 5% DMSO, for 1 hour at 25 0 C.
  • the apparent association constant revealed was 200 ⁇ M.
  • the compounds selected were immobilized on a suitable affinity chromatograpy resin support and applied both in the affinity chromatograpy of a pure sample of polyclonal human IgG Cy5 labelled and in the purification of a crude sample of a CHO (Chinese Hamster Ovary) cells supernatant spiked with plyclonal human IgG Cy5 labelled.
  • Polyclonal human IgG Cy5 labeling Polyclonal human IgG (Sigma-Aldrich-Fluka, Buchs, Switzerland) was labelled with Cy5 Mono- reactive kit (Amersham, cat.no PA25001) according to the protocol of the provider and purified over a PD10 column (GE Healthcare, cat.no 17-0851-01) as described by the supplier.
  • L2C2C40 or L2C16C40 resin were loaded on a chromatography cartridge (Glen Research, cat.no 20-0030-00) and washed 3x with 1mL PBS before loading a crude sample of CHO (Chinese Hamster Ovary) cells supernatant (60 ⁇ l_) spiked with human IgG Cy5 labelled (20 ⁇ L, 9.68 ⁇ M).
  • the flow-through, the washing steps (washing Ix with 10 mL PBS; 1x with 10 mL 500 mM NaCI, 0.5 mM EDTA; Ix with 10 mL 100 mM NaCI 1 0.1% Tween 20, 0.5 mM EDTA) and the elutate (elution 3x with 200 ⁇ L aqueous triethylamine 100 mM) were collected and eventually concentrated back to a final volume of 100 ⁇ L by centrifugation in a Vivaspin 500 tube (Vivascience, cat.no VS0101 , cut-off 10.000 MW).
  • Figure 7 shows three different experimental schemes, which could be conceived for the construction and encoding of a large library.
  • the first scheme features the stepwise addition of groups of chemical moieties onto an initial scaffold, using suitable orthogonal chemical reactions and/or protecting strategies, followed by the sequential addition of the corresponding DNA codes by an iterative ligation procedure.
  • This scheme which corresponds broadly to known methods, is conceptually simple and can be implemented experimentally, but requires 2 pre-annealed oligonucleotides for each encoding event (i.e., 200 + 200 + 200 oligonucleotides for a library containing 100 x 100 x 100 chemical groups; Figure 7a).
  • the second synthetic and encoding strategy represents an embodiment of the first aspect of the invention.
  • the mixture was centrifuged in a table-top centrifuge for 40min (16.00Og) at 4°C, the supernatant removed and the pellet washed with 300 ⁇ l_ ice-cold ethanol 90%. After a further 20min centrifugation (16.000g) at 4°C, the pellet was dried and redissolved in water.
  • Hybridization of 3 pairs (A, B, C) of oligonucleotides (A: 5'-CAT GGA ATT CGC TCA CTC CGA CTA GAG G-3 1 and 5'-(Phosphate)-CGT ACC TCT AGT CGG AGT GAG CGA ATT CCA TG-3 1 ; B: 5'-(Phosphate)-TAC GTG AGC TTG ACC TGG TGA G-3 1 and 5'- (Phosphate)-GCT TCT CAC CAG GTC AAG CTC A-3'; C: 5'-(Phosphate)-AAG CAC GTT CGC TGG ATC CTC AAC TGT G-3' and 5'-CAC AGT TGA GGA TCC AGC GAA CGT-3'.
  • IBA IBA
  • 1x ligase buffer 40 mM Tris-HCI, 10 mM MgCI2, 10 mM DTT, 0.5 mM ATP, pH 7.8
  • the ligations were performed mixing 10 ⁇ l of hybridized oligonucleotide pairs A and B with 10 ⁇ l of 1x ligase buffer and 1 ⁇ l_ of T4 ligase (Roche Applied Science, Basel, Switzerland), and incubated at room temperature for 2 hours.
  • the ligation product was purified using a Qiagen Nucleotide Removal Kit (Qiagen, cat.no 28306), and eluted with 50 ⁇ l of 10 mM Tris-HCI pH 8.0. 18 ⁇ l of the eluate was mixed with 10 ⁇ l of hybridized oligonucleotide pair C (which was present in excess), 2 ⁇ l of 10x ligase buffer, and 1 ⁇ l of T4 ligase, and incubated for 2 hours at room temperature. Aliquots of the two starting oligonucleotides, and the different ligation products were subjected to electrophoresis on a 20% TBE gel.
  • Stepwise encoding by Klenow Polymerase (Method b Underlined sequences represent coding sequences)
  • reagents were added to the respective final concentrations: a 42mer 5'-amino-C12-DNA-oligonucleotide (5'-GGA GCT TGT GAA TTC TGG ATC TTA GGA CGT GTG TGA ATT GTC-3', IBA), 2 ⁇ M, 42mer 3'-C6-biotinylated-oligonucleotide (5'-GTA GTC GGA TCC GAC CAC GTT CCT GAC AAT TCA CAC ACG TCC-3', IBA), 3 ⁇ M, Klenow buffer (NEB, cat.no B7002S), dNTPs (Roche, cat.no 11969064001), 0.5mM, Klenow Polymerase enzyme (NEB, cat.no M0210L), 5 units.
  • a 42mer 5'-amino-C12-DNA-oligonucleotide 5'-GGA GCT TGT GAA TTC TGG ATC TTA GGA CGT GTG TGA
  • the Klenow polymerization reaction was incubated at 37 0 C for 1h, purified on ion-exchange cartridge (Qiagen, cat.no 28306) and eluted in 100 ⁇ L of 4M urea. After incubating at 94°C for 2min, 50 ⁇ l of streptavidin-sepharose slurry (GE Healthcare, cat.no 17-5113-01) were added and the slurry was incubated for 1h at 4 0 C. The streptavidin sepharose resin and the supernatant were separated by centrifugation in a SpinX column (Corning Costar Incorporated). The DNA in the supernatant was ethanol precipitated as described above.
