EP1625230A2 - Selection and evolution of chemical libraries - Google Patents
Selection and evolution of chemical librariesInfo
- Publication number
- EP1625230A2 EP1625230A2 EP04731316A EP04731316A EP1625230A2 EP 1625230 A2 EP1625230 A2 EP 1625230A2 EP 04731316 A EP04731316 A EP 04731316A EP 04731316 A EP04731316 A EP 04731316A EP 1625230 A2 EP1625230 A2 EP 1625230A2
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- molecule
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- tagged
- library
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- 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
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- 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
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- 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
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- 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
- the present invention relates to a method for screening libraries of molecules showing specific interaction, such as binding activity or catalytic activity, with a target molecule.
- the method makes use of a primary library, which comprises the candidate molecules of the library marked with nucleic acid tags and' a secondary library, which is used for amplifying and identifying the nucleic acid tags of the molecules in the primary library.
- 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.
- the method comprises the steps of
- 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 the 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),
- the method may be used for screening potential drug candidates for binding activity against a certain receptor.
- the target molecule could be the receptor e.g. immobilised to a solid phase
- the tagged X-molecule species could be IO 9 peptides, each peptide carrying a specific DNA tag species
- the Y- molecule species could comprise DNA tag species complementary to the DNA tag species of the peptide and further carry one or more fixed regions which may be used as binding sites for PCR primers in step g) as mentioned above.
- the specific interaction between tagged X-molecule species and target molecules might in this case be binding.
- 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. 2A-2D illustrate schematically embodiments of a tagged X-molecule species
- 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):
- each X-tag species of at least 50% of the X-tag species of the primary library such as at least 60%, 70%, 80%, 90%, 95% or
- each X-tag species of at least 95% of the X-tag species of the primary library may be capable of hybridising to at most 5 different Y-tag species.
- the Y-tag of a Y-molecule species may hybridise to only one tagged X-molecule species of the primary library.
- the Y-tag of a Y-molecule species may be able to hybridise to at 35 least 2 different tagged X-molecule species, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000 or 10.000 such as at least 100.000 different tagged X-molecule species.
- 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 Y-tag of a Y-molecule species may be able to hybridise to at most 1000 molecules of a tagged X-molecule species at a time, such as at most 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 such as at most 1 molecule 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).
- the number of repetitions in step h), may be at least 1 times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 30 times or such as at least 40 times.
- the number of repetitions may be from 1-100 repetitions, such as 1-3 repetitions, 3-5 repetitions, 5-10 repetitions, 10-15 repetitions, or 15-25 repetitions, such as 25-100 repetitions.
- Step i) is optional, thus in an embodiment of the present invention the Y-molecule species of high prevalence are not directly identified. Alternatively, step i) is performed and the Y- molecule species of high prevalence are identified in a generation of the secondary library.
- 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).
- the primary library provided in step b) may be substantially identical in every repetition, e.g. the primary library provided may be a sample from a larger primary library stock solution or the primary library may be prepared following the same recipe in every repetition.
- Two primary libraries are considered “substantially identical" if the relative standard deviation, between the two libraries, of the weight percentage of each tagged X- molecule species is at most 10%, such as at most 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% such as at most 0.01%.
- the primary library provided in step b) may be different from the initial primary library in at least one of the repetitions, such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 of the repetitions.
- 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.
- At least one molecule of a tagged X-molecule species should be present in the 25 primary library.
- the concentration of a tagged X-molecule species may be at least IO "22 M such as at least IO "21 M, IO "19 M, IO “18 M, IO "17 M, IO “16 M, IO “15 M, IO “14 M, IO "13 M, IO "12 M, IO “11 M, 10 " ⁇ o M, IO "9 M, IO "8 M, IO "7 M, IO "6 M, IO "5 M, IO “4 M, such as at least IO "3 M.
- the concentration of a tagged X-molecule species in the primary library may be in the range of IO "22 M - IO "20 M, IO "20 M - IO "18 M, IO "18 M - IO “16 M, IO “16 M - IO “14 M, 30 IO "14 M - IO "12 M, IO "12 M - IO “10 M, IO “10 M - IO “8 M, IO "6 M - IO "4 M, or IO "4 M - IO "3 M.
- the concentration of a tagged X-molecule species in the primary library may be at most 100 mM such as at most IO "2 M, IO "3 M, IO “4 M, IO "5 M, IO “6 M, IO “7 M, IO “8 M, IO "9 M, IO "10 M, IO “11 M, IO “12 M, IO “13 M, IO “14 M, IO “15 M, IO "16 M, IO "17 M, IO "18 M, IO “18 M, IO “19 M, 10 " 35 20 M, IO "21 M, such as at most IO "22 M.
- the primary library may be on liquid form and may comprise an aqueous solvent.
- the primary library may also comprise an organic solvent and it may comprise both an organic and an aqueous phase at the same time.
- the weight percentage of water in the primary library is at least 50%, such as at least 60, 70, 80, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% such as at least 99.9%
- the primary library may also be attached to a solid phase such as particle or a microsphere.
- the particle or the microsphere may comprise a material selected from the group consisting of an organic polymer, a metal, a metal oxide, an alloy, a magnetic materiel, and a combination of these materials.
- the metal oxide may be a silicon oxide such as quartz or glass.
- the organic polymer can be selected from the group consisting of polyethylene glycol-polyacrylamide,, poly styrene, poly vinyl chloride, poly vinyl alcohol, polypeptides, poly ethylene, poly propylene and poly methamethacrylate and a combination of these materials.
- the particle or microsphere may comprise a composite material having one or more segments with a material as described above.
- the primary library may further comprise an additive selected from the group consisting of a detergent, such as Tween 20, NP 40, octylphenolpoly(ethyleneglycolether)
- a detergent such as Tween 20, NP 40, octylphenolpoly(ethyleneglycolether)
- Triton X-100 CHAPS, CHAPSO, sodium dodecylsulfate (SDS); a preservative, such as sodium azide ; a pH buffer such as a phosphate buffer, Tris, Mops or a HEPES buffer; a salt such as MgCl , NaCl, KCI, Na-glutamate or K-glutamate ; a water soluble polymer such as polyethylene glycol (PEG) or polyvinyl alchohol (PVA). Examples of other suitable additives may be found in Sambrook et al or other general text books known to the person skilled in the art.
