WO2004099441A2 - Selection et developpement de bibliotheques chimiques - Google Patents

Selection et developpement de bibliotheques chimiques Download PDF

Info

Publication number
WO2004099441A2
WO2004099441A2 PCT/DK2004/000325 DK2004000325W WO2004099441A2 WO 2004099441 A2 WO2004099441 A2 WO 2004099441A2 DK 2004000325 W DK2004000325 W DK 2004000325W WO 2004099441 A2 WO2004099441 A2 WO 2004099441A2
Authority
WO
WIPO (PCT)
Prior art keywords
species
molecule
tag
tagged
library
Prior art date
Application number
PCT/DK2004/000325
Other languages
English (en)
Other versions
WO2004099441A3 (fr
Inventor
Thorleif MØLLER
Original Assignee
Hyscite Discovery As
Moeller Thorleif
Pedersen Kim
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 Hyscite Discovery As, Moeller Thorleif, Pedersen Kim filed Critical Hyscite Discovery As
Priority to EP04731316A priority Critical patent/EP1625230A2/fr
Publication of WO2004099441A2 publication Critical patent/WO2004099441A2/fr
Publication of WO2004099441A3 publication Critical patent/WO2004099441A3/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
    • C12N15/1034—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
    • 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
    • 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
    • C12N15/1048—SELEX
    • 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
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms

Definitions

  • Screened compounds include both natural and synthetic compounds. Natural compounds originate from plants, microorganisms or other sources. Synthetic compounds are the result of tedious, organic chemical synthesis. Either way, it is not trivial to build large collections of compounds.
  • Lam et al. disclose a split-mix combinatorial synthesis of peptides on resin beads and tested the beads against labelled acceptor molecules. Beads binding acceptor molecules were found by visual inspection, physically removed, and the identity of the active peptide was determined by direct sequence analysis.
  • Houghten et al. used an iterative selection and synthesis process for the screening of combinatorial peptide libraries.
  • Hexapeptide libraries were used to synthesise 324 separate libraries, each with the first two positions fixed with one of 18 natural amino acids and the remaining 4 positions occupied by all possible combinations of 20 natural amino acids. The 324 libraries were then tested for activity to determine the optimal amino acids in the first two positions. To define the optimal third position, another 20 libraries were synthesised by varying the third position and tested for activity. Using this iterative process of synthesis and selection, an active hexapeptide was identified from a library with a total size of more than 34 million hexapeptides.
  • the identified peptide is not necessarily the most active peptide in the library, since the first selection is done on the basis of average activity (and not the presence of 1 or a few good peptides) in the 324 libraries that each contains 160.000 (20 4 ) different peptides and likewise for the subsequent selections.
  • Another screening approach is based on genetic methods.
  • the advantage of the genetic methods is that libraries can be evolved through iterated cycles of diversification (mutation), selection and amplification as illustrated in Figure IA.
  • the initial library needs only contain very tiny amounts of the individual library members, which in turn allow very large numbers of different library species, i.e. very large libraries.
  • the structure of active compounds can be decoded with little effort by DNA sequencing.
  • the power of genetic methods for the screening of large libraries is now generally appreciated and has on numerous occasions been used to find new ligands.
  • the major limitation is that only biological molecules can be screened, i.e. peptides that can be synthesised by the translational apparatus or oligonucleotides that can be copied by polymerases. Therefore various approaches have been suggested for the application of genetic screening methods for libraries composed of non-biological molecules.
  • a one-pot library of two-piece bifunctional molecules can be build.
  • a library of this type is not evolvable in the traditional sense because the tag does not specify the synthesis of the compounds, rather the tag only reports the synthesis.
  • affinity selected library members have their retrogenetic tag amplified by PCR. DNA strands that are amplified can then be used to enrich for a subset of the library by hybridization with matching tags. The enriched library subset may then be affinity selected against the target and retrogenic tags again PCR amplified for another round of enrichment of a subset of the library.
  • the number of active library members does not increase during the rounds, because active library molecules cannot be amplified/synthesised by way of their tags. Instead it is attempted to remove the non-specific binders from the library as the process proceeds. For very large libraries, though, the amounts of active library members are very tiny, and extra manipulations needed to enrich a library subset before affinity selection seems unfavourable.
  • the present invention relates to methods of screening of libraries using an information transfer to an evolvable secondary library as schematically illustrated in Figure IB.
  • a secondary library comprising a plurality of Y-molecule species, each Y-molecule species comprising a specific tag species (Y-tag species),
  • step h) optionally, repeating steps a), f) and g), wherein the secondary library provided in step a) is derived from a secondary library produced in a previous step g),
  • the peptides of the primary library that bind to the receptor molecules are selected in step d), and their corresponding Y-molecule species are selected in step f) by selecting Y-molecule species that are capable of hybridising to the DNA-tag species attached to the selected peptides.
  • the selected Y-molecule species may be used for preparing a new secondary library, which will be enriched relatively with respect to Y-molecule species that correspond to peptides that bind well to the receptor.
  • the new secondary library may be used in the next repetition of the steps a)-g) and because it is already selectively enriched, the Y-molecule species of the good binders will hybridise even more efficiently than in the first repetition.
  • the concentration of the Y-molecule species corresponding to X-molecules that are poor binders will be reduced as the repetitions progress with new secondary libraries for each repetition and therefore the Y-molecule species of poor binders will hybridise more inefficiently for each repetition.
  • the steps a)-g) are repeated a number of times and for every repetition, the secondary library is further enriched with respect to the Y-molecule species corresponding to the good binders. Finally, the latest secondary library may be analysed and the Y-molecule species of highest concentration are identified along with their corresponding peptides. The identified peptides may now be studied further in more complex models such as cellular or animal models.
  • the present methods may be used for identifying new enzymes for both industrial and therapeutic use, new antibodies and aptamers e.g. for diagnostics, new catalysts, and so forth.
  • Figure IB shows the principle of double selection and evolution
  • FIGS. 3A and 3B illustrate schematically embodiments of a Y-molecule species
  • FIGS 4A and 4B illustrate the steps of the method described in Example 1,
  • FIGS 5A, 5B and 5C illustrate the steps of the method described in Example 2,
  • FIGS 6A, 6B and 6C illustrate the steps of the method described in Example 3,
  • FIGS 7A, 7B and 7C illustrate the steps of the method described in Example 4,
  • FIGS 8A and 8B illustrate the steps of the method described in Example 5,
  • FIGS 9A, 9B and 9C illustrate the steps of the method described in Example 6,
  • FIGS 10A, 10B and 10C illustrate the steps of the method described in Example 7,
  • FIGS 11A, 11B and 11C illustrate the steps of the method described in Example 8,
  • Figure 12 shows a schematic drawing of a tagged X-molecule species having a small peptide as X-molecule species
  • FIGS 13A and 13B illustrate the steps of the method described in Example 9, and
  • the present invention relates to a method of selecting and/or identifying, among a plurality of molecules, a molecule that is capable of specifically interacting with a target molecule.
  • the method comprises the steps of a) providing a secondary library comprising a plurality of Y-molecule species, each Y-molecule species comprising a specific tag species (Y-tag species),
  • a primary library comprising a plurality of tagged X-molecule species, wherein a tagged X-molecule species comprises an X-molecule species and a specific tag species (X-tag species), and wherein at least one X-tag species of the primary library is capable of hybridising to at least one Y-tag species of the secondary library,
  • step h) optionally, repeating steps a) , f) and g), wherein the secondary library provided in step a) is derived from a secondary library produced in a previous step g),
  • step a) and step b) are performed before steps c) - i).
  • Step a) may be performed before step b) or step b) may be performed before step a).
  • Step e) and f) may be performed before step c) and d), such that Y-tag species are hybridised to X-tag species, before tagged X-molecule species are selected against the target molecule.
  • Step d) and f) may be performed simultaneously.
  • steps c) to g) may be substituted by steps c-1) to f-l):
  • the Y-tag of a Y-molecule species may hybridise to only one tagged X-molecule species of the primary library.
  • a Y-tag of a Y-molecule species may be able to hybridise to several tagged X-molecule species at a time.
  • the Y-molecule species may be able to hybridise to at least 1 molecule of a tagged X-molecule species at a time, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000 or 10.000 such as at least 100.000 molecules of a tagged X-molecule species at a time.
  • the X-tag species of a tagged X- molecule species are not homologues of the X-tag species of another tagged X-molecule species. Also, it may be preferred that the X-tags of individual molecules of the same tagged X-molecule species are identical, alternatively that they are homologues. The X-tag of identical X-molecules may also be non-homologues, that is, two different tagged X- molecule species may comprise the same X-molecule but comprise different X-tags.
  • Step e) is optional, thus in one embodiment of the present invention the step e) is not performed. In an alternative embodiment step e) is performed. Instead of performing step e), one may use intermediate libraries for transferring the information of the selected tagged X-molecule species, and consequently, one of the intermediate libraries may be hybridised to the secondary library as an alternative to hybridising the selected tagged X- molecule species to the secondary library.
  • Step h) is optional, thus in one embodiment of the present invention the step h) is not performed.
  • step h) is performed.
  • Step h) comprises the repetition of steps a), f), and g), wherein the secondary library provided in step a) is derived from a secondary library produced in a previous step g).
  • Step h) may furthermore comprise the repetition of one or more of the steps b), c), d) and e).
  • step h) may comprise the repetition of steps a)-g).
  • it is the newest secondary library, i.e. the secondary library of the latest step g) that is used in the next repetition as governed by step h).
  • it is the newest secondary library that is analysed and/or identified in step i), i.e. the secondary library of the latest step g).
  • a first primary library and a second primary library are used in different repetitions in step h).
  • the first and second libraries may differ in that the X-tags of the tagged X-molecule species of the first library are complementary to the X- tags of the corresponding tagged X-molecule species of the second primary library.
  • step d) any unwanted activity coming from the X-tag that may interfere with the primary selection of step d) will not be detected, since the X-tag of the first library is unlikely to have the same binding activity as its complimentary counterpart in the second primary library. Therefore, if a tagged X-molecule species is selected in step d) due to unwanted activity of the X-tag when the first primary library is used, it is unlikely that the same tagged X-molecule species will be selected when the second primary library with the complementary X-tags are used.
  • the method may furthermore comprise a step of monitoring the amplification product of step g) at least one time.
  • the purpose of the monitoring is to evaluate whether another repetition should be performed or whether the secondary library is ready for identification.
  • the amplification product may be analysed by standard methods as described in Sambrook et al and Abelson, e.g. by sequencing the amplification product of step g) in bulk or by cloning the amplification product and sequencing the individual clones. If the analysis reveals that the secondary library has been significantly enriched with respect to a Y-molecule species one could consider interrupting the repetitions and proceeding with steps i) and j). Depending on the actual embodiment and based on the results of the analysis, the skilled person will be able to determine the right conditions to stop repeating steps a)-g).
  • a subset of the primary library may e.g. mean the entire material primary library or it may mean a fraction of the material of the primary library, said fraction having a composition which is representative for the composition of the primary library. Also, a subset of the primary library may mean a fraction of the material of the primary library, said fraction having a composition, which is only representative for the composition of the primary 10 library with respect to some of the tagged X-molecule species.
  • the primary library comprises a plurality of tagged X-molecule species, wherein a tagged X-molecule species comprises an X-molecule species and a specific tag species (X-tag species), and wherein at least one X-tag species of the primary library is capable of 15 hybridising to at least one Y-tag species of the secondary library.
  • a tagged X-molecule species comprises an X-molecule species and a specific tag species (X-tag species)
  • X-tag species specific tag species
  • the primary library may comprise at least IO 2 tagged X-molecule species, such as at least IO 3 , IO 4 , 10 s , IO 6 , IO 7 , 10 8 , IO 9 , 10 10 , 10", IO 12 , 10 13 , IO 14 such as at least IO 15 tagged X-molecule species.
  • the primary library may comprise 10 3 -10 18 tagged X- 20 molecule species, 10 3 -10 6 tagged X-molecule species, 10 6 -10 9 tagged X-molecule species, 10 9 -10 12 tagged X-molecule species, 10 12 -10 15 tagged X-molecule species or 10 15 -10 18 tagged X-molecule species.
