WO2016109808A1 - Procédés et compositions pour une synthèse dirigées par matrices d'acide nucléique, de grandes bibliothèques de petites molécules complexes - Google Patents

Procédés et compositions pour une synthèse dirigées par matrices d'acide nucléique, de grandes bibliothèques de petites molécules complexes Download PDF

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WO2016109808A1
WO2016109808A1 PCT/US2015/068308 US2015068308W WO2016109808A1 WO 2016109808 A1 WO2016109808 A1 WO 2016109808A1 US 2015068308 W US2015068308 W US 2015068308W WO 2016109808 A1 WO2016109808 A1 WO 2016109808A1
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codon
reactive
oligonucleotides
sequence
small molecules
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PCT/US2015/068308
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Nicholas K. TERRETT
William H. CONNORS
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Ensemble Therapeutics Corporation
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR

Definitions

  • the invention relates generally to methods and compositions for performing nucleic acid-templated synthesis. More particularly, the invention relates to methods and compositions for producing small molecule libraries of greater size, scope and complexity than previously possible by nucleic acid template synthesis.
  • Nucleic acid-templated organic synthesis enables modes of controlling reactivity that are not possible in a conventional synthesis format and allows synthetic molecules to be manipulated using translation, selection, and amplification methods previously available only to biological macromolecules (Gartner et al. (2001) J. AM. CHEM. SOC. 123: 6961-3; Gartner et al. (2002) ANGEW. CHEM., INT. ED. ENGL. 123: 61796-1800; Gartner et al. (2002) J. AM. CHEM. Soc. 124: 10304-6; Calderone et al. (2002) ANGEW. CHEM., INT. ED. ENGL.
  • the present invention provides methods and compositions for producing small molecule libraries of greater size, scope and complexity than those previously possible by nucleic acid template synthesis.
  • the invention provides a method for producing a library of small molecules associated with corresponding oligonucleotides.
  • the method comprises the steps of (a) providing a plurality of templates comprising a plurality of first reactive units associated with a corresponding plurality of first oligonucleotides, (b) providing a plurality of first transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein each second oligonucleotide defines a first anti- codon sequence complementary to a first codon sequence; and (c) annealing first and second oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity, thereby producing a plurality of first small molecules associated with the corresponding first oligonucleotides.
  • Each first oligonucleotide defines at least a first codon sequence, a second codon sequence, and a third codon sequence, and each of the first, second, and third codon sequence is at least 12 bases in length and is different from one another. Each first oligonucleotide is at least 70 bases in length.
  • the method can also include dividing a plurality of templates comprising a plurality of first reactive units associated with a corresponding plurality of first oligonucleotides into a plurality of aliquots, and for each aliquot, providing a plurality of first transfer units, a plurality of second transfer units, a plurality of third transfer units, and, optionally a plurality of fourth transfer units, wherein the order of adding the first, second, third, and optionally fourth transfer units is different from any other aliquot.
  • Each first oligonucleotide defines at least a first codon sequence, a second codon sequence, and a third codon sequence, and each of the first, second, and third codon sequence is at least 12 bases in length and is different from one another. Each first oligonucleotide is at least 70 bases in length.
  • the method includes recombining two or more of the aliquots to create a library of small molecules.
  • the template may have any one of the following features.
  • at least one codon is at least 14 bases in length.
  • each of the first, second, third, and, optionally, fourth codon is at least 14 bases in length.
  • the first oligonucleotide is at least 90 bases in length.
  • each first oligonucleotide comprises a unique tag sequence that defines the linker or capping group, or any other structural modification to any small molecule that was not achieved through a DNA-templated reaction step.
  • each of the corresponding first oligonucleotides has a nucleotide sequence informative of at least a portion of the synthetic history of the small molecule associated therewith.
  • the concentration of the plurality of templates is at least 90 nM and no greater than 500 nM at each step when a reactive unit is added.
  • the small molecules may have any of the following features.
  • the second, third or fourth small molecule comprises a moiety that was added as a soluble reagent to the first oligonucleotide-associated small molecule (e.g. , to the first, second, third, or fourth small molecule); and, optionally, wherein each of the first
  • oligonucleotides comprises a nucleotide sequence that is informative of the soluble reagent- added moiety.
  • at least one of the first, second, third, fourth or fifth reactive unit, or the soluble reagent is a trivalent moiety.
  • the second, third or fourth small molecule, or the soluble reagent comprises a reactive moiety or can be further modified or deprotected to reveal a reactive functional group capable of further reaction with a plurality of chemical moieties.
  • the reactive moiety capable of further reaction with another chemical moiety can be but is not limited to a nucleophilic primary or secondary amine or a free carboxyl group.
  • the methods may further comprise the following steps.
  • the method comprises (i) splitting the library into a plurality of aliquots following addition of the reactive moiety capable of further reaction with a plurality of chemical moieties onto the first oligonucleotide-associated small molecules; and (ii) adding to each of the plurality of aliquots a different reagent that reacts with the reactive moiety present on the first oligonucleotide- associated small molecules present therein.
  • different reagents include an acylating agent, a sulfonating agent, a heteroaryl halide reagent, reductive amination reagents and an amide-forming reagent.
  • an identifying sequence e.g. , a tag
  • a tag can be ligated to the 3' terminus of each of the plurality of templates.
  • one or more of the plurality of second, third, fourth or fifth oligonucleotides is bound to a first member of a binding pair, e.g., biotin.
  • a binding pair e.g., biotin.
  • one or more of the plurality of first, second, third or fourth small molecules bound to the first member of a binding pair is purified by contact with a second binding pair member, wherein the second member of the binding pair (e.g., streptavidin) is bound to a solid support.
  • the plurality of templates is reacted with a capping reagent that differentially caps the small molecules that did not react with one or more of the prior-added reactive units or soluble reagents.
  • the capping reagent is an acid anhydride, e.g. , acetic anhydride, or acyl chloride or other activated acylating group known to those skilled in the art.
  • the invention relates to a library of compounds produced by any of the methods described herein.
  • FIG. 1 is a schematic illustration depicting an exemplary template covalently attached to a product (macrocycle) encoded by nucleic acid template synthesis.
  • the exemplary template comprises a plurality of regions including two fixed regions (10 bases), two tag regions (7 bases) and three codons (12 bases) for DPC reactions.
  • a macrocycle small molecule is synthesized on the DNA template, with the linker corresponding to Tag 2, the spacer corresponding to Tag 1, and building blocks 1, 2, and 3 corresponding to codons 1, 2 and 3, respectively.
  • FIG. 2 is a schematic illustration depicting how to efficiently create a library of templates suitable for nucleic acid template synthesis.
  • each codon position contains one of 24 variants (but more variants are possible), and each of the codon sets in each of the positions Rl, R2, and R3 has its own unique set of 24 codons, making a total of 72 different codon sequences used in total, to generate 13,824 different DNA sequences corresponding to 13,824 different templates.
  • FIG. 3 is a schematic illustration of an embodiment in which the Tag 2 position of a DNA template is kept fixed with a unique base sequence that defines one trivalent linker building block.
  • FIG. 4 is a schematic illustration of an embodiment in which a plurality of different linkers (16 as shown) are employed in a single library mixture, wherein the template oligonucleotides were synthesized in 16 mixtures denoted L-l through LI 6 each comprising 13,824 different sequences. Combining the 16 different mixtures gives a total template library complexity of 221,184 different template sequences.
  • FIG. 5 is a schematic illustration depicting the addition of a spacer, which is defined by the Tag 1 sequence in the DNA template.
  • FIG. 6 shows the design of DNA templates with four codons which increases library size. Using 24 variants for the new building block introduced by this codon will provide 331,776 different template sequences. When 16 different four-codon template mixtures are pooled, a library of 5,308,416 unique compounds is created, which is a 24-fold increase in library diversity as compared to a 16-mixture pool of three codon templates, which provides only 221,184 compounds.
  • FIG. 7 is a schematic illustration showing how changing the sequence of the chemical steps in a nucleic acid-templated library synthesis can generate diverse architectures, thereby increasing the diversity of small molecules in a DPC library.
  • the present invention facilitates the creation of large, diverse libraries of small molecules that are created by nucleic acid templated synthesis. This can be facilitated by one or more of the choice of specific templates, the choice of specific chemical reactants, capping processes, the choice of appropriate chemistries, and changing the order of synthesis steps during nucleic acid template synthesis. Each of the features is discussed in more detail below.
  • codon and anti-codon as used herein, refer to complementary oligonucleotide sequences in a template and in a transfer unit, respectively, that permit the transfer unit to anneal to the template during nucleic acid-templated synthesis.
  • soluble reagent refers to a chemical reagent or chemical moiety that is not linked to an oligonucleotide and does not participate in nucleic acid- templated synthesis.
  • the soluble reagent can directly modify the small molecule attached to the oligonucleotide by chemical reaction independent of nucleic acid-templated addition.