  • the resulting singele-stranded oligonucleotide was mixed with a 42mer unmodified DNA oligonucleotide (5'-GTC GTA TCG CCA TGG TCC AAC ATC GTA GTC GGA GAG GAC CAC-3') and a Klenow polymerization reaction was performed as described above. Aliquots of the three starting oligonucleotides, and the different Klenow products were applied on a 15% TBE-Urea gel.
  • reagents were added to the respective final concentrations: a 42mer 5'-amino-C12-DNA-oligonucleotide (5'-GGA GCT TGT GAA TTC TGG ATC TTA GGA CGT GTG TGA ATT GTC-3', IBA), 2 ⁇ M, a 42mer 5'-C6-biotinylated-oligonucleotide containing the non-palindromic BssSI restriction site (in boldface type) (5'-GTA GTC GGA CAC GAG TAC TGG TAA TCG ACA ATT CAC ACA CGT CC-3', IBA), 3 ⁇ M, klenow buffer (NEB 1 cat.no B7002S), dNTPs (Roche, cat.no 11969064001), 0.5mM, and Klenow Polymerase enzyme (NEB, cat.no M0210L), 5 units.
  • reaction mixture was purified on ion-exchange cartridge (Qiagen, cat.no 28306) and eluted in 25 ⁇ L of water. 8 units of BssSI enzyme were added to the purified Klenow product in 50 ⁇ l_ of BssSI restriction buffer (NEB, cat.no B7003S) .
  • the restriction cutting reaction was carried out at 37 0 C for 1.5h. 50 ⁇ L of streptavidin-sepharose slurry (GE Healthcare, cat.no 17-5113-01) was added and the slurry was incubated for 30min at 4 0 C.
  • Figure 8a shows a further experimental scheme, which could be conceived for the construction and encoding of a large library according to the third aspect of the invention.
  • Stepwise encoding by "Klenow-Ligation" (Underiined sequences represent coding sequences)
  • a 42mer 5'-amino-C12-DNA-oligonucleotide (5'-GGA GCT TGT GAA TTC TGG ATC TTA GGA CGT GTG TGA ATT GTC-3 1 , IBA)
  • 32OnM a 44mer oligonucleotide containing the restriction site BamHI (boldface type) (5'-GTA GTC GGA TCC GAC CAC GCA TAT AAG ACA ATT CAC ACA CGT CC-3'.IBA)
  • 60OnM klenow buffer
  • NBD cat.no B7002S
  • dNTPs (Roche, cat.no 11969064001), 0.5mM
  • Klenow Polymerase enzyme (NEB, cat.no M0210L), 5 units.
  • reaction mixture was purified on ion exchange cartridge (Qiagen, cat.no 28306) and eluted in 33 ⁇ l_ of water.
  • 10 units of BamHI enzyme were added to the purified Klenow product (10OnM) in 50 ⁇ L of BamHI restriction buffer (NEB, cat.no B7003S) containing 0.1mg/ml_ BSA.
  • the restriction cutting reaction was carried out at 37°C for 12 hours and purified on ion-exchange cartridge (Qiagen, cat.no 28306) and eluted in 50 ⁇ l_ of water.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de préparation d'une bibliothèque de produits chimiques codés par ADN comprenant des éléments qui comprennent une fraction chimique, une fraction de liaison et une fraction d'ADN. Ce procédé comprend pour chaque élément les étapes consistant à : (I) coupler un synthon initial à un oligomère simple brin codant initial par l'intermédiaire d'une fraction de liaison pour former un conjugué initial, l'oligomère simple brin codant initial comprenant une première région amorce, une région codante initiale et une première région d'hybridation; (II) éventuellement coupler un synthon intermédiaire et un oligomère simple brin codant intermédiaire audit conjugué initial pour former un conjugué intermédiaire, le couplage ayant lieu dans n'importe quel ordre et : (a) le synthon intermédiaire étant couplé au résidu du synthon initial; et (b) l'oligomère simple brin codant intermédiaire comprenant une région codante intermédiaire et une seconde région d'hybridation et étant couplé par : (i) hybridation d'un oligomère simple brin complémentaire qui comprend un modificateur chimique, une première région d'hybridation complémentaire, une région codante intermédiaire complémentaire et une seconde région d'hybridation complémentaire par interaction entre la première région d'hybridation et la première région d'hybridation complémentaire; (ii) traitement du conjugué formé avec une ADN polymérase pour allonger l'oligomère simple brin codant initial pour qu'il soit complémentaire à la région codante intermédiaire complémentaire et la seconde région d'hybridation complémentaire; (iii) retrait de l'oligomère simple brin complémentaire par dénaturation de l'ADN et capture de l'oligomère simple brin complémentaire par l'intermédiaire du modificateur chimique; (III) coupler un synthon final et un oligomère simple brin codant final au conjugué initial ou intermédiaire selon ce qui est approprié pour former un conjugué final, le couplage ayant lieu dans n'importe q
PCT/EP2008/010770 2007-12-19 2008-12-17 Bibliothèques de produits chimiques codés par adn WO2009077173A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US824907P 2007-12-19 2007-12-19
US61/008,249 2007-12-19