  • a next generation secondary library is derived from the starting material by providing a secondary library which has an information content similar to at most the 1000 Y-molecule species of highest concentration in the starting material, the starting material being the amplification product of step f).
  • concentration of amplification product e.g. by dilution or up- concentration.
  • Step 11) may also comprise one or more of the steps selected from the group consisting of amplification, dilution, restriction, ligation, purification of the coding or the anti-coding strands of the PCR-product, a purification by a standard method e.g. as described in Sambrook et al.
  • the tagged X-molecule species which is illustrated schematically in Figure 2A comprises an X-molecule species (2) linked via a linker molecule (4) to an X-tag (3).
  • the X-molecule species may be build of X-groups (16), e.g. the five X-groups E, D, C, B and A, and the X- tag (3) may be build of tag codons (5), such as the five tag codons A', B', C, D', E'.
  • the X-groups may be connected in a linear way as illustrated in Figure 2A.
  • Alternatively X- groups may form branched structures. To obtain a branched X-molecule structure at least one multifunctional X-group, said X-group comprising at least two active groups, said active groups are capable of further reaction.
  • nucleic acid should be interpreted broadly and may for example be an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • nucleic acid molecules composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as molecules having non-naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages which function similarly or combinations thereof.
  • Tagged X-molecule species may be of any stochiometry, i.e. any ratio between X-molecule and X-tag species.
  • a tagged X-molecule may comprise at least 2 molecules of an X- tag species, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10.000, 100.000 such as at least 1.000.000 molecules of an X-tag species.
  • a tagged X-molecule species may comprise at least 2 molecules of an X-molecule species, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10.000, 100.000 such as at least 1.000.000 molecules of an X-molecule species.
  • the term “functional derivatives” means derivative of the capture components, said derivatives having substantially the same or improved capture component capability as compared to the capabilities of a capture component listed above.
  • the photocleavable group may be an o-nitrobenzyl linker, such as described in Olejnik et al 1 and in Olejnik et al 2.
  • nucleotides that cannot be replicated by polymerases are LNA, PNA, 2'OH methylated RNA, morpholinos, phosphorothioate nucleotides.
  • backbone-substituted oligonucleotides of the above-mentioned may be employed.
  • Such tagged X-molecule species may be used where one desires to find an oligonucleotide that is not recognized by proteins that have evolved to interact with natural nucleic acids, e.g. it may be desirable that the particular oligonucleotide is not degraded by nucleases. Or the use of non-natural oligonucleotide may also be desired because of specific demands on chemical stability, solubility or other characteristics.
  • Step b) and c) may be performed in the same reaction mixture or in separate mixtures. It may be preferred that step b) and/or step c) comprise(s) a solid phase reaction. Alternatively, it may be preferred that step b) and/or step c) comprise(s) a liquid phase reaction. Step b) may be performed before step c) or step c) may be performed before step b).
  • the first X-group could contain e.g. three reaction sites, each allowing addition of another X-group which may or may not contain further reaction sites (functionalities capable of receiving another X-group).
  • the resulting tagged X-molecule species may be of the type shown in Figure 2A.
  • the amino acid may be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, a synthetic amino acid, a beta amino acid, a gamma amino acid and a peptoid (N- substituted glycine).
  • the X-molecule species may comprise a component selected from a group consisting of a peptide, a nucleic acid, a protein, a receptor, a receptor analogue, a polysaccharide, a drug, a hormone, a hormone analogue and an enzyme. They may also be selected from the group consisting of a synthetic molecule and a molecule isolated from nature.
  • the X-molecule species may have a molar weight of at least 500 D, such as 1000 D, 5 kD, 10 kD, 20 kD, 40 kD, 80 kD, 200 kD, 500 kD, such as at least 1000 kD. Also the X- molecule species may have a molar weight in the range of 500 D - 1000 kD, such as 500 D- 5 kD, 5 kD 1000 kD, 5 kD - 50 kD, 50 kD - 200 kD, 200 kD - 500 kD or 500 kD - 1000 kD.
  • the X-molecule species may comprise at most 500 monomer building blocks and/or X- groups such as at most 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, such as at most 3 monomer building blocks and/or X-groups.
  • the X-molecule species may comprise at least 1 monomer building blocks and/or X-groups such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, such as at least 50 monomer building blocks and/or X-groups.
  • the X-molecule species may comprise 2-100 monomer building blocks and/or X-groups, such as 2-10, 2-20, 2-10, 5-10, 5-20, or 10-50 monomer building blocks and/or X-groups.
  • the X-molecule species may be stable within the temperature range 0 to 95 degrees C such as within 0 to 10 degrees C, 10 to 20 degrees C, 20 to 30 degrees C, 30 to 40 degrees C, 35 to 38 degrees C, 40 to 50 degrees C, 50 to 60 degrees C, 55 to 65 degrees C, 60 to 70 degrees C, 70 to 80 degrees C, 80 to 90 degrees C, such as within the temperature range 90 to 95 degrees C.
  • the X-molecule species may survive 1 hour of autoclaving at 120 degrees C.
  • the tagged X-molecule species and/or the X-moiecule species may be produced by combinatorial chemistry, e.g. such as described in WO 93/20242 or in Needels et al, e.g. using the split-pool principle.
  • tagged X-molecule species may be prepared using a convergent synthesis, i.e. preparing X-tag and X-molecule (purified, synthesised, or other) separately, followed by attachment of the X-tag to the X- molecule.
  • the Y-molecule species may comprise a Y-tag species and may be capable of being amplified.
  • FIG. 3A A schematic illustration of a Y-molecule species is shown in Figure 3A and 3B.
  • the Y-tag (11) comprises the five tag codons (5), namely A', B', C, D' and E'.
  • the Y- tag (11) is flanked by a first fixed region (13) and a second fixed region (14).
  • One of the fixed regions (13) or (14) may be used as a primer binding site during a PCR process.
  • the Y-tag (11) may comprise only one fixed region (13).
  • the binding site may either be a part of the tag species or may not be a part of the tag species.
  • the Y-molecule species are selected so that the Y-molecule species have substantially no intrinsic binding activity or affinity for the tagged X-molecule species and/or the target molecule.
  • Y-molecule species may preferably have affinity against corresponding tagged X-molecule species, but not against target molecule or other tagged X-molecule species.
  • Y-molecule species which may be unsuitable for use in the present method due to a high level of non-specific or intrinsic binding may be identified by screening the Y-molecule species for intrinsic binding.
  • the target molecule can be any given molecule or structure to which one wishes to find a ligand.
  • Therapeutically relevant target molecules are mostly proteinaceous molecules.
  • the target molecules may be selected from the group consisting of a protein, a hormone, an interleukin receptor, ion channels, a ribonucleoprotein and a prion.
  • the protein may be selected from an interleukin, an antibody, an enzyme, a membrane protein, a membrane bound protein, an intracellular protein and an extracellular protein.
  • a target molecule need not necessarily be a single protein.
  • the target molecule may be a complex of several proteins, a cell membrane, a fragment of a cell membrane e.g. having a lipid double layer, or a cell organ, e.g. golgi apparatus, endoplasmatic reticulum, mitochondria, etc, an entire cell, groups of cells or a tissue.
  • it may be desirable to find molecules that are transported into a cell instead of binding to a particular place on or in the cell.
  • a molecular library may be incubated with target molecule cells for a certain time and molecules that are transported into the cell may be recovered by e.g. phenol extraction of the cells followed by ethanol precipitation.
  • the X-tag species comprise a biotin-group or a similar capture component to facilitate recovery.
  • the target molecule could also be a nucleic acid such as a RNA molecule (e.g. tRNA, rRNA, mRNA, miRNA etc.) or a given DNA sequence. Also metabolic intermediates, e.g. stabilised intermediates, may be employed as target molecules.
  • the target molecule could also be a transition-state analogue, e.g. if one wishes to find new catalysts.
  • the cell may be a eukaryote cell such as a plant cell, a mammalian cell or a yeast cell or the cell may be a prokaryote cell or the cell may be an archae.
  • the target molecule may be a virus or a fragment of a virus.
  • the concentration of the target molecule used in step c) is kept as low as possible to reduce non-specific binding, while at the same time allowing binding and selection of X-molecules binding specifically to the target molecule.
  • the appropriate concentration of target can be calculated using the law of mass action.
  • 99% of tagged X-molecules with a k d of 10 "9 M will be bound to the target at equilibrium.
  • the ratio between the average number of molecules per tagged X- molecule species and the number of target molecules may be at least li lO 1 , 1 : 10 2 , 1 : 10 3 , 1 : 10 4 , 1 : 10 s , 1: 10 6 , 1: 10 7 ,1 : 10 8 , 1: 10 9 , 1: 10 10 , 1: 10", 1: 10 12 or 1: 10 13 , such as at least l: 10 14 .
  • the ratio between the total number of molecules of all tagged X-molecules species and the number of target molecules may be at most 10 15 : 1 such as at most 10 14 : 1, 10 13 :1, 10 12 :1,10":1, 10 10 : 1,10 9 : 1,10 8 :1, 10 7 :1, 10 6 :1 10 5 : 1, or 10 4 :1, such as at most 10 3 : 1.
  • the tag species comprises a sequence of tag codons, said tag codon is capable of binding to a tag codon with a complementary sequence. The binding occurs preferably by hybridisation.
  • the tag species are capable of specific Watson-Crick basepairing and replication by polymerases in PCR.
  • a tag codon may comprise at least one nucleotide, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, such as at least 50 nucleotides.
  • the sequence of tag codons within a tag species may comprise at least 1 tag codons, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, such as at least 20 tag codons.
  • the tag species may be orthogonal meaning that tag codons and tag codon sequences are selected and/or designed so that no tag species can partly of fully hybridise to another tag species within the temperature range 55-70 degrees C.
  • Tag codons may be designed for example by employing methods described in US 5,635,400 (Minimally Cross-Hybridising Sets of Oligonucleotide Tags).
  • the tag species may be prepared by standard phosphoramidite oligonucleotide synthesis such as described in Sambrook and in Abelson. However, if a hexacodon tagging system is used, i.e. if the codons comprise six nucleotides, it may be desirable to use hexanucleotide phoshoramidites as building blocks, instead of mononucleotide phosporamidites, as this will result in sixfold fewer couplings in the oligonucleotide synthesis. The same applies if employing a pentacodon, heptacodon tagging system or similar systems.
  • the ratio between the concentration of a tagged X-molecule species in the primary library and the concentration of its corresponding Y-molecule species in the secondary library at least 1: 10 10 , such as at least 1: 10 6 , 1: 10 5 , 1: 10 4 , 1: 10 3 , 1:10 2 , li lO 1 , 1: 1, 10 ⁇ 1, 10 2 : 1, 10 3 :1, 10 4 : 1, 10 5 : 1, or 10 s : 1, such as at least 10 10 : 1.
  • the specific interaction between the target molecule and the tagged X-molecule species is an important process and many levels and combinations of specific interaction are envisioned.
  • the specific interaction is an interaction selected from the group consisting of the binding of a tagged X-molecule species to the target molecule, conformational changes of the tagged X-molecule species and/or the target molecule, the binding of an tagged X- molecule species to the target molecule, enzymatic activity from the tagged X-molecule species on the target molecule, enzymatic activity from the target molecule on the tagged X-molecule species, enzymatic activity complex of the tagged X-molecule species and target molecule, effects in cells, tissue and animals mediated by the target molecule upon binding of the tagged X-molecule species, and any combination thereof.
  • the present invention is only the X-molecule of the tagged X- molecule species that interacts specifically with the target molecule, whereas in another embodiment it is the combination of X-molecule and X-tag species that is responsible for the interaction.
  • the temperature during the selection of tagged X-molecule species capable of interacting specifically with the target molecule is preferably within the range of 0 to 100 degrees C such as within the temperature 0 to 10 degrees C, 10 to 20 degrees C, 20 to 30 degrees C, 30 to 40 degrees C, 35 to 38 degrees C, 40 to 50 degrees C, 50 to 60 degrees C, 55 to 65 degrees C, 60 to 70 degrees C, 70 to 80 degrees C, 80 to 90 degrees C, such as within the temperature range 90 to 100.