- the primary library may be a microarray and the individual spots of the array may be the different tagged X-molecule species.
- the secondary library comprises a plurality of Y-molecule species, said Y-molecule species comprising a specific tag species (Y-tag species).
- the secondary library may comprise at least 10 1 Y-molecule species, such as at least IO 3 , IO 4 , 10 5 , IO 6 , IO 7 , 10 s , IO 9 , 10 10 , 10 11 , IO 12 , IO 13 , IO 14 , such as at least 10 15 Y-molecule species.
- the secondary library may comprise 10 3 -10 18 Y-molecule species, 10 3 -10 6 Y-molecule species, 10 6 -10 9 Y-molecule species, 10 9 -10 12 Y-molecule species, 10 12 -10 15 Y-molecule species or 10 15 -10 18 Y-molecule species.
- the secondary library is enriched for Y-molecule species that correspond to tagged X- molecule species that are capable of interacting specifically with the target molecule
- the Y-molecule species corresponding to tagged X-molecule species that do not interact with the target molecule are diluted in the secondary library.
- the concentration of a Y-molecule species in the secondary library may be at least IO "23 M, such as at least IO "21 M, IO “20 M, IO “19 M, 10 "18 M, IO “17 M, 10 "16 M, IO “15 M, IO “14 M, IO "13 M, IO "12 M, IO “11 M, IO “10 M, IO “9 M, IO “8 M, IO "7 M, IO "6 M, IO “5 M, IO “4 M, such as at least IO "3 M.
- the concentration of a Y-molecule species may be at most 100 mM such as at most IO "2 M, IO "3 M,10 “4 M, IO “5 M, IO “6 M, IO “7 M, IO “8 M, IO “9 M, IO "10 M, IO “11 M, IO “12 M, IO “13 M, IO “14 M, IO “15 M, IO "16 M, IO "17 M, IO "18 M, IO “18 M, IO “19 M, IO “20 M, IO “21 M, such as at most IO "22 M.
- the concentration of a Y-molecule species in the primary library may be in the range of IO "22 M - IO "20 M, IO "20 M - IO "18 M, IO "18 M - IO “16 M, IO "16 M - IO “14 M, IO "14 M - IO "12 M, IO "12 M - IO “10 M, IO “10 M - IO “8 M, IO "6 M - IO "4 M, or in the range of IO "4 M - IO "3 M.
- the secondary library of step a) is derived from X-tag species of selected tagged X-molecule species of a previous step d).
- the term "derive" should be interpreted broadly as providing a secondary library with the same or similar information contents as the starting material, said starting material may e.g. be the X-tags of the selected tagged X-molecule species or the amplified Y-molecule species of step g).
- the information contents means the ratio or percentage of the concentration or weight of each Y-molecule species relative to the total concentration or total weight of the Y-molecule species. This may be exemplified by considering a mixture of amplified Y-molecule species comprising the three Y-molecule species Yl, Y2 and Y3 having concentrations of 2 nM, 47 nM and 1 nM, respectively.
- the information content of the mixture of amplified Y-molecule species Yl, Y2, and Y3 would thus be 2:47: 1 or if expressed as percentages: 4% of Yl, 94% of Y2 and 2% of Y3.
- the percentage of a Y molecule species in the derived secondary library is in the range of 50%-150% of percentage in the starting material, such as 60%-140%, 70%-130%, 80%-120%, 90%-110%, 95%-105%, 97%-103%, 98%-102%, 99%-101%%, 99.5%-100.5%, or 99.9%-100.1% such as in the range of 99.99%-100.01%.
- the percentage of Y2 should be in the range of 95%- 105% of the percentage in the starting material, this means that the percentage of Y2 should be in the range of 89.3% (94%*0,95) and 98.7% (94%*1,05).
- the molar percentage a Y-molecule species is within 50%-150% of the molar percentage of the Y-molecule species in the starting material.
- Deriving may also mean providing a secondary library with the same or similar information contents as the 50% Y-molecule species or X-tags of the selected tagged X-molecule species of highest concentration and/or weight% in the starting material, such as the top 40%, 30%, 20%, 10%, 5%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001% or 0.00001%, such as the top 0.000001% of the Y-molecule species of highest concentration and/or weight% in the starting material.
- a next generation secondary library is derived from the starting material by providing a secondary library which has an information content similar the 0.001% Y-molecule species of highest concentration in the starting material, the starting material being the amplification product of step f).
- Deriving may also mean providing a secondary library with the same or similar information contents as at most the 1,000,000 Y-molecule species or X-tags of the selected tagged X- molecule species of highest concentration and/or weight% in the starting material, such as at most the 100,000, 10,000, 1000, 500, 250, 100, 50, 30, 20, 15, or 10, 5, 4, 3, or 2, such as the one Y-molecule species or X-tags of the selected tagged X-molecule species of highest concentration and/or weight% in the starting material.
- 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).
- Deriving may comprise processes such as 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. Also, deriving may comprise analysing the contents of the starting material and e.g. synthesising or mixing a library with the same or similar composition.
- the result of the monitoring of the amplification product of step g) may be used for calculating or estimating the optimal dilution of the amplification product to yield the next generation secondary library.
- the secondary library may be derived e.g. using a process where X-tags, either from a non-selected or selected primary library are PCR amplified, whereafter anti-coding strands of the resulting PCR-product is purified and used as a secondary library.
- coding strand or "coding part” should be interpreted as the tag species of an X-tag species.
- anti-coding strand or “anti-coding part” is a tag species that is either complementary to the coding tag species or complementary to a tag species which is a homologue of the coding tag species.
- the X-tags of the first primary library are defined as the coding strands and X-tags of the second primary library are defined as anti-coding strands.
- the secondary library of step a) may for example be provided by a method comprising the following steps
- A-tag species amplifiable tag species
- the tagged X-molecule species are characterised by being divided into two sub- libraries of tagged Xi-molecule species and tagged X 2 -molecule species, wherein the amplifiable tag species (A ⁇ of the Xi-molecule species may be different from the amplifiable tag species (A 2 ) of the X 2 -molecule species
- step 10) selecting the anti-coding A 2 -tag species of step 9) that hybridise to selected coding Ai-tag species, and
- step 11 using the selected anti-coding A 2 -tag species of step 10) as secondary library.