  • the first and second reactive units of the template for example, and the transfer units are attached to oligonucleotides that can participate in nucleic acid-templated synthesis.
  • oligonucleotide or “nucleic acid” as used herein refer to a polymer of nucleotides.
  • the polymer may include, without limitation, natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxy adenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
  • Nucleic acids and oligonucleotides may also include other polymers of bases having a modified backbone, such as a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a threose nucleic acid (TNA) and any other polymers capable of serving as a template for an amplification reaction using an amplification technique, for example, a polymerase chain reaction, a ligase chain reaction, or non-enzymatic template-directed replication.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • TAA threose nucleic acid
  • reaction intermediate refers to a chemical reagent or a chemical moiety chemically transformed into a different reagent or chemical moiety with a soluble reagent.
  • small molecule refers to an organic compound either synthesized in the laboratory or found in nature having a molecular weight from about 300 Daltons (Da) to about 1,500 Da.
  • small molecule scaffold refers to a chemical compound having at least one site or chemical moiety suitable for functionalization.
  • the small molecule scaffold or molecular scaffold may have two, three, four, five or more sites or chemical groups suitable for functionalization. These functionalization sites may be protected or masked as would be appreciated by one of skill in this art. The sites may also be found on an underlying ring structure or backbone.
  • the small molecule scaffolds are not nucleic acids, nucleotides, or nucleotide analogs.
  • transfer unit refers to a molecule comprising an oligonucleotide having an anti-codon sequence attached to a reactive unit including, for example, but not limited to, a building block, monomer, small molecule scaffold, or other reactant useful in nucleic acid-templated chemical synthesis.
  • template refers to a molecule comprising an
  • the template optionally may comprise (i) a plurality of codon sequences, (ii) an amplification means, for example, a PCR primer binding site or a sequence
  • compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present invention also consist essentially of, or consist of, the recited components, and that the processes of the present invention also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions are immaterial so long as the invention remains operable. Moreover, unless specified to the contrary, two or more steps or actions may be conducted simultaneously. [0033] In one aspect, the invention provides a method for producing a library of small molecules associated with corresponding oligonucleotides.
  • the method comprises the steps of (a) providing a plurality of templates comprising a plurality of first reactive units associated with a corresponding plurality of first oligonucleotides, wherein (i) each first oligonucleotide defines at least a first codon sequence, a second codon sequence, and a third codon sequence; (ii) each of the first, second and third codon sequence is at least 12 bases in length; (iii) each of the first, second and third codon sequence is different from one another; and (iv) each first oligonucleotide is at least 70 bases in length; (b) providing a plurality of first transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein each second oligonucleotide defines a first anti-codon sequence complementary to a first codon sequence; (c) annealing first and second
  • oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity, thereby producing a plurality of first small molecules associated with the corresponding first oligonucleotides; (d) providing a plurality of second transfer units comprising a plurality of third reactive units covalently attached to a corresponding plurality of third oligonucleotides, wherein each third oligonucleotide defines a second anti-codon sequence complementary to the second codon sequence; (e) annealing first and third oligonucleotides having complementary codon and anti-codon sequences to bring the reaction products of step (c) and the third reactive units into reactive proximity thereby producing a plurality of second small molecules associated with the corresponding first oligonucleotides; (f) providing a plurality of third transfer units comprising a plurality of fourth reactive units covalently attached to a corresponding plurality of fourth oligonucleotides, wherein each fourth oligon
  • step (e) complementary codon and anti-codon sequences to bring the reaction products of step (e) and the fourth reactive units into reactive proximity thereby producing a plurality of third small molecules associated with the corresponding first oligonucleotides, wherein each of the corresponding first oligonucleotides has a nucleotide sequence informative of at least a portion of the synthetic history of the third small molecule associated therewith.
  • oligonucleotide is at least 70 bases in length; (b) dividing the plurality of templates into a plurality of aliquots; (c) for each aliquot (i) providing a plurality of first transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein each second oligonucleotide defines a first anti-codon sequence complementary to a first codon sequence; (ii) annealing first and second
  • oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity, thereby producing a plurality of first small molecules associated with the corresponding first oligonucleotides; (iii) providing a plurality of second transfer units comprising a plurality of third reactive units covalently attached to a corresponding plurality of third oligonucleotides, wherein each third oligonucleotide defines a second anti-codon sequence complementary to the second codon sequence; (iv) annealing first and third oligonucleotides having complementary codon and anti-codon sequences to bring the plurality of first small molecules of step (iii) and the third reactive units into reactive proximity thereby producing a plurality of second small molecules associated with the corresponding first oligonucleotides; (v) providing a plurality of third transfer units comprising a plurality of fourth reactive units covalently attached to a corresponding plurality of fourth oligonucleot
  • the first oligonucleotide comprises a fourth codon of at least 12 bases in length
  • the method comprises the additional steps of providing a plurality of fourth transfer units comprising a plurality of fifth reactive units covalently attached to a corresponding plurality of fifth oligonucleotides, wherein each fifth oligonucleotide defines a fourth anti-codon sequence complementary to the fourth codon sequence; and annealing first and fifth oligonucleotides having complementary codon and anti-codon sequences to bring the plurality of third small molecules of step (g) or (vi) and the fifth reactive units into reactive proximity thereby producing a plurality of fourth small molecules associated with the corresponding first oligonucleotides, wherein each of the corresponding first oligonucleotides has a nucleotide sequence informative of at least a portion of the synthetic history of the fourth small molecule associated therewith.
  • each codon region ensures sufficient base-pairing to give high-affinity duplex formation with a suitably high melting temperature such that the duplex will form and be maintained at ambient temperature.
  • at least one codon is at least 14 bases in length.
  • each of the first, second, third, and, when present, fourth codon is at least 14 bases in length.
  • the first oligonucleotide is at least 90 bases in length.
  • a soluble reagent e.g. , a free reactant not attached to an oligonucleotide-transfer unit
  • the third or fourth small molecule comprises a moiety that was added as a soluble reagent to the first oligonucleotide-associated small molecule of one or more of steps (c), (e), (g) or (i) (or (ii), (iv), (vi) or (viii)); and, optionally, wherein each of the first oligonucleotides comprises a nucleotide sequence that is informative of the soluble reagent-added moiety.
  • the structural complexity of small molecules generated by nucleic acid-templated synthesis can be further enhanced by the incorporation of a trivalent (i.e., trifunctional) building block, having three sites of attachment.
  • a trivalent building block having three sites of attachment.
  • at least one of the first, second, third, fourth or fifth reactive unit, or the soluble reagent, is a trivalent moiety.
  • the second, third or fourth small molecule or the soluble reagent comprises a reactive moiety or can be further modified or deprotected to reveal a reactive functional group capable of further reaction with a plurality of chemical moieties.
  • the reactive moiety capable of further reaction with another chemical moiety can be but is not limited to a nucleophilic primary or secondary amine or a free carboxyl group.
  • Diversity can also be increased by (i) splitting the library into a plurality of aliquots following addition of the reactive moiety capable of further reaction with a plurality of chemical moieties onto the first oligonucleotide-associated small molecules; and (ii) adding to each of the plurality of aliquots a different reagent that reacts with the reactive moiety present on the first oligonucleotide-associated small molecules present therein.
  • different reagents include an acylating agent, a sulfonating agent, a heteroaryl halide reagent, reductive animation reagents and an amide-forming reagent.
  • an identifying sequence e.g. , a tag
  • a tag can be ligated to the 3' terminus of each of the plurality of templates.
  • the concentration of the plurality of templates is at least 90 nM and no greater than 500 nM at each step when a reactive unit is added.
  • the concentration of the plurality of templates can be 90 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM or 500 nM.
  • one or more of the plurality of second, third, fourth or fifth oligonucleotides is bound to a first member of a binding pair, e.g. , biotin.
  • one or more of the plurality of first, second, third or fourth small molecules bound to a first member of a binding pair is purified by contact with a second member of a binding pair, wherein the second member of the binding pair (e.g. , streptavidin) is bound to a solid support.
  • Binding pair members can be any binding pairs known in the art, including, for example, biotin and avidin or streptavidin, antibody-antigen pairs, etc.
  • the plurality of templates is reacted with a capping reagent that differentially caps the small molecules that did not react with one or more of the prior-added reactive units or soluble reagents.
  • the cap renders the small molecule that did not react with the one or more of the prior-added reactive units or soluble reagents unable to react with any further reactive units or soluble reagents. In this manner, these components can no longer participate in further chemical steps in the library preparation and are removed from the library pool upon stepwise purification.
  • each first oligonucleotide comprises a unique tag sequence that defines the linker or capping group, or any other structural modification to any small molecule that was not achieved through a DNA-templated reaction step.
  • the nucleic acid template can direct a wide variety of chemical reactions without obvious structural requirements by specifically recruiting reactants linked to complementary oligonucleotides.