  • the time in which the specific interaction between the tagged X-molecule species and the target molecule occurs may be within the range 0.001 sec- 20 days such as within 0.001- 0.01 sec, 0.01-0.1 sec, 0.1-1 sec, 1-30 sec, 30-60 sec, 60 sec to 1 minute, 1 minute - 20 minutes, 20 minutes to 60 minutes, 60 minutes to 5 hours, 5 hours to 12 hours, 12 hours to 1 day, 1 day to 3 days, 3 days to 6 days, such as within 6 days to 20 days.
  • substantially all target molecules are bound in the same spatial fashion relative to the solid phase surface. In another embodiment, substantially all target molecules present the same parts, such as epitopes, moieties, sequences etc., of the target molecule to the tagged X-molecule species.
  • the primary library can be contacted to a target molecule in a number of different experimental settings. Most often the target molecule is present in the solid phase and the primary library in the liquid phase. I.e. the target molecule has been immobilised on a solid matrix. Alternatively, the target may be immobilized after contacting the primary library.
  • the target molecule may be immobilized using CNBr activated sepharose or the target molecule may be biotinylated and immobilized on streptavidin sepharose beads or magnetic streptavidin beads (e.g. Dynabeads ® M-280 Streptavidin).
  • filterbinding to can be employed, e.g. to nitrocellulose filters. A great variety of methods for immobilisation of target molecules are known to those skilled in the art.
  • the target molecule may also be present in the liquid phase together with the primary library and the primary library may be present in the solid phase with the target molecule being in the liquid phase.
  • the solid phase may be various kinds of beads as mentioned above, but also microchips/arrays and the like can be employed.
  • the liquid phase will most often be aqueous, the exact composition depending on the particular affinity selection.
  • the pH of the aqueous media can be controlled using buffer systems such as MOPS, Tris, HEPES, phosphate etc, as can the ionic strength by the addition of appropriate salts.
  • the library may be counter selected against the solid phase without target molecule, before being selected against the solid phase with target molecule.
  • specific binders may be specifically co-eluted with the target molecule, e.g. by cleaving the linker (e.g. photocleavage) that attaches the target molecule to the solid phase.
  • linker e.g. photocleavage
  • competitive elution using known ligands of the target may be used or elution with excess soluble target.
  • the liquid phase is not limited to aqueous media, as organic solvent may also be employed, those being e.g. DMF, THF, acetonitrile, and organic - aqueous mixtures as well as two phase systems.
  • organic solvent e.g. DMF, THF, acetonitrile, and organic - aqueous mixtures as well as two phase systems.
  • the binding reaction may be performed at any desired temperature. If the target molecule is e.g a therapeutically relevant human molecule, the binding reaction may be performed at 37 °C. And for target molecules from thermophilic bacteria a higher temperature can be employed, as well as low temperatures for target molecules from psychrophile organisms, not to preclude any temperature for any target molecule.
  • the time period for incubation of the binding reaction can be from minutes to hours and even days.
  • the incubation can be adjusted such that the binding reaction is at thermodynamical equilibrium.
  • fast binders large K on value
  • binders with small K off values by washing the binding reaction and selecting primary library members that stay bound after a chosen time period.
  • fast on - fast off binders can be selected by the same method of washing and selecting after a chosen (shorter) time period.
  • the present invention it is possible select for various strengths of binding between the target molecule and the tagged X-molecule species by controlling the conditions during the washing and by controlling the number of washing steps. E.g. if 10 washing steps are performed during the selection process the selected tagged X-molecule species may tend to bind more strongly to the target molecule than if only 2 washing steps were performed.
  • the selection of Y-molecule species comprises hybridising a Y-molecule species to the X-tag species of a tagged X-molecule species.
  • the hybridisation is preferably performed at stringent conditions.
  • the skilled person is readily able devise suitable conditions for the hybridization reactions, assisted by textbooks such as Sambrook, Ausubel et al and Anderson.
  • the selection may comprise a process selected from the group consisting of amplification, extraction, binding to hydroxyapatite, an enzymatic digest and a hybridisation to a strand immobilized on solid phase followed by a washing step.
  • the secondary library may be hybridized to X-tag species in a number of ways. If the selected X-molecule species have a stable interaction with their target molecules, the secondary library can be hybridized to X-tag species of tagged X-molecule species fixed to their target molecules. After washing away non-binding Y-molecule species (non- hybridized), hybridized Y-molecule species may be eluted by denaturation with high pH, high temperature or other before PCR amplification. However, it can also be feasible to use the entire binding reaction as template in the PCR reaction, i.e. the solid phase is employed directly in the PCR reaction.
  • selected X-molecule species can be eluted from the target prior to hybridization with the secondary library. Elution may be done by changing the buffer, e.g. changing ionic strength, pH, detergents, etc., or by raising the temperature. If ligands are sought that bind to the same site of the target molecule as another known ligand the latter may be used for competitive elution.
  • the eluted X-molecule species can then be hybridized to the secondary library in solution, in which case the double stranded product may be recovered by hydroxyapatite chromatography.
  • the X-tag species may be provided with a capture component such as biotin to facilitate recovery.
  • eluted X-molecule species are hybridized to Y-molecule species in solution and hybridized Y-moiecule species recovered by binding X-molecule species to streptavidin beads through a biotin capture component.
  • Eluted X-molecule species can also be immobilized before hybridization.
  • X-tag species itself may be designed to facilitate hybridization by employing modified or non-natural nucleotides such as PNA, LNA, 2'0-methylated RNA etc. Further, the sequence content of X-tag species may be designed to facilitate the hybridization reaction.
  • the resulting second-generation secondary library is purified using standard methods (spin-column, gel filtration, gel purification or other) and its concentration adjusted before hybridization with another subset of the primary library selected against the target molecule.
  • standard methods spin-column, gel filtration, gel purification or other
  • concentration adjusted before hybridization with another subset of the primary library selected against the target molecule.
  • the anti-coding strand may be purified by elution from immobilized coding strands on streptavidin or by purification from PAGE, as described in the Examples.
  • the concentration of the secondary library can be adjusted such as to have Y-molecule species corresponding to active X-molecule species in molar excess (e.g. 10, 50 or 100 fold). Otherwise, the molar ratios can be adjusted such as to reflect a 1 to 1 molar ratio.
  • the fold of enrichment in the secondary library can be estimated by measuring the part selected using e.g. radiolabelled Y-molecule species.
  • the concentration of Y-molecule species in the secondary library may be adjusted by amplification and/or dilution after each round.
  • Example 1 the part of the primary library that does not bind to the solid phase can be pre-hybridised to the Y-molecule species of the secondary library, before the selected tagged X-molecule species are hybridised to the pre-hybridised secondary library.
  • the primary and secondary library may be hybridised before selection against the solid phase.
  • a photocleavable biotin may be incorporated in the X-tag.
  • the primary library is selected against the target molecule, the non-binding tagged X-molecule species are collected and hybridised to the secondary library.
  • the hybridisation mixture is illuminated to cleave of the biotin group, whereafter the Y-molecule species of the pre-hybridised secondary library are hybridised to selected tagged X-molecule species, that may still be bound to the target molecule or more likely have been eluted using e.g. SDS, urea or high temperature.
  • the biotin group on selected tagged X-molecule species are used as affinity tag to select secondary library members that correspond to active tagged X-molecule species.
  • the amplification is performed using a technique selected from the group consisting of Polymerase Chain Reaction techniques (PCR), Strand Displacement Amplification (SDA), Ligation-Rolling Circle Amplification (L-RCA) and their combinations/modifications. These methods are well known to the person skilled in the art and are described in Sambrook.
  • PCR Polymerase Chain Reaction techniques
  • SDA Strand Displacement Amplification
  • L-RCA Ligation-Rolling Circle Amplification
  • Y-molecule species may be analysed and identified by standard methods as described in Sambrook et al and Abelson, e.g. by sequencing in bulk or by cloning the amplification product and sequencing the individual clones.
  • the identification of the Y-molecule species of high prevalence may comprise a step selected from the group consisting of identifying the Y-molecule species with the highest concentration, identifying the Y-molecule species with the highest signal in a hybridisation test, identifying one or more or all Y-molecule species with a concentration and/or signal at a certain threshold, identifying one or more or all Y-molecule species with a concentration and/or signal less than a certain threshold, identifying one or more or all Y-molecule species with a concentration and/or signal above a certain threshold and combinations thereof.
  • the Y-molecule species are identified as the Y-molecule species, which are present in the PCR product at a concentration at or above a certain concentration threshold.
  • the identification of the Y-molecule species may be performed with a method comprising the steps of
  • the tagged X-molecule species that interact specifically with the target molecule is identified from the records respective to which X-tag species that correspond to which X-molecule species.
  • the relevant X-tag species may be identified by identifying the Y-molecule species of high prevalence and either calculating, determining and/or looking up their corresponding X-tag species.
  • the records that relate Y-molecule species to X-tag species and X-tag species to X-molecule species may preferably be handled electronically, e.g. in a computer system.
  • An additional aspect of the present invention relates to the use of the methods described herein for identifying new enzymes for both industrial and therapeutic use, new antibodies and aptamers e.g. for diagnostic and/or therapeutic use, new catalysts, and so forth.
  • the methods are used for identifying pharmaceutically active compound.
  • the use comprises the preparation of a primary library where the X-molecule species of the tagged X-molecule species are molecules to be tested for pharmaceutical or therapeutic activity against a given disease.
  • the target molecule should preferably have an expected or known relation to the disease.
  • X- molecule species being capable of e.g. binding to the target molecule may be identified and these identified X-molecule species are likely to have pharmaceutical or therapeutic activity against the disease.
  • Examples 1-4 are proof of concept experiments where DNA oligonucleotide libraries are screened to demonstrate that the presented invention can be used as a screening method.
  • Examples 5-8 are extensions of Examples 1-4, which outline how libraries composed of other tagged X-molecule species can be screened. Hence, Examples 5-8 should be generally applicable to libraries composed of tagged X-molecule species.
  • Example 1 Model system using streptavidin as target molecule and a DNA oligonucleotide comprising a biotin group in a DNA oligonucleotide library as primary library
  • a model library comprising IO 6 different DNA oligonucleotide species in equimolar amounts is screened for binding activity against streptavidin immobilized on sepharose.
  • One particular oligonucleotide in the library contains a biotin-group at its 5'end and it is intended to demonstrate that the identity of this particular oligonucleotide can be found using the present invention.
  • the primary library is prepared by mixing a degenerate oligonucleotide, which has a total diversity of 10 s , with the biotinylated oligonucleotide, such that the latter is present in equimolar amounts with individual sequences of the degenerate oligonucleotide.
  • the present invention can be used to find a signal within about IO 6 fold excess noise.
  • noise is used to denote X and Y-molecules that we do not expect to have significant affinity toward the target. Strictly speaking, though, we do not know whether any X or Y- molecules have affinity toward the target, since it is well known that oligonucleotides can take up tertiary structures that bind protein targets with high-affinity and selectivity.