- the only difference between a tagged Xi-molecule species and the corresponding tagged X 2 -molecule species is the sequence of the primer binding site; the X-molecule species of the two species are preferably identical.
- the X-tags may be complementary to the X 2 -tags which could be used to prevent identification of tagged X-molecule species having X-tags with an unwanted binding activity, i.e. X-tags that, either alone or in combination with X-molecules, have affinity for the target and/or the solid phase.
- steps 8)-ll) could be performed by
- step 10) selecting the anti-coding Aj-tag species of step 9) that hybridise to selected coding A 2 -tag species, and
- step 11 using the selected anti-coding A ⁇ tag species of step 10) as secondary library.
- Step 11) of the method for providing a secondary library may furthermore comprise at least one step selected from the groups of steps consisting of
- 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 amplification product is only the amplified tag species and their complementary parts and not side products of the amplification process such as primer- dimers.
- the tagged X-molecule species comprises an X-tag species linked to an X-molecule species, said X-tag species comprising a tag species as defined herein.
- X-tag species linked to an X-molecule species, said X-tag species comprising a tag species as defined herein.
- 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.
- the X-molecule species (2) of the tagged X-molecule species (1) is a molecule such as a protein, a peptide, a oligonucleotide, a small molecule, etc., and said X-molecule species (2) is linked to the X-tag (3) via a linker molecule (4).
- the X-tag species may be linked to the X-molecule species via a linker molecule or via a direct binding.
- the bond involved in direct binding or in the linking using a linker molecule may be of a covalent character or of a non-covalent character.
- the linker molecules may be selected from the group consisting of a di-aldehyde such as a polyethylene glycol, glutaraldehyde, a polymer such as an oligosacharide, a nucleic acid and a peptide.
- the linker molecule may comprise 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.
- nucleic acid mimetics are peptide nucleic acid (PNA-), Locked Nucleic Acid (LNA-) , xylo-LNA-, phosphorothioate-, 2'-methoxy-, 2'-methoxyethoxy-, morpholino- and phosphoramidate- comprising molecules or the like.
- the polymer of the linker molecule may comprise at least 2 monomers such as at least 5, 10, 15, 20, 50, 100 such as at least 200 monomers. Also, the polymer of the linker molecule may be at least 5 A long such as at least 10 A, 15 A, 20 A, 30 A, 50 A, such as at least 1000 A long.
- the polymer of the linker molecule may be substantially linear and it may be substantially unbranched or branched.
- the linker of the tagged X-molecule species may be solid phase such as particle or a microsphere.
- the particle or the microsphere may comprise a material selected from the group consisting of an organic polymer, a metal, a metal oxide, an alloy, a magnetic materiel, and a combination of these materials.
- the metal oxide may be a silicon oxide such as quartz or glass.
- the organic polymer can be selected from the group consisting of polyethylene glycol-polyacrylamide, poly styrene, poly vinyl chloride, poly vinyl alcohol, polypeptides, poly ethylene, poly propylene and poly methamethacrylate and a combination of these materials.
- the particle or microsphere may be a composite material having one or more segments with a material as described above.
- 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 tagged X-molecule species may further comprise a capture component.
- the capture component may comprise a capture component selected from the group consisting of an amino group, carboxylic group, thiol group, oligonucleotide, peptide, biotin, imino biotin, an avidin, a streptavidin, an antibody, and functional derivatives thereof.
- 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 tagged X-molecule may comprise a release component.
- the release component may be located in the X-molecule, or between the X-molecule and the linker molecule, or in the linking molecule, or between the linker molecule and the X-tag species, or in the X- tag species, or between the capture component and the X-tag species.
- the release component may be selected from the group consisting of a selective cleavage site for an enzyme, a cleavage site for a nucleic acid restriction enzyme, a disulfide bridge, a ribonucleotide, a photocleavable group.
- the photocleavable group may be an o-nitrobenzyl linker, such as described in Olejnik et al 1 and in Olejnik et al 2.
- X-tag species cannot be replicated by polymerases.
- the X-tag species is composed of unnatural or modified nucleotides that cannot be replicated by polymerases, but are capable of specific basepairing.
- unnatural nucleotides are LNA (locked nucleic acids), PNA (peptide nucleic acids), TNA (threose nucleic acids), 2'OH methylated RNA, morpholinos, phosphorothioate nucleotides etc.
- an X-tag species composed of unnatural nucleotides may be desired to change the hybridization characteristics of the X-tag species, its chemical or biological stability, its solubility or other characteristics.
- the X-tag species may also be the X-molecule of the tagged X-molecule species. A non-limiting example thereof is shown in Figure 2C, where the X- molecule species 2 and the X-tag 3 is the same part of the tagged X-molecule species 1.
- the X-tag species may not be able to be replicated by polymerases.
- 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.
- the X-tag species of the tagged X- molecule species are hybridised to nucleic acid molecules (during step C)), said nucleic acid molecules may comprise universal nucleotides and/or a sequence complementary to the X-tag species.
- this approach may in some cases this may be advantageous, since doubled stranded nucleic acid's are less likely to have affinity against the target or exhibit non-specific binding activity than single stranded nucleic acid's.
- Figure 2D A non-limiting illustration of this embodiment is shown in Figure 2D.
- the X- molecule species (2) is linked to the X-tag (3) via the linker molecule (4).
- the X-tag (3) is furthermore hybridised to a complementary nucleic acid molecule (22).
- the X-tag species comprises a primer binding site for amplifying the X-tag species.
- An X-tag species comprising a primer binding site is called an A-tag species.
- a primer binding site may be a fixed region within an X-tag species or Y-tag species, said fixed region may be substantial identical or homologue for all the different species.
- the fixed region may be an oligonucleotide sequence that is present in all X-tag species or in all Y-tag species of a primary or secondary library.
- the term "homologue" of a given nucleic acid molecule capable of hybridising to a given target sequence means a nucleic acid molecule which is capable of hybridising to the same given target sequence at the same or similar conditions as the given nucleic acid molecule.
- a corresponding DNA molecule, RNA molecule, LNA molecule or PNA molecule would be considered homologue if it was capable to hybridise to the complementary sequence of the single stranded DNA molecule at a temperature in the temperature range 40 degrees C - 95 degrees C, such as 50 degrees C - 80 degrees C, 50 degrees C - 75 degrees C, 55 degrees C - 65 degrees C, or 55 degrees C - 62.5 degrees C, such as 55 degrees C - 60 degrees C.