Publications (2)

Publication Number Publication Date
WO2009077173A2 true WO2009077173A2 (fr) 2009-06-25
WO2009077173A3 WO2009077173A3 (fr) 2009-10-29

Family

ID=40790879

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/010770 WO2009077173A2 (fr) 2007-12-19 2008-12-17 Bibliothèques de produits chimiques codés par adn

Country Status (1)

Country Link
WO (1) WO2009077173A2 (fr)

Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100227039A1 (en) * 2009-03-03 2010-09-09 Givaudan S.A. Off-Taste Masking
WO2011127933A1 (fr) 2010-04-16 2011-10-20 Nuevolution A/S Complexes bifonctionnels et procédés de fabrication et d'utilisation de tels complexes
WO2012019765A1 (fr) * 2010-08-10 2012-02-16 European Molecular Biology Laboratory (Embl) Procédés et systèmes pour le traçage d'échantillons et de combinaisons d'échantillons
EP2748357A4 (fr) * 2011-09-07 2015-04-15 Chem Inc X Procédés de marquage de banques codées par de l'adn
WO2015091207A1 (fr) * 2013-12-20 2015-06-25 Philochem Ag Production de bibliothèques chimiques codées
WO2015135856A1 (fr) * 2014-03-14 2015-09-17 Philochem Ag Purification de produits conjugués d'adn
WO2015185067A1 (fr) * 2014-06-06 2015-12-10 Herlev Hospital Détermination de reconnaissance d'antigène par l'intermédiaire d'un marquage par code-barres de multimères du cmh
CN105218405A (zh) * 2015-11-04 2016-01-06 上海泰坦科技股份有限公司 (r,s)-2-[[5-(9-芴甲氧羰基氨基)二苯并[a,d]环庚烷-2-基]氧]乙酸的合成工艺
EP2872680A4 (fr) * 2012-07-13 2016-01-27 Chem Inc X Banques encodées par l'adn possédant des liaisons oligonucléotidiques codantes ne pouvant être lues par les polymérases
WO2016074683A1 (fr) 2014-11-11 2016-05-19 Lundorf Pedersen Materials Aps Procédé d'identification de molécules présentant des caractéristiques souhaitées
US9359601B2 (en) 2009-02-13 2016-06-07 X-Chem, Inc. Methods of creating and screening DNA-encoded libraries
WO2017027973A1 (fr) * 2015-08-18 2017-02-23 Novation Pharmaceuticals Inc. Dérivés de benzopyrane et utilisations associées
CN108178790A (zh) * 2018-01-18 2018-06-19 上海药明康德新药开发有限公司 一种将细胞膜分离以便对跨膜蛋白进行dna编码化合物库筛选的方法
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US10077440B2 (en) 2002-10-30 2018-09-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
CN110093408A (zh) * 2018-01-31 2019-08-06 成都先导药物开发股份有限公司 一种dna编码化合物库及化合物筛选方法
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
WO2019236644A1 (fr) * 2018-06-05 2019-12-12 Arrakis Therapeutics, Inc. Bibliothèques codées et procédés d'utilisation pour le criblage de cibles d'acide nucléique
WO2020047095A1 (fr) * 2018-08-28 2020-03-05 The Scripps Research Institute Utilisation d'une immobilisation non covalente dans des banques codées par l'adn
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
EP3538669A4 (fr) * 2016-11-10 2020-05-20 The Scripps Research Institute Codage de bibliothèque de composés en phase solide avec codage à barres à base de polynucléotides
US10669538B2 (en) 2001-06-20 2020-06-02 Nuevolution A/S Templated molecules and methods for using such molecules
WO2020128064A1 (fr) 2018-12-21 2020-06-25 Philochem Ag Bibliothèques chimiques codées par l'acide nucléique
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US10731151B2 (en) 2002-03-15 2020-08-04 Nuevolution A/S Method for synthesising templated molecules
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
CN112575389A (zh) * 2019-09-30 2021-03-30 成都先导药物开发股份有限公司 一种dna编码化合物库的液相色谱纯化方法
US11118215B2 (en) 2003-09-18 2021-09-14 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US11186836B2 (en) 2016-06-16 2021-11-30 Haystack Sciences Corporation Oligonucleotide directed and recorded combinatorial synthesis of encoded probe molecules
EP3928796A1 (fr) 2014-02-03 2021-12-29 Eidgenöessische Technische Hochschule Zurich Conjugués de médicaments à petites molécules
WO2022084486A1 (fr) 2020-10-23 2022-04-28 Eth Zurich Bibliothèques codées par l'acide nucléique auto-purifié
US11402373B2 (en) 2014-06-13 2022-08-02 Immudex Aps General detection and isolation of specific cells by binding of labeled molecules
US11702652B2 (en) 2005-12-01 2023-07-18 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US11795580B2 (en) 2017-05-02 2023-10-24 Haystack Sciences Corporation Molecules for verifying oligonucleotide directed combinatorial synthesis and methods of making and using the same
US11807623B2 (en) 2017-11-30 2023-11-07 Arrakis Therapeutics, Inc. Nucleic acid-binding photoprobes and uses thereof
US11987904B2 (en) 2016-03-04 2024-05-21 Aptamer Group Limited Oligonucleotides and methods for preparing
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE450609T1 (de) 2002-12-19 2009-12-15 Nuevolution As Durch quasizufallsstrukturen und funktionen geführte synthesemethode
US20070026397A1 (en) 2003-02-21 2007-02-01 Nuevolution A/S Method for producing second-generation library