  • biotin group serves two roles in Example 1 to 4; the role of a specific interaction in the library relative to the target molecule and the role of a capture component used to manipulate DNA-strands.
  • the steps of Example 1 are illustrated in Figure 4A and 4B.
  • the two Figures are meant to be combined.
  • the primary library comprises a plurality of tagged X-molecule species (1), one of which is the active tagged X-molecule species (6).
  • the active tagged X-molecule species (5) is marked with a large "X” and the inactive tagged X-molecule species are marked with a small "x".
  • the active X-molecule species is a biotin group. Where the biotin group is used as an affinity handle (capture group) for manipulation of DNA strands, the biotin group is indicated by "b”.
  • streptavidin sepharose (8) adopts the role of the target molecule it is denoted solid phase bound target and where it is used for manipulation of DNA, it is denoted streptavidin sepharose (18).
  • Step a) Providing the primary library
  • the primary library is prepared such as to contain about 10 s different sequences. This is accomplished using redundant positions during DNA synthesis. To achieve a library with
  • 5'bCAGTAG TAGCCA ACGGCT AGTA AGCTAG TCGGAG CGAAAC GGATCC GCTATA ACCTCG ATCG TTAGAC GCTATC CGAGTA
  • 126 ⁇ l is aliquoted into 63 standard PCR reactions each containing: 10 ⁇ l Optibuffer, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCl 2 , 2 ⁇ l 20 ⁇ M upstream PCR-primer, 2 ⁇ l 20 ⁇ M downstream PCR-primer, 61 ⁇ l H 2 0 and 1 ⁇ l BIO-X-ACTTM Short DNA polymerase (4 units). The reaction is cycled 10 times with 94°C for 30 sec, 55 °C for 30 sec, 72 °C for 90 sec followed by 10 minutes extension at 72 °C.
  • Step 2 Contacting the primary libraries with the target molecule
  • the solid phase is washed twice with 1000 ⁇ l binding buffer to select tagged X-molecule species interacting with the solid phase bound target. Moreover, tagged 5 X-molecule species bound specifically are eluted using the photocleavable biotin linker; the solid phase is resuspended in 75 ⁇ l binding buffer and placed on a sheet of parafilm whereafter the sample is illuminated for 6 minutes as described (Olejnik J, Krzymanska- Olejnik E, Rothschild KJ. Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides. Nucleic Acids Res 1996 Jan 10 15;24(2):361-6). The samples are then spinfiltered and the liquid phase collected.
  • the liquid phase containing specifically eluted tagged X-molecule species is aliquoted into standard 60 PCR reactions each containing: 10 ⁇ l optibuffer buffer, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCI 2 , 2 ⁇ l 20 ⁇ M upstream PCR-primer, 2 ⁇ l 20 ⁇ M downstream PCR-primer 2, 62 ⁇ l H 2 0 and 1 ⁇ l BIO-X-ACTTM Short DNA polymerase (4 units).
  • the reaction is cycled 10 times with 94°C for 30 sec, 55 °C for 30 sec, 72 °C for 60 sec followed by 10 minutes at
  • PCR primers 1 and 5 are employed for amplification of the psn2 primary library. Because PCR primer 5 is biotinylated in its 5'end, the resulting PCR product is biotinylated at the 5'end of the coding strand. Likewise, for amplification of psn3, PCR primers 6 and 7 are employed which biotinylates the resulting PCR product at the 5' end of the anti-coding
  • Step 5 Providing the secondary library
  • the secondary library can be increasingly diluted, because it evolves to contain a larger fraction of signal oligo (ss3), i.e. if the secondary library is 10000 fold enriched in signal oligonucleotides, a 10.000 fold shortage in total amount of the secondary library can be used for hybridisation.
  • the amount of secondary library can also be adjusted to have ss3 in moderate excess (5 -50 fold) over psi for the hybridisation reaction. Further, number of cycles in the PCR reactions can be adjusted in later rounds and carrier nucleic acids may be employed.
  • Step 8) Identifying molecules of high prevalence
  • Example 4 is a modification of Example 2, the major difference being that the hybridisation reaction is performed with both the anti-coding and the coding strand in solution, as opposed to Example 2 and 3, where one strand is immobilised during the hybridisation reaction.
  • the steps of Example 4 are illustrated in Figure 7A-C. The three should be combined so that Figure 7A and 7B run in parallel and continue in Figure 7C. Step 1) Providing the primary libraries
  • the two primary libraries employed are identical to the libraries of Example 2.
  • PCR-primer 1 5' GATGAT AGTAGT TCGTCG TCAC
  • PCR-primer 2 5'bGCAGCA ACTACT CATCAT GACT PCR primer-3 5' TACTCG GATAGC GTCTAA CGAT PCR primer-4 5'bCAGTAG TAGCCA ACGGCT AGTA
  • PCR primer-8 5' CAGTAG TAGCCA ACGGCT AGTA
  • the second nucleotide from the 3' end in PCR primer-8 is a ribonucleotide.
  • Step 2 Contacting the primary libraries with the target molecule
  • Step 3 Selecting tagged X-molecule species that interact with the target molecule
  • Step 4) Amplifying the selected A-tag species
  • PCR primers 4 and 8 are used for PCR amplification of selected psn3 molecules.
  • Step 5 Providing the secondary library
  • the volume is increased to 100 ⁇ l by addition of binding buffer + 2 ⁇ g/ ⁇ l tRNA, whereafter the sample is added to 6 ⁇ l pre-equilibrated streptavidin sepharose and incubated for 30 minutes at 55 °C with mixing.
  • the psn3 primary library is again selected against the solid phase bound target and selected X-tags PCR amplified.
  • the anti-coding strand from the resulting PCR product is hydrolysed with NaOH and the coding strand purified from PAGE.
  • Purified psn3 coding strands are hybridized to complementary anti-coding Y-molecule species of the first generation secondary library in solution, where after hybridised Y-molecule species (psn2 strands) are selected on streptavidin sepharose.
  • selected Y-molecule species are PCR amplified to generate the second-generation secondary library.
  • the secondary library can be increasingly diluted, because is evolves to contain a larger fraction of signal oligo (ss3), i.e. if the secondary library is 10000 fold enriched in signal oligonucleotides, a 10.000 fold shortage in total amount of the secondary library can be used for hybridisation.
  • the amount of secondary library can also be adjusted to have ss3 in moderate excess (10 -100 fold) over psi for the hybridisation reaction. Further, number of cycles in the PCR reactions can be adjusted in later rounds and carrier nucleic acids may be employed.
  • Step 7) Monitoring the evolution of the secondary library
  • Step 8) Identifying molecules of high prevalence
  • Step 9) Identifying tagged X-molecule species with an X-tag species corresponding to the high prevalence Y-molecule species
  • Example 5 In this Example, a (hypothetical) library composed of beta-peptides is screened for specific interaction of the beta-peptide versus a target molecule.
  • the primary library contains IO 6 beta-peptide tagged X-molecule species.
  • the steps of Example 5 are illustrated in Figures 8A-8B. The two figures should be combined. It is important to note that the screening method used in this Example would apply for other tagged X-molecule species as well.
  • tagged X-molecule species could have been intrinsic to the X-tag species (one-piece bifunctional tagged X-molecule species) or could have been any chemical entity (d-peptide, gamma-peptide, peptoid, sugar, LNA oligonucleotide, PNA oligomer, small molecule, natural compound, mixed compounds, etc.) with an appended X-tag species (two-piece bifunctional tagged X-molecule species).
  • the steps of Example 5 are illustrated in figure 8 and 9.
  • Step a) Providing the primary library
  • the tagged X-molecule species are prepared by performing two alternating parallel syntheses such that a DNA tag species is being chemically linked to the peptide being synthesised (figure 8A).
  • the chemistry for the implementation of this synthesis has been outlined in several publications such as in Nielsen et al. and WO 93/20242.
  • the library (pbl) is built by the combinatorial synthesis of a hexameric peptides formed from 10 different beta-amino acids, which brings the overall diversity of the library to IO 6 .
  • Each beta-amino acid is encoded by a particular hexacodon.
  • the employed hexacodons are provided as hexameric phosphoramidites, to reduce the number of couplings in the synthesis of the DNA-tag.
  • the hexacodons could also have been formed using six couplings.
  • 10 orthogonal codons are used to encode the corresponding beta-aa.
  • the use of orthogonal codons is preferred to reduce faulty hybridisation. (This is particular important for the rate of hybridisation, as it minimizes the time a given X-tag species uses on sampling Y-tag species, before it makes a productive encounter with a 100% complementary Y-tag species. )
  • a biotin group is added at a final coupling step during synthesis, to generate tagged X-molecule species as outlined below.
  • the biotin group is added as an affinity handle to facilitate later manipulations of selected tagged X-molecule species.
  • a schematic structure of primary Pbl (primary beta-peptide) molecules is shown in Figure 12.
  • the primary library is used at a concentration of 100 ⁇ M in binding buffer.
  • the secondary library (pb2) can by synthesised using redundancies as described in Example 1, i.e. that instead of using mono phosporamidites mixtures, hexacodon phosphoramidite mixtures would be used. However, then the coupling efficiencies of individual hexacodon phosphoramidites will have to be further examined to ensure similar coupling efficiency for different hexacodon phoshoramidites.
  • the secondary library is prepared in a split-mix combinatorial DNA oligonucleotide synthesis using hexameric anticodons as building blocks, such that each X-tag species will have a complementary counterpart (Y-molecule) in the secondary library.
  • Hexacodon anticodons may also be added using six couplings of mono phosphoramidites.
  • Y-molecule species corresponding to the tagged X-molecule species outlined above will be:
  • Two primers are used for PCR amplification, one of which incorporates a biotin-group into the 5'end of the coding strand of the PCR product:
  • PCR-primer 1 5 ' GATGAT AGTAGT TCGTCG TCAC PCR-primer 2: 5' bGCAGCA ACTACT CATCAT GACT
  • the first generation secondary library is used at a concentration of 100 ⁇ M.
  • Step c) Contacting the target molecule with the primary library
  • the primary library is contacted with the solid phase bound target molecule (e.g. Tumour Necrosis factor alfa) immobilized on sepharose, henceforth also denoted the solid phase.
  • the solid phase bound target molecule e.g. Tumour Necrosis factor alfa
  • Six ⁇ l solid phase (20 ⁇ l 30% suspension) is equilibrated in 1000 ⁇ l binding buffer 2 (200 mM KCI, 25 mM Tris-HCI, pH 8, 0.01 % Triton X-100) in an eppendorf tube for 5 minutes at 37°C with mixing, whereafter the sample is centrifuged and the binding buffer disposed. This washing procedure is repeated twice to equilibrate the solid phase for incubation with the library.
  • the primary library (100 ⁇ l) is then added 100 ⁇ l 2xbinding buffer before being incubated with the solid phase at 37°C for 60 minutes with mixing.
  • Step d) Selecting tagged X-molecule species that interact with the solid
  • the solid phase is washed twice as described above with 1000 ⁇ l binding buffer 2 to select tagged X-molecule species interacting with the solid phase.
  • Step e Hybridising selected tagged X-molecule species to the secondary library
  • the secondary library (lOO ⁇ l) is added 1 volume 2xhybridisation buffer, before being added to the solid phase bound target with bound tagged X-molecule species. Next, the sample is heated to 85 °C for 5 minutes, followed by incubation at 65 °C for 12 hours.
  • Step f) Selecting Y-molecule species hybridised to selected tagged X-molecule species
  • composition of the secondary library is analysed by batch sequencing of the double stranded secondary library. By comparison with the first generation secondary library, it can be determined whether sequence pool is still completely random or whether it has evolved as compared to the starting pool (see also Example 1, step j)
  • Step k Identifying molecules of high prevalence
  • Step I) Identifying tagged X-molecule species with an X-tag species corresponding to the high prevalence Y-molecule species
  • Example 6 a library composed of IO 9 beta-peptides is screened for activity. Two primary libraries are employed and the secondary library is provided using the alternative method also described in Example 2. Again, it is important to note that the screening method used in this Example would apply for other tagged X-molecule species as well.
  • the steps of Example 6 are illustrated in Figures 9A-9C. The three figures should be combined so that Figure 9A and 9B runs in parallel and continue in Figure 9C.
  • Step 1) Providing the primary libraries
  • Tagged X-molecule species are prepared as described in Example 5, except that fixed regions for PCR amplification are added in both ends of the X-tag.
  • 32 orthogonal hexameric codons are used for each position, i.e. a total of 192 hexameric codons are employed.
  • Pb2 (primary beta-peptide):
  • Pb3 (primary beta-peptide):
  • Hexameric betapeptide-CAGTAG TAGCCA ACGGCT AGTA AGCTAG TCGGAG CGAAAC GGTTTA GCTATA ACCTCG ATCG TTAGAC GCTATC CGAGTA-3'
  • the primary libraries are used at a concentration of 500 ⁇ M in binding buffer.
  • the following PCR primers are used:
  • PCR-primer 1 5' GATGAT AGTAGT TCGTCG TCAC PCR-primer 2 5'bGCAGCA ACTACT CATCAT GACT
  • Step 3 Selecting tagged X-molecule species that interact with the solid phase.
  • the solid phase is washed twice with 1000 ⁇ l binding buffer to select tagged X-molecule species interacting with the solid phase bound target.
  • Step 4) Amplifying the selected A-tags
  • Second strands are eluted from the solid phase with bound tagged X- molecule species, before serving as templates for PCR amplification; the solid phase is resuspended in 60 ⁇ l 100 mM NaOH and spinfiltered, whereafter the eluate is neutralised by the addition of 60 ⁇ l 100 mM HCI and 15 ⁇ l 900 mM Tris-HCI pH 8.5.
  • 126 ⁇ l is aliquoted into 63 standard PCR reactions each containing: 10 ⁇ l Optibuffer, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCI 2 , 2 ⁇ l 20 ⁇ M upstream PCR-primer, 2 ⁇ l 20 ⁇ M downstream PCR-primer 2, 61 ⁇ l H 2 0 and 1 ⁇ l BIO-X-ACTTM Short DNA polymerase (4 units). The reaction is cycled 10 times with 94°C for 30 sec, 55 °C for 30 sec, 72°C for 90 sec followed by 10 minutes extension at 72 °C.
  • PCR primers 1 and 2 are employed for amplification of the pbl primary library. Because PCR primer 2 is biotinylated in its 5'end, the resulting PCR product is biotinylated at the 5'end of the coding strand. Likewise, for amplification of pb2, PCR primers 3 and 4 are employed which biotinylates the resulting PCR product at the 5' end of the coding
  • Step 5 Providing the secondary library
  • the anti-coding strand of the pbl PCR product is batch eluted by adding 400 ⁇ l 100 mM NaOH to the solid phase followed by centrifugation of the eppendorf tube. After elution, the streptavidin sepharose containing the pbl coding strand is washed twice with 1000 ⁇ l hybridization buffer.
  • the anti-coding strand of the pb2 PCR product is eluted with 400 ⁇ l 100 mM NaOH using spinfiltration. The eluate is subsequently neutralized, whereafter the ssDNA is ethanol precipitated and redissolved in 400 ⁇ l binding buffer.
  • streptavidin sepharose is washed two times with 1000 ⁇ l lxhybridisation buffer followed by one wash with wash-buffer (lxSSC+0.01% Triton X- 100) buffer for 5 minutes at 65°C to select hybridised pb2 anti-coding strands (Y-molecule species)
  • the secondary library can be increasingly diluted, because is evolves to contain a larger fraction of Y-molecule species corresponding to active tagged X-molecule species, i.e. if the secondary library is 10000 fold enriched in Y- molecule species corresponding to active tagged X-molecule species, a 10.000 fold shortage in total amount of the secondary library can be used for hybridisation.
  • the amount of secondary library can also be adjusted to have Y-molecule species corresponding to active tagged X-molecule species in moderate excess (5-50 fold) over active tagged X-molecule species for the hybridisation reaction. Further, the number of cycles in the PCR reactions can be adjusted in later rounds and carrier nucleic acids may be employed.
  • Step 7) Monitoring the evolution of the secondary library
  • composition of the secondary library is analysed by batch sequencing of the double stranded secondary library. By comparison with the first generation secondary library, it can be determined whether sequence pool is still completely random or whether it has evolved as compared to the starting pool.
  • Step 8) Identifying molecules of high prevalence
  • Step 9) Identifying tagged X-molecule species with an X-tag species corresponding to the high prevalence Y-molecule species
  • Step 2 Contacting the primary libraries with the target molecule
  • Step 8) Identifying molecules of high prevalence
  • PCR-primer 1 5' GATGAT AGTAGT TCGTCG TCAC
  • PCR-primer 2 5'bGCAGCA ACTACT CATCAT GACT PCR primer-3 5' TACTCG GATAGC GTCTAA CGAT PCR primer-4 5'bCAGTAG TAGCCA ACGGCT AGTA
  • PCR primer-8 5' CAGTAG TAGCCA ACGGCT AGTA
  • the second nucleotide from the 3' end in PCR primer 8 is a ribonucleotide (in bold type).
  • Second-strand synthesis is performed as described in Example 2.
  • Step 2) Contacting the target molecule with the primary library
  • Step 3 Selecting tagged X-molecule species that interact with the solid phase
  • Step 4) Amplifying the selected A-tags
  • 126 ⁇ l is aliquoted into 63 standard PCR reactions each containing: 10 ⁇ l Optibuffer, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCI 2 , 2 ⁇ l 20 ⁇ M upstream PCR-primer, 2 ⁇ l 20 ⁇ M downstream PCR-primer 2, 61 ⁇ l H 2 0 and 1 ⁇ l BIO-X-ACTTM Short DNA polymerase (4 units). The reaction is cycled 10 times with 94°C for 30 sec, 55 °C for 30 sec, 72 °C for 90 sec followed by 10 minutes extension at 72 °C.
  • the pbl primary library is again selected against the solid phase and selected X-tags PCR amplified.
  • the anti-coding strand from the resulting PCR product is hydrolysed with NaOH and the coding strand purified from PAGE.
  • Purified pbl coding strands are hybridized to complementary anti-coding Y-molecule species of the first generation secondary library in solution, whereafter hybridised Y-molecule species (pb2 strands) are selected on streptavidin sepharose.
  • hybridised Y-molecule species pb2 strands
  • selected Y-molecule species are PCR amplified to generate the second-generation secondary library.
  • the secondary library can be increasingly diluted, because it evolves to contain a larger fraction of Y-molecule species corresponding to active tagged X-molecule species, i.e. if the secondary library is 10000 fold enriched in Y- molecule species corresponding to active tagged X-molecule species, a 10.000 fold shortage in total amount of the secondary library can be used for hybridisation.
  • the amount of secondary library can also be adjusted to have Y-molecule species corresponding to active tagged X-molecule species in moderate excess (5 -50 fold) over active tagged X-molecule species for the hybridisation reaction. Further, the number of cycles in the PCR reactions can be adjusted in later rounds and carrier nucleic acids may be employed.
  • Step 8) Identifying molecules of high prevalence See Example 1, step k.
  • the two libraries were prepared as outlined in Example 1 and screened in parallel for binding against a solid phase bound target, in this case streptavidin sepharose.
  • a solid phase bound target in this case streptavidin sepharose.
  • X-molecule was designed with a photocleavable linker between X-tag and X-molecules.
  • the active oligonucleotide containing a 5'biotin, PS-BamHI to be present in the primary library was synthesised separately with the following sequence PS-BamHI 5'pbCGCTAT GTTGAC TAGCAG GGATCC ATTCTG ATCGCT
  • PS-BamHI was diluted into PL-10e5 to create the IO 5 library.
  • the underlined sequence indicates a BamHI restriction site used to monitor evolution of the secondary library.
  • Step b) Providing the secondary library
  • the secondary library oligonucleotides For each coding DNA oligonucleotide in the primary libraries (tagged X-molecule species), there is a complementary anti-coding DNA oligonucleotide in the secondary libraries (Y- molecule species). Additionally, the secondary library oligonucleotides have fixed regions in both ends to enable PCR amplification.
  • SL-10e5 were diluted into SS-BamHI to create the IO 6 secondary library.
  • SL-10e6 were diluted into SS-NcoI to create the 10 secondary library.
  • sequences in bold are the anti-coding sequences and the flanking sequences are fixed regions for PCR amplification. Again restriction sites are underlined.
  • PCR-12 GCCTGTTGTGAG CCTCCT GTCGAA
  • PCR-11 bGGGAG ACAAGA ATAACC TCAGC
  • Step c-1) Hybridising Y-molecule species of the secondary library with X-tag species of the primary library
  • Negative control omitting signal in secondary library otherwise as A: 27 ⁇ l 20x SSC 5.4 ⁇ l 0.15% Triton X-100 5 25 ⁇ l 200 ⁇ M PL-10e5 (MWG, 180304)
  • Step d-1) Contacting the target molecule with at least a subset of the primary library hybridised to the secondary library
  • Performance beads in 20% EtOH, Amersham, 17-5113-01 was centrifuged to pellet the solid phase.
  • the supernatant was disposed and 600 ⁇ l 6xSSC, 0.01% Triton X-100 added. After resuspension of the solid phase, it was again pelleted by centrifugation and the supernatant disposed.
  • the solid phase was then resuspended in 600 ⁇ l 6xSSC, 2 ⁇ g/ ⁇ l tRNA (170 ⁇ l 7 ⁇ g/ ⁇ l tRNA (tRNA from Roche, 109 541, phenol extracted;)+ 180 ⁇ l 20x SSC + 250 ⁇ l H 2 0), centrifugated and the supernatant disposed.
  • Step e-1) Selecting the tagged X-molecule species of the primary library that interact 10 specifically with the target molecule, thereby also selecting Y-tags hybridised to selected X-tags
  • Step f-l amplifying the selected Y-molecule species, the product of the amplification process being a secondary library
  • Amplification was performed according to the following program: Initial denaturation: 94 °C, 5 min. 10 30 cycles: 94°C, 30 sec
  • Step j) Monitoring the evolution of the secondary library
  • Step h) Preparation of the next generation secondary library Only the anticoding strand of the PCR product from above is desired and was therefore purified. 100 ⁇ l 30% Streptavidin Sepharose High Performance beads in 20% EtOH were centrifuged, the supernatant disposed and 600 ⁇ i 6x SSC, 0.01% Triton X-100 added. After resuspension of the streptavidin sepharose, it was again pelleted by centrifugation and the supernatant disposed. The streptavidin sepharose was then resuspended in 70 ⁇ l 6x SSC, 0.01% Triton X-100 to give a total volume of app. 100 ⁇ l.
  • sample A-D from step g were added 200 ⁇ l 20x SSC + 20 ⁇ l of the above equilibrated streptavidin sepharose.
  • samples A-D were incubated at RT for 20 minutes with mixing. Then the samples were transferred to spin-off filters (2x 370 ⁇ l) and centrifuged at 3000 rpm for 2x 1 minute.
  • samples were added 300 ⁇ l lOxwash buffer + 0.01% Triton X-100 and centrifuged at 3000 rpm for 2x 1 minute.
  • second wash the samples were added 300 ⁇ l lxwash buffer + 0.01% Triton X-100 and centrifuged at 3000 rpm for 2x 1 minute.
  • samples A-D were resuspended in 40 ⁇ l 100 mM NaOH by pipetting up and down a few times and then incubated at RT for 5 minutes. The anticoding strands were then collected by centrifugation at 13000 rpm for 1 minute. 40 ⁇ l of the eluted samples were neutralised by adding 40 ⁇ l 100 mM HCI + 18 ⁇ l 1 M Tris pH 8 + 2 ⁇ l 0.5% Triton X-100. Next, the samples were desalted by gel-filtration on G25 columns (MicroSpin G-25 columns, Amersham, 27-5325-01).
  • A-2 and A-3 were as A-l, except that 10-fold and 100-fold diluted Sample A was used.
  • 300 ⁇ l solid phase bound target suspension (30% Streptavidin Sepharose High Performance beads) was centrifuged to pellet the solid phase. The supernatant was disposed and 1800 ⁇ l 6xSSC, 0.01% Triton X-100 added. After resuspension of the solid phase, it was again pelleted by centrifugation and the supernatant disposed. The solid phase was resuspended in 1800 ⁇ l 6xSSC + 2 ⁇ g/ ⁇ l tRNA and after centrifugation the supernatant disposed. Finally, the solid phase was resuspended in 210 ⁇ l 6xSSC, 0.01% Triton X-100 to give a total volume of app. 300 ⁇ l.
  • Step e-1) Selecting the tagged X-molecule species of the primary library that interact specifically with the target molecule, thereby also selecting Y-tags hybridised to selected X-tags
  • samples were transferred to spin-off filters and centrifuged at 3000 rpm for 2x 1 minute.
  • samples were added 300 ⁇ l lOxwash buffer (1 M NaCl, 100 mM Tris-HCI pH 8) + 0.01% Triton X-100 and centrifuged at 3000 rpm for 2x 1 minute.
  • samples were added 300 ⁇ l lxwash buffer + 0.01% Triton X-100 and centrifuged at 3000 rpm for 2x 1 minute.
  • Step f-l amplifying the selected Y-molecule species, the product of the amplification process being a secondary library
  • the solid phase from above might be used directly in PCR, but to enhance selection of hybridised Y-molecules, X-molecules with hybridised Y-tags were photocleaved of the solid phase: The solid phase were resuspended in 100 ⁇ l lxwash buffer, 0.01% Triton X-100 and placed on the UV table for 3 minutes. The released complexes (Y-tags hybridised to X- molecules) were collected by centrifugation at 3000 rpm for 2x 1 minute.
  • Amplification was performed according to the following program: Initial denaturation: 94 °C, 5 min. 30 cycles: 94°C, 30 sec
  • Step j) Monitoring the evolution of the secondary library
  • samples with 1 ⁇ l H 2 0 instead of restriction enzyme were also prepared. All were incubated at 37 °C for 2 hours and then added 3 ⁇ l 30% glycerol and resolved on a 4% GTG agarose gel.
  • Figure 15 shows +/- restriction enzyme of sample A-l to B-2 and 25 bp DNA ladder (2.5 ⁇ l).
  • Figure 16 shows +/- restriction enzyme of sample B-3, neg. PCR Control (BamHI), C-1 to C-3 and 25 bp DNA ladder (2.5 ⁇ l).
  • Figure 17 shows +/- restriction enzyme of sample D-1 to D-3, neg. PCR Control (Ncol) and 25 bp DNA ladder (2.5 ⁇ l).
  • sample Cl Approximately 5% of sample Cl and about 20% of sample C-2 and C-3 could be restricted by Ncol. This means that the secondary library had evolved from containing 1 SS-NcoI oligonucleotides per 3.000.000 library oligonucleotides into containing between 5 and 20 SS-NcoI oligonucleotides per 100 library oligonucleotides. This reflects an enrichment of app. 130.000 (for Cl) and 520.000 fold (for C2 and C3). Importantly, no restriction was seen in any of the controls.
  • Lam et al. Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ.
  • Lam et al. Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmierski WM, Knapp RJ.
  • a new type of synthetic peptide library for identifying ligand-binding activity Nature 1991 Nov 7;354(6348) :82-4