- the tagged X-molecule species may be prepared using a method comprising the steps of
- a linker molecule comprising at least a first functional group and a second functional group, said first functional group is capable of receiving a tag codon group, said second functional group is capable of receiving an X-group
- 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 X-group may comprise at least one component selected from the group consisting of an amino acid, a nucleotide, a monosaccharide, a disaccharide, a carbohydrate, derivatives thereof, dimers, trimers and oligomers thereof and any combinations thereof.
- 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 most 5.000 kD (kiloDalton) such as at most l.OOOkD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 50 kD, 25 kD, 10 kD, 2000 D, 1000 D, 500 D, 250 D, 100 D such as at most 50 D.
- the X-molecule species may have a molar weight in the range of 50- 2000 D, such as e.g. 150-1500 D, 200-1300 D, 500-1000 D, 50-500 D, 250-1000 D, 1000-1500 D or 1500-2000 D.
- 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.
- the Y-molecule species may furthermore comprise a binding site for a PCR primer, e.g. located at the 3' end of the Y-molecule species at the 5'end or at both ends.
- 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 may further comprise a capture component selected from the group consisting of an amino group, a carboxylic group, a thiol group, a peptide, an oligonucleotide, a biotin, an avidin, a streptavidin, an antibody, and functional derivatives thereof.
- a capture component selected from the group consisting of an amino group, a carboxylic group, a thiol group, a peptide, an oligonucleotide, a biotin, an avidin, a streptavidin, an antibody, and functional derivatives thereof.
- the capture component is located at the end of the Y-molecule species.
- the Y-molecule species may comprise detectable groups such as radiolabelled groups or fluorescent markers.
- the Y-molecule species may further comprise a release component.
- the release component may be located in the Y-tag species, between the capture component and the Y-molecule, or between the Y-tag species and the binding site for the PCR primer, or at the end of the Y-molecule species.
- the release component may be selected from the group consisting of a selective cleavage site for an enzyme, a cleavage site for a nucleic acid restriction enzyme, a ribonucleotide, and a photocleavable group.
- the photocleavable group may be an o-nitrobenzyl linker.
- 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.
- target molecule concentrations may be used such that the primary library is first selected against a relatively low target concentration, and then successively selected against increasing concentrations of target. For each target concentration, a separate secondary library is used. In this way, tagged X-molecules may be identified according to their binding affinity (k d ).
- 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 will vary from application to application and it will furthermore vary during the repetitions of the method.
- 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 methods of selection may be any suitable methods known in the art of screening and selection, e.g. as described in Abelson.
- Binding conditions can be adjusted such as to minimize unspecific binding of the tagged X- molecule species in the selection process.
- 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
- M A or C
- R A or G
- W A or T
- S C or G
- Y C or T
- K G or T
- V A or C or G
- H A or C or T
- D A or G or T
- B G or C or T
- N A or G or C or T.
- Oligonucleotide pnl (primary noise) has a total diversity of 3.0xl0 6 . The redundancy of each position is indicated below the sequence. pnl 5' MRDTAA KYHGAG YRBAAC RRBTCT RYVATC MYDTCA
- the active oligonucleotide containing a 5'biotin, psi (primary signal) to be present in the primary library is synthesised separately with the following sequence
- oligonucleotides are synthesised using standard DNA oligonucleotide synthesis such as described in (Oligonucleotide Synthesis: A Practical Approach, M.J Gait) and can consequently be purchased from commercial suppliers such as DNA technology A/S, Forskerparken/Science Park Aarhus, Gustaw Wieds Vej 10A, DK-8000 Aarhus C, Denmark, www.dna-technology.com
- psi 100 ⁇ M is diluted 3xl0 4 times in TE buffer (10 mM Tris-HCI pH 8, 1 mM EDTA) + 0.01 % Triton X-100 and 1 ⁇ l of this dilution added to 99 ⁇ l pnl having a total oligonucleotide concentration of 100 ⁇ M.
- Step b) Providing the secondary library
- the secondary library is composed of 3xl0 6 different DNA sequences in equimolar amounts synthesised using redundancies during DNA-synthesis. This is schematically illustrated as the Y-molecule species 11 of Figure 4A.
- the secondary library oligonucleotides For each coding DNA oligonucleotide in the primary library (tagged X-molecule species), there is a complementary anti-coding DNA oligonucleotide in the secondary library (Y-molecule species).
- the secondary library oligonucleotides have fixed regions in both ends to enable PCR amplification.
- the noise in the secondary library is represented by oligonucleotide snl and the signal is represented by oligonucleotide ssl:
- sequence in bold is the anti-coding sequence and the flanking sequences are fixed regions for PCR amplification. Again the underlined sequence is the BamHI restriction site.
- PCR primer 1 and PCR primer 2 are used for PCR amplification, the latter PCR primer comprises a biotin group and incorporates the 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
- ssl 100 ⁇ M is diluted 3xl0 4 times in Te-buffer+ 0.01% Triton X-100 and 1 ⁇ l of this dilution added to 99 ⁇ l snl oligonucleotide stock (100 ⁇ M). Note that another 100 ⁇ l primary library will be prepared for each round of double selection and evolution, whereas the secondary library will only be prepared once.
- Step c) Contacting the primary library with the target molecule
- the primary library is contacted with streptavidin immobilized on sepharose (Streptavidin Sepharose High Performance, Cat. No. 17-5113-01, Amersham Biosciences, henceforth also denoted the “solid phase” or “solid phase bound target” when adopting the role of the target and "streptavidin sepharose” when used for manipulations of DNA strands.).
- streptavidin Sepharose High Performance Cat. No. 17-5113-01, Amersham Biosciences, henceforth also denoted the "solid phase” or “solid phase bound target” when adopting the role of the target and "streptavidin sepharose” when used for manipulations of DNA strands.
- Six ⁇ l solid phase (20 ⁇ l 30% suspension) is equilibrated in 1000 ⁇ l binding buffer of 6xSSC + 0.01% Triton X-100 (YxSSC means Y*150 mM NaCl and Y*15 mM trisodium citrate pH
- Step e Hybridising selected tagged X-molecule species to the secondary library
- the secondary library (lOO ⁇ l) is added 1 volume 2xhybridisation buffer (12xSSC + 0.02% Triton X-100 + 4 ⁇ g/ ⁇ l tRNA), before being added to the solid phase with bound tagged X- molecule species.