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004083427A2 (fr) * 2003-03-20 2004-09-30 Nuevolution A/S Codage par ligature de petites molecules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004083427A2 (fr) * 2003-03-20 2004-09-30 Nuevolution A/S Codage par ligature de petites molecules

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BULLER F ET AL: "Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, ELSEVIER SCIENCE, GB, vol. 18, no. 22, 15 November 2008 (2008-11-15), pages 5926-5931, XP025627172 ISSN: 0960-894X [retrieved on 2008-07-15] *
GARTNER Z J ET AL: "DNA-templated organic synthesis and selection of a library of macrocycles" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, WASHINGTON, DC, vol. 305, no. 5690, 10 September 2004 (2004-09-10), pages 1601-1605, XP002397753 ISSN: 0036-8075 *
HALPIN DAVID R ET AL: "DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution" PLOS BIOLOGY, PUBLIC LIBRARY OF SCIENCE, US, vol. 2, no. 7, 1 July 2004 (2004-07-01), pages 1022-1030, XP002424416 ISSN: 1544-9173 *
MANNOCCI LUCA ET AL: "High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 105, no. 46, November 2008 (2008-11), pages 17670-17675, XP002541365 ISSN: 0027-8424 *
MELKKO ET AL: "Lead discovery by DNA-encoded chemical libraries" DRUG DISCOVERY TODAY, ELSEVIER, RAHWAY, NJ, US, vol. 12, no. 11-12, 1 June 2007 (2007-06-01), pages 465-471, XP022095609 ISSN: 1359-6446 *
MELKKO S ET AL: "Encoded self-assembling chemical libraries" NATURE BIOTECHNOLOGY MAY 2004 NATURE PUBLISHING GROUP US, vol. 22, no. 5, May 2004 (2004-05), pages 568-574, XP002541364 cited in the application *
SCHEUERMANN J ET AL: "DNA-encoded chemical libraries" JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 126, no. 4, 1 December 2006 (2006-12-01), pages 568-581, XP024956589 ISSN: 0168-1656 [retrieved on 2006-12-01] *
TENG S F ET AL: "A STRATEGY FOR THE GENERATION OF BIOMIMETIC LIGANDS FOR AFFINITY CHROMATOGRAPHY. COMBINATORIAL SYNTHESIS AND BIOLOGICAL EVALUATION OF AN IGG BINDING LIGAND" JOURNAL OF MOLECULAR RECOGNITION, HEYDEN & SON LTD., LONDON, GB, vol. 12, no. 1, 1 January 1999 (1999-01-01), pages 67-75, XP001084821 ISSN: 0952-3499 *

Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10669538B2 (en) 2001-06-20 2020-06-02 Nuevolution A/S Templated molecules and methods for using such molecules
US10731151B2 (en) 2002-03-15 2020-08-04 Nuevolution A/S Method for synthesising templated molecules
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
US11001835B2 (en) 2002-10-30 2021-05-11 Nuevolution A/S Method for the synthesis of a bifunctional complex
US10077440B2 (en) 2002-10-30 2018-09-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US11118215B2 (en) 2003-09-18 2021-09-14 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US11965209B2 (en) 2003-09-18 2024-04-23 Nuevolution A/S Method for obtaining structural information concerning an encoded molecule and method for selecting compounds
US11702652B2 (en) 2005-12-01 2023-07-18 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US9359601B2 (en) 2009-02-13 2016-06-07 X-Chem, Inc. Methods of creating and screening DNA-encoded libraries
US11168321B2 (en) 2009-02-13 2021-11-09 X-Chem, Inc. Methods of creating and screening DNA-encoded libraries
US20100227039A1 (en) * 2009-03-03 2010-09-09 Givaudan S.A. Off-Taste Masking
US8329239B2 (en) * 2009-03-03 2012-12-11 Givandan S.A. Off-taste masking
CN102413713A (zh) * 2009-03-03 2012-04-11 奇华顿股份有限公司 异味掩蔽
WO2011127933A1 (fr) 2010-04-16 2011-10-20 Nuevolution A/S Complexes bifonctionnels et procédés de fabrication et d'utilisation de tels complexes
US11225655B2 (en) 2010-04-16 2022-01-18 Nuevolution A/S Bi-functional complexes and methods for making and using such complexes
EP3540059A1 (fr) 2010-04-16 2019-09-18 Nuevolution A/S Complexes bifonctionnels et procédés de fabrication et d'utilisation de tels complexes
WO2012019765A1 (fr) * 2010-08-10 2012-02-16 European Molecular Biology Laboratory (Embl) Procédés et systèmes pour le traçage d'échantillons et de combinaisons d'échantillons
US10865409B2 (en) 2011-09-07 2020-12-15 X-Chem, Inc. Methods for tagging DNA-encoded libraries
EP3351631A3 (fr) * 2011-09-07 2018-08-29 X-Chem, Inc. Procédés de marquage de bibliothèques de codage adn
EP2748357A4 (fr) * 2011-09-07 2015-04-15 Chem Inc X Procédés de marquage de banques codées par de l'adn
EP3392338A1 (fr) * 2012-07-13 2018-10-24 X-Chem, Inc. Bibliothèques de codage adn ayant des liaisons oligonucléotidiques de codage non lisibles par les polymérases
EP2872680A4 (fr) * 2012-07-13 2016-01-27 Chem Inc X Banques encodées par l'adn possédant des liaisons oligonucléotidiques codantes ne pouvant être lues par les polymérases
EP3851529A1 (fr) * 2012-07-13 2021-07-21 X-Chem, Inc. Bibliothèques de codage adn ayant des liaisons oligonucléotidiques de codage non lisibles par les polymérases
US11674135B2 (en) 2012-07-13 2023-06-13 X-Chem, Inc. DNA-encoded libraries having encoding oligonucleotide linkages not readable by polymerases
EP3083957B1 (fr) 2013-12-20 2018-05-23 Philochem AG Production de bibliothèques chimiques codées
WO2015091207A1 (fr) * 2013-12-20 2015-06-25 Philochem Ag Production de bibliothèques chimiques codées
US10240147B2 (en) 2013-12-20 2019-03-26 Philochem Ag Production of encoded chemical libraries
EP3928796A1 (fr) 2014-02-03 2021-12-29 Eidgenöessische Technische Hochschule Zurich Conjugués de médicaments à petites molécules