  • the template hybridizes or anneals to one or more transfer units to direct the synthesis of a reaction intermediate that can subsequently be converted by further chemical reaction into a reaction product (e.g., a small molecule).
  • the reaction product then is selected or screened based on certain criteria, such as the ability to bind to a preselected target molecule.
  • the associated template can then be sequenced to decode the synthetic history of the reaction intermediate and/or the reaction product.
  • the length of the template may vary greatly depending upon the type of the nucleic acid-templated synthesis contemplated.
  • the template may be from 20 to 400 nucleotides in length, from 30 to 300 nucleotides in length, from 40 to 200 nucleotides in length, or from 50 to 100 nucleotides in length, from 40 to 400 nucleotides or from 40 to 100 nucleotides in length.
  • the template may be 40, 50, 60, 70, 80, 90, or 100 nucleotides in length.
  • the length of the template will of course depend on, for example, the length of the codons, the complexity of the library, the complexity and/or size of a reaction product (e.g., a small molecule), the use of spacer sequences, etc.
  • the sequence of the template may be designed in a number of ways. For example, the length of the codon must be determined and the codon sequences must be set. If a codon length of two is used, then using the four naturally occurring bases only 16 possible combinations are available to be used in encoding the library. If the length of the codon is increased to three (the number Nature uses in encoding proteins), the number of possible combinations increases to 64. If the length of the codon is increased to four, the number of possible combinations increases to 256. Other factors to be considered in determining the length of the codon are mismatching, frame-shifting, complexity of library, etc. As the length of the codon is increased up to a certain point the number of mismatches is decreased; however, excessively long codons likely will hybridize despite mismatched base pairs.
  • the codons may range from 3 to 50 nucleotides, from 3 to 40 nucleotides, from 3 to 30 nucleotides, from 3 to 20 nucleotides, from
  • nucleotides 3 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides,
  • nucleotides from 5 to 50 nucleotides, from 5 to 40 nucleotides, from 5 to 30 nucleotides, from 5 to 20 nucleotides, from 5 to 15 nucleotides, from 5 to 10 nucleotides, from 6 to 50 nucleotides, from 6 to 40 nucleotides, from 6 to 30 nucleotides, from 6 to 20 nucleotides, from 6 to 15 nucleotides, from 6 to 10 nucleotides, from 7 to 50 nucleotides, from 5 to 50 nucleotides, from 5 to 40 nucleotides, from 5 to 30 nucleotides, from 5 to 20 nucleotides, from 5 to 15 nucleotides, from 5 to 10 nucleotides, from 6 to 50 nucleotides, from 6 to 40 nucleotides, from 6 to 30 nucleotides, from 6 to 20 nucleotides, from 6 to 15 nucleotides, from 6 to 10 nucleotides
  • 7 to 40 nucleotides from 7 to 30 nucleotides, from 7 to 20 nucleotides, from 7 to 15 nucleotides, from 7 to 10 nucleotides, from 8 to 50 nucleotides, from 8 to 40 nucleotides, from
  • 8 to 30 nucleotides from 8 to 20 nucleotides, from 8 to 15 nucleotides, from 8 to 10 nucleotides, from 9 to 50 nucleotides, from 9 to 40 nucleotides, from 9 to 30 nucleotides, from
  • 9 to 20 nucleotides from 9 to 15 nucleotides, from 9 to 10 nucleotides.
  • codons are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the set of codons used in the template preferably maximizes the number of mismatches between any two codons within a codon set to ensure that only the proper anti-codons of the transfer units anneal to the codon sites of the template. Furthermore, it is important that the template has mismatches between all the members of one codon set and all the codons of a different codon set to ensure that the anti-codons do not inadvertently bind to the wrong codon set.
  • the choice of exemplary codon sets and methods of creating functional codon sets are described, for example, in U.S. Patent Nos. 7,491,494; 7,771,935; and 8,206,914, by Liu et al. Using this and other approaches, different sets of codons can be generated so that no codons are repeated.
  • nucleic acid identifiers may be incorporated into the template to identify a spacer moiety, linker moiety, capping reagent, or soluble reagent used in such synthesis.
  • additional nucleic acid (e.g., DNA) tag sequences are used to identify the reaction product covalently attached to the template at the end of the library synthesis, but they do not engage in DPC-catalyzed reactions. Instead, they are used to identify subsets of the library that have a particular linker, spacer, capping reagent, or soluble reagent.
  • a template oligonucleotide can be synthesized with a given tag sequence that corresponds to a specific linker moiety.
  • the individual linker moieties are chemically attached to the 5 '-amino terminus of the template, maintaining a direct relationship between the tag sequence and the linker moiety.
  • the template-linker conjugates can then be mixed and used in DPC reactions directly.
  • the final library products that are synthesized can be identified ultimately by sequencing the DNA revealing the structure of the small molecule by a consideration of both the codon regions and the tag region, which defines the linker moiety.
  • the linker, spacer, capping moiety or soluble reagent can be added independently to a template mixture and then an identifying tag DNA sequence for such added reagent added to the 3 '-end of the nucleic acid template by a ligation reaction.
  • the tag is unique for the linker, spacer, capping moiety or soluble reagent.
  • the sequence of the added tag can be identified and the identity of the non-DPC linker, spacer, capping moiety or soluble reagent can be determined.
  • an exemplary template can comprise one or more (e.g., two) tag regions (e.g., a 7 base tag) encoding a linker, spacer, capping moiety or soluble reagent.
  • Tag 2 corresponds to the linker of the attached small molecule, and Tag 1 to the spacer, although this relationship is not fixed and can be reversed (see also FIG. 3, showing that the fixed Tag 2 region defines the linker building block).
  • the sequence encoding a tag e.g., Tag 1 or Tag 2 stays constant for a given template.
  • FIG. 1 e.g., Tag 1 or Tag 2
  • the identity of the linker attached to a template can be determined by determining the sequence of Tag 2.
  • the identity of the spacer attached to a given template can be determined by determining the sequence of Tag 1.
  • the tag regions may range from 3 to 30 nucleotides, from 3 to 20 nucleotides, from 3 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 4 to 10 nucleotides, from 5 to 30 nucleotides, from 5 to 20 nucleotides, from 5 to 15 nucleotides, from 5 to 10 nucleotides, from 6 to 30 nucleotides, from 6 to 20 nucleotides, from 6 to 15 nucleotides, from 6 to 10 nucleotides, from 7 to 30 nucleotides, from 7 to 20 nucleotides, from 7 to 15 nucleotides, from 7 to 10 nucleotides, from 8 to 30 nucleotides, from 8 to 20 nucleotides, from 8 to 15 nucleotides, from 8 to 10 nucle
  • tag regions are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the 3'- and 5'-ends of the nucleic template optionally may comprise "fixed sequence regions” or “fixed regions” of bases (e.g., 10 bases), which represent polymerase chain reaction (PCR) primer sites. These sites can be used to facilitate PCR amplification of the DNA sequences the specific base sequence of the DNA is used to directly identify library components.
  • a PCR primer site comprises bases from both the fixed sequence region and an adjacent region of the template, e.g. , a portion of a tag or codon.
  • the fixed sequence regions can range from 2 to 50 nucleotides, from 2 to 40 nucleotides, from 2 to 30 nucleotides, from 2 to 20 nucleotides, from 2 to 15 nucleotides, from 2 to 10 nucleotides, from 3 to 50 nucleotides, from 3 to 40 nucleotides, from 3 to 30 nucleotides, from 3 to 20 nucleotides, from 2 to 50 nucleotides, from 2 to 40 nucleotides, from 2 to 30 nucleotides, from 2 to 20 nucleotides, from 2 to 50 nucleotides, from 2 to 40 nucleotides, from 2 to 30 nucleotides, from 2 to 20 nucleotides, from 2 to 50 nucleotides, from 2 to 40 nucleotides, from 2 to 30 nucleotides, from 2 to 20 nucleotides, from 2 to 50 nucleotides, from 2 to 40 nucleotides, from 2 to 30 nucleot
  • nucleotides 3 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides, from 3 to 10 nucleotides, from 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 30 nucleotides, from 4 to 20 nucleotides, from 4 to 15 nucleotides,
  • nucleotides from 5 to 50 nucleotides, from 5 to 40 nucleotides, from 5 to 30 nucleotides, from 5 to 20 nucleotides, from 5 to 15 nucleotides, from 5 to 10 nucleotides, from
  • 6 to 50 nucleotides from 6 to 40 nucleotides, from 6 to 30 nucleotides, from 6 to 20 nucleotides, from 6 to 15 nucleotides, from 6 to 10 nucleotides, from 7 to 50 nucleotides, from
  • 7 to 40 nucleotides from 7 to 30 nucleotides, from 7 to 20 nucleotides, from 7 to 15 nucleotides, from 7 to 10 nucleotides, from 8 to 50 nucleotides, from 8 to 40 nucleotides, from 8 to 30 nucleotides, from 8 to 20 nucleotides, from 8 to 15 nucleotides, from 8 to 10 nucleotides, from 9 to 50 nucleotides, from 9 to 40 nucleotides, from 9 to 30 nucleotides, from 9 to 20 nucleotides, from 9 to 15 nucleotides, from 9 to 10 nucleotides.