- 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
- the particular strong interaction between biotin and streptavidin means that the secondary library can be hybridised directly to selected X-tagged molecules bound to the solid phase. Had the interaction been less strong and the target molecule not been stable during the hybridisation reaction, selected tagged X-molecules could have been immobilized on streptavidin sepharose after selection, as described in Example 5.
- Step g Amplifying the selected Y-molecule species
- the washed solid phase may be used directly as template in the amplification step.
- hybridised Y-molecule species are elut ⁇ d using spin filtration; the solid phase is suspended in 20 ⁇ l 100 mM NaOH, and again separated from the liquid phase using a spin column (Quantum Prep Mini Spin Filters, Cat. No. 732-6027, Bio-Rad).
- the dried precipitate is dissolved in 28 ⁇ l H 2 0 of which 25 ⁇ l is aliquoted into 25 standard PCR reactions each containing: 10 ⁇ l OptiBuffer, supplied with the enzyme, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCl 2 , 2 ⁇ l 20 ⁇ M PCR-primer 1, 2 ⁇ l 20 ⁇ M PCR-primer 2 (comprising a biotin group as a capture component), 63 ⁇ l H 2 0 and 1 ⁇ l BIO- X-ACTTM (4 units) Short DNA polymerase (Bioline GmbH, Im Proceedingspark, TGZ-2, D-
- the PCR product from above is added 1 volume 2x binding buffer and immobilised on 40 ⁇ l pre-equilibrated streptavidin sepharose (in Figure 4B it is the streptavidin sepharose 18) by way of the 5'biotin capture component that was incorporated into the coding strand by PCR primer 2.
- the immobilized PCR product is
- the concentration of the second-generation secondary library is estimated by UV-absorption and adjusted to a suitable concentration, preferably 10-50 fold lower than the previous generation secondary
- This second generation library is now ready for next round, where another subset of primary library is selected against the solid phase bound target and selected primary library members hybridized to the second-generation secondary library.
- a total of 10 pmol (i.e. 1000 fold less than in the first round) second generation secondary library can be employed in the next round, in which case ssl will have the same concentration in the first and second generation secondary library.
- a successively lower total concentration of the secondary library can be employed because it evolves to contain a larger fraction of ssl.
- the amount of secondary library can also be adjusted to have ssl in moderate excess (5 - 10 50 fold) over psi for the hybridisation reaction. This provides a safety margin securing information transfer, as well as increasing the rate of hybridisation. If the amount of secondary library is adjusted such as to have ssl in excess, hybridization times can be adjusted accordingly.
- carrier nucleic acids e.g. 2 ⁇ g/ ⁇ l tRNA
- the number of cycles in the PCR reactions can be adjusted in later rounds, 20 because the number of secondary library members selected will gradually decrease. The reason for this is that a smaller amount of secondary library is employed for hybridization resulting in less non-specific binding to the solid phase and less specific hybridisation to non-specific tagged X-molecule species.
- Step j) Monitoring the evolution of the secondary library
- Approximately 0.2 ug (3-4 pmol) of the double stranded secondary library is digested with BamHI to monitor its fraction of oligonucleotide ssl. Digestion is performed with 20 units 30 BamHI in reaction buffer supplied with the enzyme (New England Biolabs, www.neb.com) with incubation for 60 minutes at 37°C.
- the digested secondary library is resolved on a 4% agarose gel using lxTBE (0.089 M Tris, 0.089 M Boric acid and 0.002 M EDTA) as running buffer.
- the fraction of ssl is estimated by comparing full-length fragments with the fragments resulting from digestion.
- a fraction of the double stranded secondary library is bulk-sequenced by standard techniques such as described in Sambrook et al.
- Step k Identifying molecules of high prevalence
- a fraction of the double stranded secondary library can be further PCR amplified with cloning primers 1 (5' GCAG CTCGAG GATGAT AGTAGT TCGTCG TCAC) and 2 (5' GCAG CTCCAG GCAGCA ACTACT CATCAT GACT), which allows directed cloning of the PCR product into pLITMUSTM28i (New England Biolabs, #N3528S) using Pstl and Xhol restriction sites.
- the identities of a number e.g. 100, individual clones are determined by sequencing (Litmus forward sequencing primer S1250S, Litmus reverse sequencing primer, S1251S, New England Biolabs), which indicates the composition of the secondary library of the given generation. If all sequenced clones, e.g. 100 clones, are different, more clones may be sequenced, but preferably, the selection process should be continued.
- the Y-molecule species of high prevalence are the three Y- molecule species whose sequences occur the most among the sequenced clones.
- Step I) Identifying tagged X-molecule species with an X-tag species corresponding to the high prevalence Y-molecule species
- the links between Y-molecule species and the X-tag species and between the X-tag species and the tagged X-molecule species can be stored in a database of a computer.
- the Y-tag sequence of the Y-molecule species of high prevalence are submitted to the computer, the computer tracks the relevant relationships in the database and the corresponding tagged X-molecule species and X-molecule species are presented on the monitor of the computer.
- the first generation secondary library is prepared from a first and a second primary library.
- the tagged X-molecule species of these two libraries comprise A- tags (X-tags comprising at least one fixed region for PCR amplification) and the libraries only differ in that their A-tags contain different fixed regions for PCR amplification.
- Both the first and the second primary library are separately selected against the solid phase and A-tags amplified by PCR.
- A-tags of first primary library is hybridized to A-tags of the second primary library whereafter hybridized and selected A-tags of the latter are amplified by PCR to generate the secondary library.
- Step 1) Providing the primary libraries
- Two primary libraries are prepared, each with a diversity of about IO 9 .
- the coding sequence (shown in bold) of the signal oligonucleotides employed are the same as in Example 1, and again the underlined sequence is a restriction site for BamHI, used to monitor the evolution of the secondary library.
- 5'bCAGTAG TAGCCA ACGGCT AGTA AGCTAG TCGGAG CGAAAC GGATCC GCTATA ACCTCG ATCG TTAGAC GCTATC CGAGTA
- every third position of the noise oligonucleotides has a redundancy that excludes identity with the signal oligonucleotides.