WO2015135856A1 (fr) * 2014-03-14 2015-09-17 Philochem Ag Purification de produits conjugués d'adn
EP3628684A1 (fr) * 2014-06-06 2020-04-01 Herlev Hospital Détermination de reconnaissance d'antigène par l'intermédiaire d'un marquage par code-barres de multimères du cmh
EP3152232B1 (fr) 2014-06-06 2019-11-13 Herlev Hospital Détermination de reconnaissance d'antigène par l'intermédiaire d'un marquage par code-barres de multimères du cmh
WO2015185067A1 (fr) * 2014-06-06 2015-12-10 Herlev Hospital Détermination de reconnaissance d'antigène par l'intermédiaire d'un marquage par code-barres de multimères du cmh
US11402373B2 (en) 2014-06-13 2022-08-02 Immudex Aps General detection and isolation of specific cells by binding of labeled molecules
US11668705B2 (en) 2014-06-13 2023-06-06 Immudex Aps General detection and isolation of specific cells by binding of labeled molecules
US11585806B2 (en) 2014-06-13 2023-02-21 Immudex Aps General detection and isolation of specific cells by binding of labeled molecules
WO2016074683A1 (fr) 2014-11-11 2016-05-19 Lundorf Pedersen Materials Aps Procédé d'identification de molécules présentant des caractéristiques souhaitées
US11576892B2 (en) 2015-08-18 2023-02-14 Novation Pharmaceuticals Inc. Benzopyran derivatives and uses thereof
WO2017027973A1 (fr) * 2015-08-18 2017-02-23 Novation Pharmaceuticals Inc. Dérivés de benzopyrane et utilisations associées
CN105218405A (zh) * 2015-11-04 2016-01-06 上海泰坦科技股份有限公司 (r,s)-2-[[5-(9-芴甲氧羰基氨基)二苯并[a,d]环庚烷-2-基]氧]乙酸的合成工艺
US11987904B2 (en) 2016-03-04 2024-05-21 Aptamer Group Limited Oligonucleotides and methods for preparing
US11186836B2 (en) 2016-06-16 2021-11-30 Haystack Sciences Corporation Oligonucleotide directed and recorded combinatorial synthesis of encoded probe molecules
EP3538669A4 (fr) * 2016-11-10 2020-05-20 The Scripps Research Institute Codage de bibliothèque de composés en phase solide avec codage à barres à base de polynucléotides
US11795580B2 (en) 2017-05-02 2023-10-24 Haystack Sciences Corporation Molecules for verifying oligonucleotide directed combinatorial synthesis and methods of making and using the same
US11807623B2 (en) 2017-11-30 2023-11-07 Arrakis Therapeutics, Inc. Nucleic acid-binding photoprobes and uses thereof
CN108178790A (zh) * 2018-01-18 2018-06-19 上海药明康德新药开发有限公司 一种将细胞膜分离以便对跨膜蛋白进行dna编码化合物库筛选的方法
CN110093408B (zh) * 2018-01-31 2023-05-02 成都先导药物开发股份有限公司 一种dna编码化合物库及化合物筛选方法
CN110093408A (zh) * 2018-01-31 2019-08-06 成都先导药物开发股份有限公司 一种dna编码化合物库及化合物筛选方法
WO2019236644A1 (fr) * 2018-06-05 2019-12-12 Arrakis Therapeutics, Inc. Bibliothèques codées et procédés d'utilisation pour le criblage de cibles d'acide nucléique
WO2020047095A1 (fr) * 2018-08-28 2020-03-05 The Scripps Research Institute Utilisation d'une immobilisation non covalente dans des banques codées par l'adn
WO2020128064A1 (fr) 2018-12-21 2020-06-25 Philochem Ag Bibliothèques chimiques codées par l'acide nucléique
CN112575389B (zh) * 2019-09-30 2023-09-19 成都先导药物开发股份有限公司 一种dna编码化合物库的液相色谱纯化方法
CN112575389A (zh) * 2019-09-30 2021-03-30 成都先导药物开发股份有限公司 一种dna编码化合物库的液相色谱纯化方法
WO2022084486A1 (fr) 2020-10-23 2022-04-28 Eth Zurich Bibliothèques codées par l'acide nucléique auto-purifié