  • fixed sequence regions are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. While primers generally must be about 6 nucleotides in length, a fixed region can be less than 6 nucleotides if a PCR primer site comprises bases from both the fixed sequence region and an adjacent region of the template, e.g., a portion of a tag or codon.
  • the templates can be synthesized using methodologies well known in the art. These methods include both in vivo and in vitro methods including PCR, plasmid preparation, endonuclease digestion, solid phase synthesis (for example, using an automated synthesizer), in vitro transcription, strand separation, etc.
  • the template when desired can be attached (for example, covalently or non-covalently attached) with a reactive unit of interest using standard coupling chemistries known in the art.
  • a linker is used to attach a reactive unit of interest to the template.
  • the linker can be bivalent (have two functional groups for attachment) or trivalent (have three functional groups for attachment).
  • the linker can be defined by a tag sequence on the template, as described above.
  • oligonucleotides are synthesized using standard phosphoramidite 3' to 5' chemistries, although alternatively, synthesis in the 5' to 3' direction can be performed.
  • the constant 3' end is synthesized. This is then split into n different vessels, where n is the number of different codons to appear at that position in the template. For each vessel, one of the n different codons is synthesized on the (growing) 5' end of the constant 3' end. Thus, each vessel contains, from 5' to 3', a different codon attached to a constant 3' end.
  • n vessels then are pooled, so that a single vessel contains n different codons attached to the constant 3' end. Any constant bases adjacent the 5' end of the codon are now synthesized.
  • the pool then is split into m different vessels, where m is the number of different codons to appear at the next (more 5') position of the template.
  • a different codon is synthesized (at the 5' end of the growing oligonucleotide) in each of the m vessels.
  • the resulting oligonucleotides are pooled in a single vessel. Splitting, synthesizing, and pooling are repeated as required to synthesize all codons and constant regions in the oligonucleotides.
  • a template can be constructed comprising, e.g., a fixed region 3' and 5' of the template, two tags (Tag 1 and Tag 2), and three codons. Each of the three codons has 24 possible variants, for a total of 13,824 different DNA sequences.
  • larger libraries can be produced by combining multiple mixtures of templates encoding different DNA sequences. For example (and as shown in FIG. 4), 16 mixtures of templates encoding 13,824 different DNA sequences can be combined to produce a single library of , e.g., 221,184 different DNA sequences. As shown in FIG. 6, even larger libraries can be produced by using a fourth codon.
  • a transfer unit comprises an oligonucleotide containing an anti-codon sequence and a reactive unit.
  • the anti-codons are designed to be complementary to the codons present in the template. Accordingly, the sequences used in the template and the codon lengths should be considered when designing the anti-codons. Any molecule complementary to a codon used in the template may be used, including natural or non-natural nucleotides. In certain
  • the codons include one or more bases found in nature (i.e. , thymidine, uracil, guanidine, cytosine, and adenine).
  • the anti-codon is associated with a particular type of reactive unit to form a transfer unit.
  • the anti-codon can be associated with a reactive unit or reactant that is used to modify a small molecule scaffold.
  • the reactant is linked to the anti-codon via a connector long enough to allow the reactant to come into reactive proximity with the small molecule scaffold.
  • the connector preferably has a length and composition to permit a specific reaction between the annealed template and reactant, while minimizing and preferably preventing the occurrence of non-specific reactions (e.g. , non-specific intramolecular reactions).
  • the reactants include a variety of reagents as demonstrated by the wide range of reactions that can be utilized in nucleic acid-templated synthesis and can be any chemical group, catalyst (e.g., organometallic compounds), or reactive moiety (e.g., electrophiles, nucleophiles) known in the chemical arts.
  • catalyst e.g., organometallic compounds
  • reactive moiety e.g., electrophiles, nucleophiles
  • the anti-codon can be associated with the reactant through a cleavable connector.
  • the connector can be cleavable by light, oxidation, hydrolysis, exposure to acid, exposure to base, reduction, etc.
  • Fruchtel et al. (1996) ANGEW. CHEM. INT. ED. ENGL. 35 : 17 describes a variety of linkages useful in the practice of the invention.
  • the linker facilitates contact of the reactant with the small molecule scaffold and in certain embodiments, depending on the desired reaction, positions DNA as a leaving group ("autocleavable” strategy), or may link reactive groups to the template via the "scarless” connector strategy (which yields product without leaving behind an additional atom or atoms having chemical functionality), or a "useful scar” strategy (in which a portion of the connector is left behind to be functionalized in subsequent steps following connector cleavage).
  • the DNA-reactive group bond is cleaved as a natural consequence of the reaction.
  • DNA- templated reaction of one reactive group is followed by cleavage of the connector attached through a second reactive group to yield products without leaving behind additional atoms capable of providing chemical functionality.
  • a "useful scar” may be utilized on the theory that it may be advantageous to introduce useful atoms and/or chemical groups as a consequence of connector cleavage.
  • a "useful scar” is left behind following connector cleavage and can be functionalized in subsequent steps.
  • transfer units can be used at submillimolar concentrations (e.g. less than 100 ⁇ , less than 10 ⁇ , less than 1 ⁇ , less than 100 nM, or less than 10 nM).
  • a linker can be defined by a tag, which can be used to identify the final reaction product covalently attached to the template.
  • the linker preferably is multivalent, and can be bivalent, trivalent, tetravalent, etc.
  • Exemplary linkers include a diamino acid, an azido-amino acid, an acetylenic amino acid, a haloaromatic functionalized amino acid, or any other similar multivalent building block, with the functionality either present or in a protected or precursor form.
  • Exemplary protected or precursor forms include but are not limited to Boc, Alloc, or Fmoc carbamate forms, or amines can be generated from azides by using a reduction step.
  • Spacer moieties can be used to add additional diversity to small molecule libraries, to increase the size of small molecules (e.g. , macrocycles), or increase the spacing between other moieties in molecules, or to introduce new and diverse functionality.
  • a spacer can be defined by a tag (see FIGS. 1, 5 and 6), which can be used to identify the final reaction product covalently attached to the template.
  • Exemplary spacers include but are not limited to amino acids, carboxy alkynes, bis-carboxylic acids, amino aldehydes, and bromo carboxylic acids.
  • a variety of small molecule compounds and/or libraries can be prepared using the methods described herein.
  • compounds that are not, or do not resemble, nucleic acids or analogs thereof, are synthesized according to the method of the invention. It is contemplated that the small molecules can include macrocycles.
  • an evolvable template can be used.
  • the template can include a small molecule scaffold upon which the small molecule is to be built (e.g. , a first reactive unit), or a small molecule scaffold may be added to the template.
  • the small molecule scaffold can be any chemical compound with two or more sites for functionalization.
  • the small molecule scaffold can include a ring system (e.g. , the ABCD steroid ring system found in cholesterol) with functionalizable groups coupled to the atoms making up the rings.
  • the small molecule may be the underlying core scaffold structure of a pharmaceutical agent such as morphine, epothilone or a cephalosporin antibiotic.
  • the sites or groups to be functionalized on the small molecule scaffold may be protected using methods and protecting groups known in the art.
  • the protecting groups used in a small molecule scaffold may be orthogonal to one another so that protecting groups can be removed one at a time.
  • the transfer units comprise an anti-codon associated with a reactant or a building block for use in modifying, adding to, or taking away from the small molecule scaffold.
  • the reactants or building blocks may be, for example, electrophiles (e.g. , anhydrides, acid chlorides, esters, nitriles, imines), nucleophiles (e.g. , amines, hydroxyl groups, thiols), catalysts (e.g. , organometallic catalysts), or side chains.
  • the transfer units are allowed to contact the template under hybridizing conditions.
  • the attached reactant or building block is allowed to react with a site on the small molecule scaffold to produce one or more reaction intermediates.
  • protecting groups on the small molecule template are removed one at a time from the sites to be functionalized so that the reactant of the transfer unit will react at only the desired position on the scaffold.
  • reaction conditions, linker, reactant, and site to be functionalized are chosen to avoid unwanted side reactions and accelerate desired intramolecular reactions. Sequential or simultaneous contacting of the template with transfer units can be employed depending on the particular compound to be synthesized.
  • the newly synthesized small molecule remains associated with the template that encoded its synthesis.
  • Decoding the sequence of the template permits the deconvolution of the synthetic history and thereby the structure of the small molecule.
  • nucleic acid-tempi ated reactions Known chemical reactions for synthesizing polymers, small molecules, or other molecules can be used in nucleic acid-tempi ated reactions.