- PCR-primer 1 5 ' GATGAT AGTAGT CGTCG 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
- Oligonucleotide ps2 (500 ⁇ M) is diluted l.lxlO 7 times in TE buffer + 0.01% Triton X-100 and 5 ⁇ l of this dilution added to 495 ⁇ l of pn2 (500 ⁇ M) to give 500 ⁇ l of the first primary library (comprising ps2 and pn2) and likewise for the preparation of the psn3 library.
- second-strand synthesis is performed, because dsDNA is less prone to interfere with selection than ssDNA.
- PCR-primer 1 is used and for psn3, PCR-primer 3 is used.
- the primary library is split into 10 aliquots of 50 ⁇ l each, to which the following is added: 100 ⁇ l 300 ⁇ M downstream primer, 1000 ⁇ l Optibuffer, 600 ⁇ l 25mM MgCI 2 , 160 ⁇ l 25 mM dNTP, 100 ⁇ l (400-units) Bio-X-ACTTM Short DNA polymerase and 8040 ⁇ l H 2 0.
- the ten tubes are incubated in a 94°C water bath for 6 minutes, transported to an 84 °C water bath for 6 minutes, next to 74°C for 6 minutes, 64°C for 6 minutes, and 54 °C for 10 minutes. After annealing of the downstream primer, second strand synthesis is performed at 72°C for 60 minutes in a water bath.
- the samples are precipitated by addition of 1/10 volume 3 M Na-acetate pH 4.5 and 3 volumes 96% ethanol and incubation for 30 minutes at -20 °C.
- the samples are then centrifuged 60 min at lO.OOOg, the supernatant disposed, and the pellet gently washed twice with 1 ml ice-cold 70% ethanol and air-dried.
- the dry pellets are redissolved in 100 ⁇ l binding buffer and all samples are pooled into a primary library of 1000 ⁇ l, that is extracted twice with 200 ⁇ l phenol, followed by one extraction with 200 ⁇ l chloroform, whereafter the primary library is ready for selection.
- Step 2 Contacting the primary libraries with the target molecule
- the two primary libraries, psn2 and psn3, are separately contacted with pre-equilibrated solid phase bound target as described in Example 1, step c Step 3) Selecting tagged X-molecule species that interact with the target molecule
- 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 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.
- PCR primers 1 and 2 are employed for amplification of the psn2 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 psn3, PCR primers 3 and 4 are employed. Similar to PCR primer 2, PCR primer 4 is biotinylated and consequently the resulting PCR product is biotinylated at the 5' end of the coding strand.
- Step 5 Providing the secondary library
- the anti-coding strand of the psn2 PCR product is batch eluted by adding 400 ⁇ l 100 mM NaOH to the streptavidin sepharose followed by centrifugation of the eppendorf tube. After elution, the streptavidin sepharose containing the psn2 coding strand is washed twice with 1000 ⁇ l hybridization buffer.
- the anti-coding strand of the psn3 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 + 2 ⁇ g/ ⁇ l tRNA.
- the streptavidin sepharose immobilised coding strands of the psn2 PCR product are now hybridised to complementary anti-coding strands from the psn3 PCR product, which are next added. Hybridisation is performed by heating the sample to 85 °C for 5 minutes, followed by incubation at 65° for 12 hours.
- streptavidin sepharose is washed two times with 1000 ⁇ l lxhybridisation buffer followed by one wash with wash-buffer (lxSSC+0.01% Triton X- 100) for 5 minutes at 65°C to select hybridised psn3 anti-coding strands (Y-molecule species)
- the new secondary library may be used as first generation secondary library in Example 1, thus replacing step b) of Example 1. Furthermore, the first primary library of Example 2 may be used as primary library of Example 1, thus replacing step a) of Example 1.
- the first primary library is again selected against the solid phase and selected A-tags PCR amplified and immobilized on streptavidin sepharose.
- the anti-coding strands are then eluted and coding strands hybridized to complementary anti-coding Y- molecule species of the first generation secondary library.
- 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 first generation 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, 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
- 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 3 is a modification of Example 2, the major difference being the use of photocleavable biotin groups.
- the biotin group adopts the role of X-molecule species
- the photocleavable linker allows specific elution of selected tagged X-molecule species.
- the biotin group serves as an affinity handle (capture group) that allows simple manipulations of DNA strands
- the photocleavable linker adds the possibility of eluting DNA strands that have been immobilized on streptavidin sepharose.
- the steps of Example 3 are illustrated in Figures 6A-6C. The three figures should be combined so that Figure 6A and 6B run in parallel and continue in Figure 6C.
- Step 1) Providing the primary libraries Two primary libraries are prepared, each with a diversity of about IO 9 .
- the signal oligonucleotides employed are the same as in Example 2, except that a photocleavable linker has been inserted between the biotin group and the X-tag species. This combination of photocleavable linker and biotin is abbreviated pcb.
- the coding sequence of the noise oligonucleotides is identical the pn2 and 3, Example 2
- PCR-primer 1 5'GATGAT AGTAGT TCGTCG TCAC PCR-primer 3 5' TACTCG GATAGC GTCTAA CGAT PCR-primer 5 5'pcb GCAGCA ACTACT CATCAT GACT PCR-primer 6 5' CAGTAG TAGCCA ACGGCT AGTA PCR-primer 7 5'pcb TACTCG GATAGC GTCTAA CGAT
- Oligonucleotide ps2 (500 ⁇ M) is diluted l.lxlO 7 times in 0.01% Triton X-100 and 5 ⁇ l of this dilution added to 495 ⁇ l of pn2 (500 ⁇ M) to give 500 ⁇ l of the psn2 primary library and likewise for the preparation of the psn3 library.
- Second-strand synthesis is performed as described in Example 2, step 1.
- Step 2 Contacting the primary libraries with the target molecule
- the two primary libraries, psn2 and psn3, are separately contacted with pre-equilibrated solid phase bound target as described in Example 1, step c 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. 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.
- Step 4) Amplifying the selected A-tag species
- 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 anti-coding strand is then cleaved of the streptavidin sepharose using the photocleavable biotin linker; the streptavidin sepharose is resuspended in 400 ⁇ l binding buffer, placed on a sheet of parafilm and illuminated for five minutes at 325 nm, wherafter the sample is spinfiltered and the eluate collected.