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (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)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Ecology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention a trait à un procédé pour le criblage de bibliothèques de molécules présentant une interaction spécifique, telle qu'une activité de liaison ou une activité catalytique, avec une molécule cible. Le procédé met en oeuvre une bibliothèque primaire, comportant les molécules candidates de la bibliothèque marquées avec des étiquettes d'acides nucléiques et une bibliothèque secondaire, utilisée pour l'amplification et l'identification des étiquettes d'acides nucléiques des molécules dans la bibliothèque primaire. Le procédé peut, par exemple, être utilisé pour l'identification de nouveaux produits pharmaceutiques, dans lequel cas la molécule cible pourrait être une molécule réceptrice.
PCT/DK2004/000325 2003-05-09 2004-05-06 Selection et developpement de bibliotheques chimiques WO2004099441A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP04731316A EP1625230A2 (fr) 2003-05-09 2004-05-06 Selection et developpement de bibliotheques chimiques

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200300711 2003-05-09
DKPA200300711 2003-05-09

Publications (2)

Publication Number Publication Date
WO2004099441A2 true WO2004099441A2 (fr) 2004-11-18
WO2004099441A3 WO2004099441A3 (fr) 2005-02-03

Family

ID=33426924

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2004/000325 WO2004099441A2 (fr) 2003-05-09 2004-05-06 Selection et developpement de bibliotheques chimiques

Country Status (2)

Country Link
EP (1) EP1625230A2 (fr)
WO (1) WO2004099441A2 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7413854B2 (en) 2002-03-15 2008-08-19 Nuevolution A/S Method for synthesising templated molecules
WO2010043418A2 (fr) * 2008-10-17 2010-04-22 Febit Holding Gmbh Amplification intégrée, traitement et analyse de biomolécules dans un support de réaction microfluidique
US7704925B2 (en) 2004-03-22 2010-04-27 Nuevolution A/S Ligational encoding using building block oligonucleotides
US7727713B2 (en) 2001-06-20 2010-06-01 Nuevolution A/S Templated molecules and methods for using such molecules
US7915201B2 (en) 2003-03-20 2011-03-29 Nuevolution A/S Ligational encoding of small molecules
US7935658B2 (en) 2003-12-17 2011-05-03 Praecis Pharmaceuticals, Inc. Methods for synthesis of encoded libraries
EP2336315A2 (fr) 2005-12-01 2011-06-22 Nuevolution A/S Procédés de codage enzymatique destinés à la synthèse efficace de bibliothèques importantes
US7972994B2 (en) 2003-12-17 2011-07-05 Glaxosmithkline Llc Methods for synthesis of encoded libraries
US7989395B2 (en) 2005-10-28 2011-08-02 Glaxosmithkline Llc Methods for identifying compounds of interest using encoded libraries
WO2011127933A1 (fr) 2010-04-16 2011-10-20 Nuevolution A/S Complexes bifonctionnels et procédés de fabrication et d'utilisation de tels complexes
US8722583B2 (en) 2002-10-30 2014-05-13 Nuevolution A/S Method for selecting a chemical entity from a tagged library
US8791053B2 (en) 2002-09-27 2014-07-29 Mpm-Holding Aps Spatially encoded polymer matrix
WO2016080838A1 (fr) * 2014-11-21 2016-05-26 Piculet Biosciences Technologies B.V. Bibliothèques de complexes de ligand bivalent auto-assemblés (sablc) et procédés permettant de cribler lesdites bibliothèques
US10730906B2 (en) 2002-08-01 2020-08-04 Nuevolutions A/S Multi-step synthesis of templated molecules
CN112562789A (zh) * 2019-09-24 2021-03-26 成都先导药物开发股份有限公司 一种确定dna编码化合物库筛选中特定序列与生物靶标结合的方法
US11104942B2 (en) * 2015-10-30 2021-08-31 Guangdong Maijinjia Biotechnologies, Co. Ltd. Method for identification of the most abundant oligonucleotide species in a library of oligonucleotides
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
WO2022081934A1 (fr) * 2020-10-15 2022-04-21 Genentech, Inc. Analyse de criblage de candidats de liaison à une cible multiplexée