  • reactions such as those listed in March 's Advanced Organic Chemistry, Organic Reactions, Organic Syntheses, organic text books, journals such as Journal of the American Chemical Society, Journal of Organic Chemistry, Tetrahedron, etc., and Carruther's Some Modern Methods of Organic Chemistry can be used.
  • the chosen reactions preferably are compatible with nucleic acids such as DNA or RNA or are compatible with the modified nucleic acids used as the template.
  • Reactions useful in nucleic-acid templated chemistry include, for example, substitution reactions, carbon-carbon bond forming reactions, elimination reactions, acylation reactions, and addition reactions. An illustrative but not exhaustive list of aliphatic
  • nucleophilic substitution reactions useful in the present invention includes, for example, S N 2 reactions, SNI reactions, SNI reactions, allylic rearrangements, nucleophilic substitution at an aliphatic trigonal carbon, and nucleophilic substitution at an aromatic carbon.
  • Specific aliphatic nucleophilic substitution reactions with oxygen nucleophiles include, for example, hydrolysis of alkyl halides, hydrolysis of gem-dihalides, hydrolysis of 1,1,1 -trihalides, hydrolysis of alkyl esters or inorganic acids, hydrolysis of diazo ketones, hydrolysis of acetal and enol ethers, hydrolysis of epoxides, hydrolysis of acyl halides, hydrolysis of anhydrides, hydrolysis of carboxylic esters, hydrolysis of amides, alkylation with alkyl halides (Williamson Reaction), epoxide formation, alkylation with inorganic esters, alkylation with diazo compounds, dehydration of alcohols, transetherification, alcoholysis of epoxides, alkylation with onium salts, hydroxylation of silanes, alcoholysis of acyl halides, alcoholysis of anhydrides, esterification of carboxylic acids, alcoholy
  • Specific aliphatic nucleophilic substitution reactions with sulfur nucleophiles include, for example, attack by SH at an alkyl carbon to form thiols, attack by S at an alkyl carbon to form thioethers, attack by SH or SR at an acyl carbon, formation of disulfides, formation of Bunte salts, alkylation of sulfinic acid salts, and formation of alkyl thiocyanates.
  • Aliphatic nucleophilic substitution reactions with nitrogen nucleophiles include, for example, alkylation of amines, N-arylation of amines, replacement of a hydroxy by an amino group, transamination, transamidation, alkylation of amines with diazo compounds, amination of epoxides, amination of oxetanes, amination of aziridines, amination of alkanes, formation of isocyanides, acylation of amines by acyl halides, acylation of amines by anhydrides, acylation of amines by carboxylic acids, acylation of amines by carboxylic esters, acylation of amines by amides, acylation of amines by other acid derivatives, N-alkylation or N-arylation of amides and imides, N-acylation of amides and imides, formation of aziridines from epoxides, formation of nitro compounds, formation of azides, formation
  • Aliphatic nucleophilic substitution reactions with halogen nucleophiles include, for example, attack at an alkyl carbon, halide exchange, formation of alkyl halides from esters of sulfuric and sulfonic acids, formation of alkyl halides from alcohols, formation of alkyl halides from ethers, formation of halohydrins from epoxides, cleavage of carboxylic esters with lithium iodide, conversion of diazo ketones to a-halo ketones, conversion of amines to halides, conversion of tertiary amines to cyanamides (the von Braun reaction), formation of acyl halides from carboxylic acids, and formation of acyl halides from acid derivatives.
  • Aliphatic nucleophilic substitution reactions using hydrogen as a nucleophile include, for example, reduction of alkyl halides, reduction of tosylates, other sulfonates, and similar compounds, hydrogenolysis of alcohols, hydrogenolysis of esters (Barton-McCombie reaction), hydrogenolysis of nitriles, replacement of alkoxyl by hydrogen, reduction of epoxides, reductive cleavage of carboxylic esters, reduction of a C-N bond, desulfurization, reduction of acyl halides, reduction of carboxylic acids, esters, and anhydrides to aldehydes, and reduction of amides to aldehydes.
  • aliphatic nucleophilic substitution reactions using carbon nucleophiles include, for example, coupling with silanes, coupling of alkyl halides (the Wurtz reaction), the reaction of alkyl halides and sulfonate esters with Group I (I A) and II (II A) organometallic reagents, reaction of alkyl halides and sulfonate esters with organocuprates, reaction of alkyl halides and sulfonate esters with other organometallic reagents, allylic and propargylic coupling with a halide substrate, coupling of organometallic reagents with esters of sulfuric and sulfonic acids, sulfoxides, and sulfones, coupling involving alcohols, coupling of organometallic reagents with carboxylic esters, coupling
  • Reactions which involve nucleophilic attack at a sulfonyl sulfur atom may also be used in the present invention and include, for example, hydrolysis of sulfonic acid derivatives (attack by OH), formation of sulfonic esters (attack by OR), formation of sulfonamides (attack by nitrogen), formation of sulfonyl halides (attack by halides), reduction of sulfonyl chlorides (attack by hydrogen), and preparation of sulfones (attack by carbon).
  • Aromatic electrophilic substitution reactions may also be used in nucleotide- templated chemistry. Hydrogen exchange reactions are examples of aromatic electrophilic substitution reactions that use hydrogen as the electrophile. Aromatic electrophilic substitution reactions which use nitrogen electrophiles include, for example, nitration and nitro-de- hydrogenation, nitrosation of nitroso-de-hydrogenation, diazonium coupling, direct introduction of the diazonium group, and animation or amino-de-hydrogenation. Reactions of this type with sulfur electrophiles include, for example, sulfonation, sulfo-de-hydrogenation, halosulfonation, halosulfo-de-hydrogenation, sulfurization, and sulfonylation. Reactions using halogen electrophiles include, for example, halogenation, and halo-de-hydrogenation.
  • Aromatic electrophilic substitution reactions with carbon electrophiles include, for example, Friedel-Crafts alkylation, alkylation, alkyl-de-hydrogenation, Friedel-Crafts arylation (the Scholl reaction), Friedel-Crafts acylation, formylation with disubstituted formamides, formylation with zinc cyanide and HC1 (the Gatterman reaction), formylation with chloroform (the Reimer-Tiemann reaction), other formylations, formyl-de-hydrogenation, carboxylation with carbonyl halides, carboxylation with carbon dioxide (the Kolbe-Schmitt reaction), amidation with isocyanates, N-alkylcarbamoyl-de-hydrogenation, hydroxyalkylation, hydroxyalkyl-de-hydrogenation, cyclodehydration of aldehydes and ketones, haloalkylation, halo-de-hydrogenation, aminoalkylation, amidoalkylation, dial
  • dialkylamino-de-hydrogenation dialkylamino-de-hydrogenation, thioalkylation, acylation with nitriles (the Hoesch reaction), cyanation, and cyano-de-hydrogenation.
  • Reactions using oxygen electrophiles include, for example, hydroxylation and hydroxy-de-hydrogenation.
  • Rearrangement reactions include, for example, the Fries rearrangement, migration of a nitro group, migration of a nitroso group (the Fischer-Hepp Rearrangement), migration of an arylazo group, migration of a halogen (the Orton rearrangement), migration of an alkyl group, etc.
  • Other reactions on an aromatic ring include the reversal of a Friedel-Crafts alkylation, decarboxylation of aromatic aldehydes, decarboxylation of aromatic acids, the Jacobsen reaction, deoxygenation, desulfonation, hydro-de-sulfonation, dehalogenation, hydro-de- halogenation, and hydrolysis of organometallic compounds.
  • Aliphatic electrophilic substitution reactions are also useful. Reactions using the SEI , SE2 (front), SE2 (back), SEI, addition-elimination, and cyclic mechanisms can be used in the present invention. Reactions of this type with hydrogen as the leaving group include, for example, hydrogen exchange (deuterio-de-hydrogenation, deuteriation), migration of a double bond, and keto-enol tautomerization. Reactions with halogen electrophiles include, for example, halogenation of aldehydes and ketones, halogenation of carboxylic acids and acyl halides, and halogenation of sulfoxides and sulfones.
  • Reactions with nitrogen electrophiles include, for example, aliphatic diazonium coupling, nitrosation at a carbon bearing an active hydrogen, direct formation of diazo compounds, conversion of amides to a-azido amides, direct amination at an activated position, and insertion by nitrenes.
  • Reactions with sulfur or selenium electrophiles include, for example, sulfenylation, sulfonation, and selenylation of ketones and carboxylic esters.
  • Reactions with carbon electrophiles include, for example, acylation at an aliphatic carbon, conversion of aldehydes to ⁇ -keto esters or ketones, cyanation, cyano-de- hydrogenation, alkylation of alkanes, the Stork enamine reaction, and insertion by carbenes.
  • Reactions with metal electrophiles include, for example, metalation with organometallic compounds, metalation with metals and strong bases, and conversion of enolates to silyl enol ethers.