- the streptavidin sepharose is then washed with another 400 ⁇ l binding buffer, spinfiltered and the eluate added to the first eluate.
- the combined eluate is now ethanol precipitated and redissolved in 400 ⁇ l hybridisation buffer.
- streptavidin sepharose is washed two times with 1000 ⁇ l lxhybridisation buffer followed by one wash with wash-buffer (3xSSC+0.01% Triton X- 100) buffer for 5 minutes at 65°C to select hybridised psn3 anti-coding strands (Y- molecule species)
- psn3 strands selected by hybridisation are eluted by photocleavage as described in step b, whereafter the eluate is ethanol precipitated.
- the dried precipitate is dissolved in 22 ⁇ l H 2 0 of which 20 ⁇ l is aliquoted into 10 PCR reactions each containing: 10 ⁇ l optibuffer, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCI 2 , 2 ⁇ l 20 ⁇ M PCR-primer 6, 2 ⁇ l 20 ⁇ M PCR-primer 7, 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 72°C.
- the psn2 primary library is again selected against the solid phase bound target, specifically eluted, selected X-tags PCR amplified and immobilized on streptavidin sepharose.
- the anti-coding strands are then eluted and coding strands hybridized to complementary anti-coding Y-molecule species of the first generation secondary library.
- 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 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 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 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
- Step g Amplifying the selected Y-molecule species
- the washed streptavidin sepharose may be used directly as template in the amplification step.
- hybridised Y-molecule species are eluted with 50 ⁇ l 100 mM NaOH using spin filtration, neutralised, ethanol precipitated and dissolved in in 28 ⁇ l H 2 0 of which 25 is aliquoted into 25 standard PCR reactions each containing: 10 ⁇ l OptiBuffer, supplied with enzyme, 16 ⁇ l 2.5mM dNTP, 6 ⁇ l 25 mM MgCI 2 , 2 ⁇ l 20 ⁇ M PCR-primer 1, 2 ⁇ l 20 ⁇ M PCR- primer 2, 63 ⁇ l H 2 0 and 1 ⁇ l BIO-X-ACTTM (4 units) Short DNA polymerase (Bioline Cat.
- BIO-21064 The reaction is cycled 10 times with 94 ° for 30 sec, 55 °C for 30 sec, 68 °C for 60 sec followed by 10 minutes extension at 68 °C. After amplification, all reactions are pooled and the PCR product is gel purified from a 4% agarose gel according to manufacturers instructions (QIAEX II Gel Extraction Kit, Cat. No. 20021, Qiagen). 400 ⁇ l H 2 0 is used to elute the PCR product from Qiaex II beads.
- 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 1000 fold enriched in Y- molecule species corresponding to active tagged X-molecule species, a 1000 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 j) 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 (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 2) Contacting the target molecule with the primary library
- 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 pbl primary library is again selected against the solid phase and selected X-tags PCR amplified and immobilized on streptavidin sepharose.
- the anti-coding strands are then eluted and coding strands hybridized to complementary anti-coding Y- molecule species of the first generation secondary library.
- 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 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
- Example 7 is an extension of Example 3. Hence, selected tagged X-molecule species are specifically eluted by competition with soluble target molecule. Moreover, a photocleavable biotin linker is used for manipulation of DNA strands. 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 7 are illustrated in Figures lOA-lOC. The three figures should be combined so that Figure 10A and 10B runs in parallel and continue in Figure IOC.
- Step 1) Providing the primary libraries
- PCR-primer 1 5 ' GATGAT AGTAGT TCGTCG TCAC PCR-primer 3 5 ' TACTCG GATAGC GTCTAA CGAT PCR-primer 5 5 ' pcb GCAGCA ACTACT CATCAT GACT PCR-primer 6 5 ' CAGTAG TAGCCA ACGGCT AGTA PCR-primer 7 5 ' pcb TACTCG GATAGC GTCTAA CGAT
- Step 2 Contacting the primary libraries with the target molecule
- Step 3 Selecting tagged X-molecule species with a specific target molecule interaction
- 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 X-molecule species bound specifically are eluted using competitive elution; the solid phase is resuspended in 500 ⁇ l binding buffer + ImM soluble target molecule and incubated at 37°C for 5 hours, whereafter the samples are spinfiltered and the liquid phase collected. Subsequently, the the liquid phase is extracted twice with 200 ⁇ l phenol, one time with 200 ⁇ l chloroform, ethanol precipitated and redissolved in 75 ⁇ l binding buffer.
- Step 4) Amplifying the selected A-tags
- Selected A-tags are PCR amplified as described in Example 3. Step 5) Providing the secondary library
- Step 7) Monitoring the evolution of the secondary library
- step j See Example 5, step j.
- Step 8) Identifying molecules of high prevalence
- Step 9) Identifying tagged X-molecule species with an X-tag (A-tag) species corresponding to the high prevalence Y-molecule species
- Example 8 is an extension of Example 6, the only difference being that the hybridisation reaction is performed in solution as also described in Example 4. 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 8 are illustrated in Figures IIA-IIC. The three figures should be combined so that Figure 11A and 11B run in parallel and continue in Figure llC.
- Step 1) Providing the primary libraries
- 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
- 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 4 and 8 are employed which biotinylates the resulting PCR product at the 5' end of the coding and introduces a ribonucleotide in the anti-coding strand. Step 5) Providing the secondary library
- pb2 PCR product is added 1/10 volume 1 M NaOH and incubated at 80 °C 15 for 5 minutes, which cleaves the anti-coding strand at the ribonucleotide reside in
- PCR primer-10 Next, the sample is neutralised, ethanol precipitated and redissolved in 500 ⁇ l formamide loading buffer. The sample is now heated to 94° for 3 minutes and loaded on a 6% denaturing (8 M urea) polyacrylamide gel and the fragments are resolved until the coding strand has reached the middle of the 20 gel. Hereafter, the positions of fragments are determined by UV-shadowing and the gel-piece containing the coding strand is cut out for subsequent passive elution. After elution, the coding strand is ethanol precipitated and redissolved in 10 ⁇ l hybridisation buffer.