Also Published As

Publication number Publication date
WO2009077173A3 (fr) 2009-10-29

Similar Documents

Publication Publication Date Title
WO2009077173A2 (fr) Bibliothèques de produits chimiques codés par adn
JP7010875B2 (ja) Ｄｎａコード化ライブラリを作製およびスクリーニングする方法
JP4803933B2 (ja) インビトロ翻訳タンパク質の符号化方法および選別方法
US7125669B2 (en) Solid-phase immobilization of proteins and peptides
JP2003508761A5 (fr)
JPH04504409A (ja) 免疫親和技術を用いた連続ペプチド及びオリゴヌクレオチド合成
JP2000044590A (ja) オリゴヌクレオチド合成用担体
JP2002520008A (ja) Ｄｎａ配列ハイブリッド形成を最適化するための改良されたペプチド核酸汎用ライブラリーの使用法
JP2003500081A (ja) ５’核酸−タンパク質結合体の生成法
AU2015230180B2 (en) Purification of DNA-conjugate products
JP2023548062A (ja) 自己精製された核酸コードライブラリー
WO2020128064A1 (fr) Bibliothèques chimiques codées par l'acide nucléique
WO2023100976A1 (fr) Banque de billes immobilisée par un peptide
JP4054683B2 (ja) 固体支持体上へのオリゴヌクレオチドの固定化
JP2005535567A (ja) 天然物精製のための高分子担持試薬

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08862565

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08862565

Country of ref document: EP

Kind code of ref document: A2