  • Aliphatic electrophilic substitution reactions with metals as leaving groups include, for example, replacement of metals by hydrogen, reactions between organometallic reagents and oxygen, reactions between organometallic reagents and peroxides, oxidation of trialkylboranes to borates, conversion of Grignard reagents to sulfur compounds, halo-de-metalation, the conversion of organometallic compounds to amines, the conversion of organometallic compounds to ketones, aldehydes, carboxylic esters and amides, cyano-de-metalation, transmetalation with a metal, transmetalation with a metal halide, transmetalation with an organometallic compound, reduction of alkyl halides, metallo-de-halogenation, replacement of a halogen by a metal from an organometallic compound, decarboxylation of aliphatic acids, cleavage of alkoxides, replacement of a carboxyl group by an acyl group, basic
  • Electrophilic substitution reactions at nitrogen include, for example, diazotization, conversion of hydrazines to azides, N-nitrosation, N-nitroso-de-hydrogenation, conversion of amines to azo compounds, N-halogenation, N-halo-de-hydrogenation, reactions of amines with carbon monoxide, and reactions of amines with carbon dioxide.
  • Aromatic nucleophilic substitution reactions may also be used in the present invention. Reactions proceeding via the S N Ar mechanism, the SNI mechanism, the benzyne mechanism, the SRNI mechanism, or other mechanism, for example, can be used. Aromatic nucleophilic substitution reactions with oxygen nucleophiles include, for example, hydroxy-de- halogenation, alkali fusion of sulfonate salts, and replacement of OR or OAr. Reactions with sulfur nucleophiles include, for example, replacement by SH or SR. Reactions using nitrogen nucleophiles include, for example, replacement by NH 2 , NHR, or NR 2 , and replacement of a hydroxy group by an amino group.
  • Reactions with halogen nucleophiles include, for example, the introduction of halogens.
  • Aromatic nucleophilic substitution reactions with hydrogen as the nucleophile include, for example, reduction of phenols and phenolic esters and ethers, and reduction of halides and nitro compounds.
  • Reactions with carbon nucleophiles include, for example, the Rosenmund-von Braun reaction, coupling of organometallic compounds with aryl halides, ethers, and carboxylic esters, arylation at a carbon containing an active hydrogen, conversions of aryl substrates to carboxylic acids, their derivatives, aldehydes, and ketones, and the Ullmann reaction.
  • Reactions with hydrogen as the leaving group include, for example, alkylation, arylation, and animation of nitrogen heterocycles.
  • Reactions with N 2 + as the leaving group include, for example, hydroxy-de-diazoniation, replacement by sulfur-containing groups, iodo-de-diazoniation, and the Schiemann reaction.
  • Rearrangement reactions include, for example, the von Richter rearrangement, the Sommelet-Hauser rearrangement, rearrangement of aryl hydroxylamines, and the Smiles rearrangement.
  • Reactions involving free radicals can also be used, although the free radical reactions used in nucleotide-templated chemistry should be carefully chosen to avoid modification or cleavage of the nucleotide template.
  • free radical substitution reactions can be used in the present invention.
  • Particular free radical substitution reactions include, for example, substitution by halogen, halogenation at an alkyl carbon, allylic halogenation, benzylic halogenation, halogenation of aldehydes, hydroxylation at an aliphatic carbon, hydroxylation at an aromatic carbon, oxidation of aldehydes to carboxylic acids, formation of cyclic ethers, formation of hydroperoxides, formation of peroxides, acyloxylation, acyloxy-de- hydrogenation, chlorosulfonation, nitration of alkanes, direct conversion of aldehydes to amides, amidation and animation at an alkyl carbon, simple coupling at a susceptible position, coupling of alkynes, arylation of aromatic compounds by diazonium salts, arylation of activated alkenes by diazonium salts (the Meerwein arylation), arylation and alkylation of alkenes
  • Free radical substitution reactions with metals as leaving groups include, for example, coupling of Grignard reagents, coupling of boranes, and coupling of other organometallic reagents. Reaction with halogen as the leaving group are included.
  • Other free radical substitution reactions with various leaving groups include, for example, desulfurization with Raney Nickel, conversion of sulfides to organolithium compounds, decarboxylative dimerization (the Kolbe reaction), the Hunsdiecker reaction, decarboxylative allylation, and decarbonylation of aldehydes and acyl halides.
  • reactions involving additions to carbon-carbon multiple bonds are also used in nucleotide-templated chemistry. Any mechanism may be used in the addition reaction including, for example, electrophilic addition, nucleophilic addition, free radical addition, and cyclic mechanisms. Reactions involving additions to conjugated systems can also be used. Addition to cyclopropane rings can also be utilized.
  • Particular reactions include, for example, isomerization, addition of hydrogen halides, hydration of double bonds, hydration of triple bonds, addition of alcohols, addition of carboxylic acids, addition of H 2 S and thiols, addition of ammonia and amines, addition of amides, addition of hydrazoic acid, hydrogenation of double and triple bonds, other reduction of double and triple bonds, reduction of the double and triple bonds of conjugated systems, hydrogenation of aromatic rings, reductive cleavage of cyclopropanes, hydroboration, other hydrometalations, addition of alkanes, addition of alkenes and/or alkynes to alkenes and/or alkynes (e.g., pi-cation cyclization reactions, hydro-alkenyl- addition), ene reactions, the Michael reaction, addition of organometallics to double and triple bonds not conjugated to carbonyls, the addition of two alkyl groups to an alkyne, 1,4-addition of organometallic compounds to activated
  • acylamidation (addition of oxygen, carbon or nitrogen, carbon), 1,3-dipolar cycloaddition (addition of oxygen, nitrogen, carbon), Huisgen reaction of azides and acetylenes, Diels-Alder reaction, heteroatom Diels-Alder reaction, all carbon 3 +2 cycloadditions, dimerization of alkenes, the addition of carbenes and carbenoids to double and triple bonds, trimerization and tetramerization of alkynes, and other cycloaddition reactions. [0087] In addition to reactions involving additions to carbon-carbon multiple bonds, addition reactions to carbon-hetero multiple bonds can be used in nucleotide-templated chemistry.
  • Exemplary reactions include, for example, the addition of water to aldehydes and ketones (formation of hydrates), hydrolysis of carbon-nitrogen double bond, hydrolysis of aliphatic nitro compounds, hydrolysis of nitriles, addition of alcohols and thiols to aldehydes and ketones, reductive alkylation of alcohols, addition of alcohols to isocyanates, alcoholysis of nitriles, formation of xanthates, addition of H 2 S and thiols to carbonyl compounds, formation of bisulfite addition products, addition of amines to aldehydes and ketones, addition of amides to aldehydes, reductive alkylation of ammonia or amines, the Mannich reaction, the addition of amines to isocyanates, addition of ammonia or amines to nitriles, addition of amines to carbon disulfide and carbon dioxide, addition of hydrazine derivative to carbonyl compounds, formation of oximes,
  • carbodiimides the conversion of carboxylic acid salts to nitriles, the formation of epoxides from aldehydes and ketones, the formation of episulfides and episulfones, the formation of ⁇ - lactones and oxetanes (e.g. , the Paterno-Buchi reaction), the formation of ⁇ -lactams, etc.
  • Reactions involving addition to isocyanides include the addition of water to isocyanides, the Passerini reaction, the Ug reaction, and the formation of metalated aldimines.
  • Elimination reactions including ⁇ , ⁇ , and ⁇ eliminations, as well as extrusion reactions, can be performed using nucleotide-templated chemistry, although the strength of the reagents and conditions employed should be considered.
  • Preferred elimination reactions include reactions that go by El, E2, ElcB, or E2C mechanisms.
  • Exemplary reactions include, for example, reactions in which hydrogen is removed from one side (e.g., dehydration of alcohols, cleavage of ethers to alkenes, the Chugaev reaction, ester decomposition, cleavage of quaternary ammonium hydroxides, cleavage of quaternary ammonium salts with strong bases, cleavage of amine oxides, pyrolysis of keto-ylids, decomposition of toluene-p- sulfonylhydrazones, cleavage of sulfoxides, cleavage of selenoxides, cleavage of sulfones, dehydrohalogenation of alkyl halides, dehydrohalogenation of acyl halides,
  • reactions in which hydrogen is removed from one side e.g., dehydration of alcohols, cleavage of ethers to alkenes, the Chugaev reaction, ester decomposition, cleavage of
  • Extrusion reactions include, for example, extrusion of N 2 from pyrazolines, extrusion of N 2 from pyrazoles, extrusion of N 2 from triazolines, extrusion of CO, extrusion of CO2, extrusion of SO2, the Story synthesis, and alkene synthesis by twofold extrusion.