- the volume is increased to 100 ⁇ l by addition of binding buffer, 30 whereafter the sample is added to 6 ⁇ l pre-equillibrated streptavidin sepharose and incubated for 30 minutes at 55 °C with mixing.
- 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 7) Monitoring the evolution of the secondary library
- Step 8) Identifying molecules of high prevalence See Example 1, step k.
- Step 9) Identifying tagged X-molecule species with an X-tag species corresponding to the high prevalence Y-molecule species
- Two primary libraries were prepared with a diversity of respectively 2,6xl0 5 and 3xl0 6 .
- 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.
- Example 9 This approach could be used generally to allow specific elution of X-tags corresponding to active X-molecules.
- the target could be attached to the solid phase by way of a photocleavable linker or active X-molecules could be eluted by competitive elution.
- the steps of Example 9 are illustrated in Figures 13A-13B. The two figures should be combined.
- Step a) Providing the primary library
- 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.
- the active oligonucleotide containing a 5'biotin, PS-NcoI to be present in the primary library was synthesised separately with the following sequence
- PS-NcoI was diluted into PL-10e6 to create the IO 6 library.
- the underlined sequence indicates an Ncol 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 primer 11 and PCR primer 12 were used for PCR amplification of the secondary libraries, the latter comprises a biotin and incorporates the biotin-group at the 5'end of the coding strand of the PCR-product, which allows purification of the anti-coding strand, i.e. the next generation secondary library.
- 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
- DNA oligonucleotides were mixed according to the scheme below to create both the primary and the secondary libraries in a total volume of 90 ⁇ l (6xSSC, 0.01% Triton X- 100). Two negative controls omitting signal oligonucleotides in secondary libraries were also prepared.
- 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.
- the equilibrated solid phase was resuspended in 70 ⁇ l 6xSSC, 0.01% Triton X-100 to give a total volume of app. 100 ⁇ l.
- 20 ⁇ l equilibrated solid phase was added to samples A-D from step e. The 5 samples were then incubated at 65 °C for 20 minutes with mixing in a table shaker to allow interaction between the primary library and the solid phase.
- 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
- the samples were transferred to spin-off filters (Ultrafree-MC filter microporous 0.22 micron, Millipore, UFC3 0GV NB) and centrifuged at 15 3000 rpm for 2x 1 minute.
- spin-off filters Ultrafree-MC filter microporous 0.22 micron, Millipore, UFC3 0GV NB
- 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.
- the 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
- 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).
- Step c-1) Hybridising Y-molecule species of the secondary library with X-tag species of the primary library
- A-2 and A-3 were as A-l, except that 10-fold and 100-fold diluted Sample A was used.
- B-2 and B-3 were as B-l, except that 10-fold and 100-fold diluted Sample B was used.
- C-2 and C-3 were as C-1, except that 10-fold and 100-fold diluted Sample C was used.
- D-2 and D-3 were as D-1, except that 10-fold and 100-fold diluted Sample D was used.
- Step d-1) Contacting the target molecule with at least a subset of the primary library hybridised to the secondary library
- 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
- Needels et al. Needels MC, Jones DG, Tate EH, Heinkel GL, Kochersperger LM, Dower WJ, Barrett RW, Gallop MA Generation and screening of an oligonucleotide-encoded synthetic peptide library. Proc Natl Acad Sci U S A 1993 Nov 15;90(22) : 10700-4.
- Olejnik et al 1 Olejnik J, Krzymanska-Olejnik E, Rothschild KJ. Photocleavable affinity tags for isolation and detection of biomolecules. Methods Enzymol 1998;291: 135-54.
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EP1597395A2 (en) | 2003-02-21 | 2005-11-23 | Nuevolution A/S | Method for producing second-generation library |
DE602004019764D1 (en) | 2003-03-20 | 2009-04-16 | Nuevolution As | LIGATION-RELATED CODING OF SMALL MOLECULES |
ATE447626T1 (en) | 2003-09-18 | 2009-11-15 | Nuevolution As | METHOD FOR OBTAINING STRUCTURAL INFORMATION FROM ENCODED MOLECULES AND FOR SELECTING COMPOUNDS |
EP2366705A1 (en) | 2003-12-17 | 2011-09-21 | Praecis Pharmaceuticals Incorporated | Methods for synthesis of encoded libraries |
US7972994B2 (en) | 2003-12-17 | 2011-07-05 | 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 |
AU2006309096B2 (en) | 2005-10-28 | 2013-07-04 | Glaxosmithkline Llc | Methods for identifying compounds of interest using encoded libraries |
PT3305900T (en) | 2005-12-01 | 2021-10-27 | Nuevolution As | Enzymatic encoding methods for efficient synthesis of large libraries |
WO2010043418A2 (en) * | 2008-10-17 | 2010-04-22 | Febit Holding Gmbh | Integrated amplification, processing and analysis of biomolecules in a microfluidic reaction medium |
EP3540059A1 (en) | 2010-04-16 | 2019-09-18 | Nuevolution A/S | Bi-functional complexes and methods for making and using such complexes |
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. |
DK3368666T3 (en) * | 2015-10-30 | 2020-12-14 | Guangdong Maijinjia Biotechnologies Co Ltd | Methods for identifying oligonucleotides |
CN112562789A (en) * | 2019-09-24 | 2021-03-26 | 成都先导药物开发股份有限公司 | Method for determining combination of specific sequence and biological target in screening of DNA coding compound library |
JP2023545506A (en) * | 2020-10-15 | 2023-10-30 | ジェネンテック, インコーポレイテッド | Multiplexed target binding candidate screening analysis |
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US5573905A (en) * | 1992-03-30 | 1996-11-12 | The Scripps Research Institute | Encoded combinatorial chemical libraries |
WO1994013623A1 (en) * | 1992-12-11 | 1994-06-23 | Chiron Corporation | Synthesis of encoded polymers |
US6087103A (en) * | 1998-03-04 | 2000-07-11 | Lifespan Biosciences, Inc. | Tagged ligand arrays for identifying target-ligand interactions |
US7479472B1 (en) * | 1998-10-19 | 2009-01-20 | The Board Of Trustees Of The Leland Stanford Junior University | DNA-templated combinatorial library chemistry |
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CN102796730A (en) * | 2012-07-18 | 2012-11-28 | 海南广陵高科实业有限公司 | Method for recycling nucleic acid molecules from polyacrylamide gel |
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