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056994A2 (fr) 2002-12-19 2004-07-08 Nuevolution A/S Procedes de synthese guidee a fonction et structure quasi-selectives
WO2004074429A2 (fr) 2003-02-21 2004-09-02 Nuevolution A/S Procede de production d'une banque de deuxieme generation
CN102796730A (zh) * 2012-07-18 2012-11-28 海南广陵高科实业有限公司 一种从聚丙烯酰胺凝胶中回收核酸分子的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020242A1 (fr) * 1992-03-30 1993-10-14 The Scripps Research Institute Bibliotheques chimiques combinatoires codees
WO1994013623A1 (fr) * 1992-12-11 1994-06-23 Chiron Corporation Synthese de polymeres codes
WO1999045149A1 (fr) * 1998-03-04 1999-09-10 Lifespan Biosciences, Inc. greffes thermiques de polyamides
WO2000023458A1 (fr) * 1998-10-19 2000-04-27 The Board Of Trustees Of The Leland Stanford Junior University Bibliotheque de chimie combinatoire a matrice d'adn

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020242A1 (fr) * 1992-03-30 1993-10-14 The Scripps Research Institute Bibliotheques chimiques combinatoires codees
WO1994013623A1 (fr) * 1992-12-11 1994-06-23 Chiron Corporation Synthese de polymeres codes
WO1999045149A1 (fr) * 1998-03-04 1999-09-10 Lifespan Biosciences, Inc. greffes thermiques de polyamides
WO2000023458A1 (fr) * 1998-10-19 2000-04-27 The Board Of Trustees Of The Leland Stanford Junior University Bibliotheque de chimie combinatoire a matrice d'adn

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JEONG SUNJOO ET AL: "In vitro selection of the RNA aptamer against the sialyl Lewis X and its inhibition of the cell adhesion" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 281, no. 1, 16 February 2001 (2001-02-16), pages 237-243, XP002309129 ISSN: 0006-291X *
NANTHAKUMAR A ET AL: "Solid-phase oligonucleotide synthesis and flow cytometric Analysis with Microspheres Encoded with Covalently Attached Fluorophores" BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, WASHINGTON, US, vol. 11, 2000, pages 282-288, XP002159465 ISSN: 1043-1802 *
NEEDELS M C ET AL: "GENERATION AND SCREENING OF AN OLIGONUCLEOTIDE-ENCODED SYNTHETIC PEPTIDE LIBRARY" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 90, 1 November 1993 (1993-11-01), pages 10700-10704, XP000652260 ISSN: 0027-8424 *

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7727713B2 (en) 2001-06-20 2010-06-01 Nuevolution A/S Templated molecules and methods for using such molecules
US10669538B2 (en) 2001-06-20 2020-06-02 Nuevolution A/S Templated molecules and methods for using such molecules
US7413854B2 (en) 2002-03-15 2008-08-19 Nuevolution A/S Method for synthesising templated 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
US8791053B2 (en) 2002-09-27 2014-07-29 Mpm-Holding Aps Spatially encoded polymer matrix
US10077440B2 (en) 2002-10-30 2018-09-18 Nuevolution A/S Method for the synthesis of a bifunctional complex
US8722583B2 (en) 2002-10-30 2014-05-13 Nuevolution A/S Method for selecting a chemical entity from a tagged library
US11001835B2 (en) 2002-10-30 2021-05-11 Nuevolution A/S Method for the synthesis of a bifunctional complex
US7915201B2 (en) 2003-03-20 2011-03-29 Nuevolution A/S Ligational encoding of small molecules
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
US7935658B2 (en) 2003-12-17 2011-05-03 Praecis Pharmaceuticals, Inc. Methods for synthesis of encoded libraries
US7972992B2 (en) 2003-12-17 2011-07-05 Praecis Pharmaceuticals, Inc. Methods for synthesis of encoded libraries
US7972994B2 (en) 2003-12-17 2011-07-05 Glaxosmithkline Llc Methods for synthesis of encoded libraries
US8410028B2 (en) 2003-12-17 2013-04-02 Glaxosmithkline Llc Methods for synthesis of encoded libraries
US7704925B2 (en) 2004-03-22 2010-04-27 Nuevolution A/S Ligational encoding using building block oligonucleotides
US7989395B2 (en) 2005-10-28 2011-08-02 Glaxosmithkline Llc Methods for identifying compounds of interest using encoded libraries
EP3305900A1 (fr) 2005-12-01 2018-04-11 Nuevolution A/S Procédés de codage enzymatique destinés à la synthèse efficace de bibliothèques importantes
EP2341140A1 (fr) 2005-12-01 2011-07-06 Nuevolution A/S Procédés de codage enzymatique destinés à la synthèse efficace de bibliothèques importantes
EP2336315A2 (fr) 2005-12-01 2011-06-22 Nuevolution A/S Procédés de codage enzymatique destinés à la synthèse efficace de bibliothèques importantes
US11702652B2 (en) 2005-12-01 2023-07-18 Nuevolution A/S Enzymatic encoding methods for efficient synthesis of large libraries
WO2010043418A3 (fr) * 2008-10-17 2010-07-08 Febit Holding Gmbh Amplification intégrée, traitement et analyse de biomolécules dans un support de réaction microfluidique
WO2010043418A2 (fr) * 2008-10-17 2010-04-22 Febit Holding Gmbh Amplification intégrée, traitement et analyse de biomolécules dans un support de réaction microfluidique
WO2011127933A1 (fr) 2010-04-16 2011-10-20 Nuevolution A/S Complexes bifonctionnels et procédés de fabrication et d'utilisation de tels complexes
EP3540059A1 (fr) 2010-04-16 2019-09-18 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
WO2016080838A1 (fr) * 2014-11-21 2016-05-26 Piculet Biosciences Technologies B.V. Bibliothèques de complexes de ligand bivalent auto-assemblés (sablc) et procédés permettant de cribler lesdites bibliothèques
NL2013857B1 (en) * 2014-11-21 2016-10-11 Piculet Biosciences Tech B V Self-assembled bivalent ligand complex (SABLC) libraries and methods for screening such libraries.
US11104942B2 (en) * 2015-10-30 2021-08-31 Guangdong Maijinjia Biotechnologies, Co. Ltd. Method for identification of the most abundant oligonucleotide species in a library of oligonucleotides
CN112562789A (zh) * 2019-09-24 2021-03-26 成都先导药物开发股份有限公司 一种确定dna编码化合物库筛选中特定序列与生物靶标结合的方法
WO2022081934A1 (fr) * 2020-10-15 2022-04-21 Genentech, Inc. Analyse de criblage de candidats de liaison à une cible multiplexée
US11834709B2 (en) 2020-10-15 2023-12-05 Genentech, Inc. Multiplexed target-binding candidate screening analysis

Also Published As

Publication number Publication date
EP1625230A2 (fr) 2006-02-15
WO2004099441A3 (fr) 2005-02-03

Similar Documents

Publication Publication Date Title
WO2004099441A2 (fr) Selection et developpement de bibliotheques chimiques
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
Lee et al. Advances in CLIP technologies for studies of protein-RNA interactions
US7883848B2 (en) Regulation analysis by cis reactivity, RACR
EP1477561B1 (fr) Molécule permettant d'homologuer un génotype et un phénotype, et utilisation de celle-ci
US7544793B2 (en) Making nucleic acid sequences in parallel and use
US20110263459A1 (en) Direct selection of structurally defined aptamers
AU2775402A (en) Nucleic acid ligands to complex targets
AU2008276001A1 (en) Improved selex and photoselex
JP2022516821A (ja) 複合体が表面結合されたトランスポソーム複合体
JP2008253176A (ja) 高親和性分子取得のためのリンカー
WO2014094620A1 (fr) Méthode de synthèse et de criblage d'un composé 'tête de série' et kit de réactif pour analyse
WO2014094621A1 (fr) Kit et méthode de synthèse et de criblage d'un composé 'tête de série'
WO2017072330A2 (fr) Procédés pour l'identification d'oligonucléotides
NL2013857B1 (en) Self-assembled bivalent ligand complex (SABLC) libraries and methods for screening such libraries.
US10132798B2 (en) Multiplexed microcolumn devices and processes for selection of nucleic acid aptamers
US8883411B2 (en) Making nucleic acid sequences in parallel and use
WO2024036284A1 (fr) Génération et sélection de réactifs d'affinité
EP4204566A1 (fr) Nouveaux aptamères x destinés à être utilisés dans la détection de snap25
WO2022155282A1 (fr) Procédés de production et de quantification de billes marquées par un identifiant moléculaire unique
CN116875668A (zh) 一种捕获特定rna结合蛋白介导的rna-rna相互作用的方法
WO2003027286A1 (fr) Procede de synthese de polynucleotide par traduction inverse a partir d'une proteine, et oligonucleotide utilise dans ce procede

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2004731316

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2004731316

Country of ref document: EP