  • Rearrangements including, for example, nucleophilic rearrangements, electrophilic rearrangements, prototropic rearrangements, and free-radical rearrangements, can also be performed using nucleotide-templated chemistry. Both 1,2 rearrangements and non-1,2 rearrangements can be performed. Exemplary reactions include, for example, carbon-to-carbon migrations of R, H, and Ar (e.g., Wagner-Meerwein and related reactions, the Pinacol rearrangement, ring expansion reactions, ring contraction reactions, acid-catalyzed
  • Villiger rearrangement and rearrangment of hydroperoxides nitrogen-to-carbon, oxygen-to- carbon, and sulfur-to-carbon migration (e.g., the Stevens rearrangement, and the Wittig rearrangement), boron-to-carbon migrations (e.g., conversion of boranes to alcohols (primary or otherwise), conversion of boranes to aldehydes, conversion of boranes to carboxylic acids, conversion of vinylic boranes to alkenes, formation of alkynes from boranes and acetylides, formation of alkenes from boranes and acetylides, and formation of ketones from boranes and acetylides), electrocyclic rearrangements (e.g., of cyclobutenes and 1,3-cyclohexadienes, or conversion of stilbenes to phenanthrenes), sigmatropic rearrangements (e.g., (l,j) sigmatropic migrations of hydrogen,
  • Oxidative and reductive reactions may also be performed using nucleotide-templated chemistry.
  • Exemplary reactions may involve, for example, direct electron transfer, hydride transfer, hydrogen-atom transfer, formation of ester intermediates, displacement mechanisms, or addition-elimination mechanisms.
  • Exemplary oxidations include, for example, eliminations of hydrogen (e.g., aromatization of six-membered rings, dehydrogenations yielding carbon- carbon double bonds, oxidation or dehydrogenation of alcohols to aldehydes and ketones, oxidation of phenols and aromatic amines to quinones, oxidative cleavage of ketones, oxidative cleavage of aldehydes, oxidative cleavage of alcohols, ozonolysis, oxidative cleavage of double bonds and aromatic rings, oxidation of aromatic side chains, oxidative decarboxylation, and bisdecarboxylation), reactions involving replacement of hydrogen by oxygen (e.g., oxidation of methylene to carbonyl, oxidation of methylene to OH, C0 2 R, or OR, oxidation of
  • arylmethanes oxidation of ethers to carboxylic esters and related reactions, oxidation of aromatic hydrocarbons to quinones, oxidation of amines or nitro compounds to aldehydes, ketones, or dihalides, oxidation of primary alcohols to carboxylic acids or carboxylic esters, oxidation of alkenes to aldehydes or ketones, oxidation of amines to nitroso compounds and hydroxylamines, oxidation of primary amines, oximes, azides, isocyanates, or nitroso compounds, to nitro compounds, oxidation of thiols and other sulfur compounds to sulfonic acids), reactions in which oxygen is added to the subtrate (e.g., oxidation of alkynes to a- diketones, oxidation of tertiary amines to amine oxides, oxidation of thioesters to sulfoxides and sulfones, and oxidation of
  • Exemplary reductive reactions include, for example, reactions involving replacement of oxygen by hydrogen (e.g., reduction of carbonyl to methylene in aldehydes and ketones, reduction of carboxylic acids to alcohols, reduction of amides to amines, reduction of carboxylic esters to ethers, reduction of cyclic anhydrides to lactones and acid derivatives to alcohols, reduction of carboxylic esters to alcohols, reduction of carboxylic acids and esters to alkanes, complete reduction of epoxides, reduction of nitro compounds to amines, reduction of nitro compounds to hydroxylamines, reduction of nitroso compounds and hydroxylamines to amines, reduction of oximes to primary amines or aziridines, reduction of azides to primary amines, reduction of nitrogen compounds, and reduction of sulfonyl halides and sulfonic acids to thiols), removal of oxygen from the substrate (e.g., reduction of amine oxides and az
  • nucleic acid-templated functional group interconversions permit the generation of library diversity by sequential unmasking.
  • the sequential unmasking approach offers the major advantage of enabling reactants that would normally lack the ability to be linked to a nucleic acid (for example, simple alkyl halides) to contribute to library diversity by reacting with a sequence-specified subset of templates in an intermolecular, non-templated reaction mode. This advantage significantly increases the types of structures that can be generated.
  • One embodiment of the invention involves deprotection or unmasking of functional groups present in a reactive unit.
  • a nucleic acid-template is associated with a reactive unit that contains a protected functional group.
  • a transfer unit comprising an oligonucleotide complementary to the template codon region and a reagent capable of removing the protecting group, is annealed to the template, and the reagent reacts with the protecting group, removing it from the reactive unit.
  • the exposed functional group then is subjected to a reagent not linked to a nucleic acid.
  • the reactive unit contains two or more protected functional groups.
  • the protecting groups are orthogonal protecting groups that are sequentially removed by iterated annealing with reagents linked to transfer units.
  • Another embodiment of the invention involves interconversions of functional groups present on a reactive unit.
  • a transfer unit associated with a reagent that can catalyze a reaction is annealed to a template bearing the reactive unit.
  • a reagent not linked to a nucleic acid is added to the reaction, and the transfer unit reagent catalyzes the reaction between the unlinked reagent and the reactive unit, yielding a newly functionalized reactive unit.
  • the reactive unit contains two or more functional groups which are sequentially interconverted by iterative exposure to different transfer unit-bound reagents.
  • Nucleic acid-templated reactions can occur in aqueous or non-aqueous (i.e. , organic) solutions, or a mixture of one or more aqueous and non-aqueous solutions.
  • aqueous solutions reactions can be performed at pH ranges from about 2 to about 12, or preferably from about 2 to about 10, or more preferably from about 4 to about 10.
  • the reactions used in DNA- templated chemistry preferably should not require very basic conditions (e.g. , pH > 12, pH > 10) or very acidic conditions (e.g., pH ⁇ 1, pH ⁇ 2, pH ⁇ 4), because extreme conditions may lead to degradation or modification of the nucleic acid template and/or molecule (for example, the polymer, or small molecule) being synthesized.
  • the aqueous solution can contain one or more inorganic salts, including, but not limited to, NaCl, Na 2 SC>4, KC1, Mg +2 , Mn +2 , etc., at various concentrations.
  • Organic solvents suitable for nucleic acid-templated reactions include, but are not limited to, methylene chloride, chloroform, dimethylformamide, and organic alcohols, including methanol and ethanol.
  • quatemized ammonium salts such as, for example, long chain
  • tetraalkylammonium salts can be added (Jost et al. (1989) NUCLEIC ACIDS RES. 17: 2143; Mel'nikov et al. (1999) LANGMUIR 15: 1923-1928).
  • Nucleic acid-templated reactions may require a catalyst, such as, for example, homogeneous, heterogeneous, phase transfer, and asymmetric catalysis. In other embodiments, a catalyst is not required.
  • a catalyst is not required.
  • additional, accessory reagents not linked to a nucleic acid are preferred in some embodiments.
  • Useful accessory reagents can include, for example, oxidizing agents (e.g., NaI0 4 ); reducing agents (e.g., NaCNBH 3 ); activating reagents (e.g., EDC, NHS, and sulfo-NHS); transition metals such as nickel (e.g., Ni(N0 3 ) 2 ), rhodium (e.g.
  • RJ1CI3 ruthenium
  • copper e.g. Cu(N0 3 ) 2
  • cobalt e.g. CoCl 2
  • iron e.g. Fe(N03) 3
  • osmium e.g. OSO 4
  • titanium e.g. T1CI4 or titanium tetraisopropoxide
  • palladium e.g. NaPdC
  • Ln transition metal ligands (e.g., phosphines, amines, and halides); Lewis acids; and Lewis bases.
  • Reaction conditions preferably are optimized to suit the nature of the reactive units and oligonucleotides used.
  • reaction products e.g., small molecules
  • desired activities such as catalytic activity, binding affinity, or a particular effect in an activity assay
  • affinity selections may be performed according to the principles used in library-based selection methods such as phage display, polysome display, and mRNA-fusion protein displayed peptides.
  • Selection for catalytic activity may be performed by affinity selections on transition- state analog affinity columns (Baca et al. (1997) PROC. NATL. ACAD. SCI. USA 94(19): 10063- 8) or by function-based selection schemes (Pedersen et al. (1998) PROC. NATL. ACAD. SCI.
  • the templates and reaction products can be selected (or screened) for binding to a target molecule.
  • selection or partitioning means any process whereby a library member bound to a target molecule is separated from library members not bound to target molecules. Selection can be accomplished by various methods known in the art.
  • the templates of the present invention contain a built-in function for direct selection and amplification.
  • binding to a target molecule preferably is selective, such that the template and the resulting reaction product (e.g. , a small molecule) bind preferentially with a specific target molecule, perhaps preventing or inducing a specific biological effect.
  • a binding molecule identified using the present invention may be useful as a therapeutic and/or diagnostic agent.
  • the selected templates optionally can be amplified and sequenced.
  • the selected reaction products if present in sufficient quantity, can be separated from the templates, purified (e.g., by HPLC, column chromatography, or other chromatographic method), and further characterized.
  • Binding assays provide a rapid means for isolating and identifying reaction products (e.g. , a small molecule) that bind to, for example, a surface (such as metal, plastic, composite, glass, ceramics, rubber, skin, or tissue); a polymer; a catalyst; or a target biomolecule such as a nucleic acid, a protein (including enzymes, receptors, antibodies, and glycoproteins), a signal molecule (such as cAMP, inositol triphosphate, peptides, or prostaglandins), a carbohydrate, or a lipid. Binding assays can be advantageously combined with activity assays for the effect of a reaction product on a function of a target molecule.
  • the selection strategy can be carried out to allow selection against almost any target. Importantly, the selection strategy does not require any detailed structural information about the target molecule or about the molecules in the libraries. The entire process is driven by the binding affinity involved in the specific recognition and binding of the molecules in the library to a given target. Examples of various selection procedures are described below.
  • the libraries of the present invention can contain molecules that could potentially bind to any known or unknown target.
  • the binding region of a target molecule could include a catalytic site of an enzyme, a binding pocket on a receptor (for example, a G-protein coupled receptor), a protein surface area involved in a protein-protein or protein-nucleic acid interaction (preferably a hot-spot region), or a specific site on DNA (such as the major groove).
  • the natural function of the target could be stimulated (agonized), reduced (antagonized), unaffected, or completely changed by the binding of the reaction product (e.g., a small molecule). This will depend on the precise binding mode and the particular binding site the reaction product occupies on the target.
  • Functional sites such as protein-protein interaction or catalytic sites
  • proteins often are more prone to bind molecules than are other more neutral surface areas on a protein.
  • these functional sites normally contain a smaller region that seems to be primarily responsible for the binding energy: the so-called "hot-spot regions" (Wells, et al. (1993) RECENT PROG. HORMONE RES. 48: 253- 262). This phenomenon facilitates selection for molecules affecting the biological function of a certain target.
  • the linkage between the template molecule and reaction product allows rapid identification of binding molecules using various selection strategies.
  • This invention broadly permits identifying binding molecules for any known target molecule.
  • novel unknown targets can be discovered by isolating binding molecules against unknown antigens (epitopes) and using these binding molecules for identification and validation.
  • the target molecule is designed to mimic a transition state of a chemical reaction; one or more reaction products resulting from the selection may stabilize the transition state and catalyze the chemical reaction.
  • the template-directed synthesis of the invention permits selection procedures analogous to other display methods such as phage display (Smith (1985) SCIENCE 228: 1315- 1317). Phage display selection has been used successfully on peptides (Wells et al. (1992) CURR. OP. STRUCT. BIOL. 2: 597-604), proteins (Marks et al. (1992) J. BIOL. CHEM. 267: 16007-16010) and antibodies (Winter et al. (1994) ANNU. REV. IMMUNOL. 12: 433-455). Similar selection procedures also are exploited for other types of display systems such as ribosome display Mattheakis et al. (1994) PROC. NATL.
  • ACAD. SCI. 91 : 9022-9026 and mRNA display (Roberts, et al. (1997) PROC. NATL. ACAD. SCI. 94: 12297-302).
  • the libraries of the present invention allow direct selection of target-specific molecules without requiring traditional ribosome-mediated translation.
  • the present invention also allows the display of small molecules which have not previously been synthesized directly from a nucleic acid template.
  • binding molecules from a library can be performed in any format to identify optimal binding molecules. Binding selections typically involve immobilizing the desired target molecule, adding a library of potential binders, and removing non-binders by washing. When the molecules showing low affinity for an immobilized target are washed away, the molecules with a stronger affinity generally remain attached to the target.
  • the enriched population remaining bound to the target after stringent washing is preferably eluted with, for example, acid, chaotropic salts, heat, competitive elution with a known ligand or by proteolytic release of the target and/or of template molecules.
  • the eluted templates are suitable for PCR, leading to many orders of amplification, whereby essentially each selected template becomes available at a greatly increased copy number for cloning, sequencing, and/or further enrichment or diversification.
  • the fraction of ligand bound to target is determined by the effective concentration of the target protein.
  • selection stringency is controllable by varying the effective concentration of target.
  • the target molecule (peptide, protein, DNA or other antigen) can be immobilized on a solid support, for example, a container wall, a wall of a microtiter plate well.
  • the library preferably is dissolved in aqueous binding buffer in one pot and equilibrated in the presence of immobilized target molecule. Non-binders are washed away with buffer. Those molecules that may be binding to the target molecule through their attached DNA templates rather than through their synthetic moieties can be eliminated by washing the bound library with unfunctionalized templates lacking PCR primer binding sites. Remaining bound library members then can be eluted, for example, by denaturation.
  • the target molecule can be immobilized on beads, particularly if there is doubt that the target molecule will adsorb sufficiently to a container wall, as may be the case for an unfolded target eluted from an SDS-PAGE gel.
  • the derivatized beads can then be used to separate high-affinity library members from nonbinders by simply sedimenting the beads in a benchtop centrifuge.
  • the beads can be used to make an affinity column. In such cases, the library is passed through the column one or more times to permit binding. The column then is washed to remove nonbinding library members.
  • Magnetic beads are essentially a variant on the above; the target is attached to magnetic beads which are then used in the selection.
  • Sepharose beads and the integrity of known properties of the target molecule can be verified.
  • Activated beads are available with attachment sites for -NH 2 or -COOH groups (which can be used for coupling).
  • the target molecule is blotted onto nitrocellulose or PVDF.
  • the blot should be blocked (e.g. , with BSA or similar protein) after immobilization of the target to prevent nonspecific binding of library members to the blot.
  • Library members that bind a target molecule can be released by denaturation, acid, or chaotropic salts.
  • elution conditions can be more specific to reduce background or to select for a desired specificity. Elution can be accomplished using proteolysis to cleave a connector between the target molecule and the immobilizing surface or between the reaction product (e.g. , a small molecule) and the template. Also, elution can be accomplished by competition with a known competitive ligand for the target molecule. Alternatively, a PCR reaction can be performed directly in the presence of the washed target molecules at the end of the selection procedure.
  • the binding molecules need not be elutable from the target to be selectable since only the template is needed for further amplification or cloning, not the reaction product itself. Indeed, some target molecules bind the most avid ligands so tightly that elution would be difficult.
  • the cells themselves can be used as the selection agent.
  • the library preferably is first exposed to cells not expressing the target molecule on their surfaces to remove library members that bind specifically or non specifically to other cell surface epitopes.
  • cells lacking the target molecule are present in large excess in the selection process and separable (by fluorescence-activated cell sorting (FACS), for example) from cells bearing the target molecule.
  • FACS fluorescence-activated cell sorting
  • a recombinant DNA encoding the target molecule can be introduced into a cell line; library members that bind the transformed cells but not the untransformed cells are enriched for target molecule binders.
  • This approach is also called subtraction selection and has successfully been used for phage display on antibody libraries (Hoogenboom et al. (1998) IMMUNOTECH 4: 1- 20).
  • a selection procedure can also involve selection for binding to cell surface receptors that are internalized so that the receptor together with the selected binding molecule passes into the cytoplasm, nucleus, or other cellular compartment, such as the Golgi or lysosomes.
  • Internalized library members can be distinguished from molecules attached to the cell surface by washing the cells, preferably with a denaturant. More preferably, standard subcellular fractionation techniques are used to isolate the selected library members in a desired subcellular compartment.
  • An alternative selection protocol also includes a known, weak ligand affixed to each member of the library.
  • the known ligand guides the selection by interacting with a defined part of the target molecule and focuses the selection on molecules that bind to the same region, providing a cooperative effect. This can be particularly useful for increasing the affinity of a ligand with a desired biological function but with too low a potency.
  • the selection process is well suited for optimizations, where the selection steps are made in series, starting with the selection of binding molecules and ending with an optimized binding molecule.
  • the procedures in each step can be automated using various robotic systems.
  • the invention permits supplying a suitable library and target molecule to a fully automatic system which finally generates an optimized binding molecule. Under ideal conditions, this process should run without any requirement for external work outside the robotic system during the entire procedure.
  • the selection methods of the present invention can be combined with secondary selection or screening to identify reaction products (e.g. , small molecules) capable of modifying target molecule function upon binding.
  • reaction products e.g. , small molecules
  • the methods described herein can be employed to isolate or produce binding molecules that bind to and modify the function of any protein or nucleic acid.
  • nucleic acid-templated chemistry can be used to identify, isolate, or produce binding molecules (1) affecting catalytic activity of target enzymes by inhibiting catalysis or modifying substrate binding; (2) affecting the functionality of protein receptors, by inhibiting binding to receptors or by modifying the specificity of binding to receptors; (3) affecting the formation of protein multimers by disrupting the quaternary structure of protein subunits; or (4) modifying transport properties of a protein by disrupting transport of small molecules or ions.
  • Functional assays can be included in the selection process. For example, after selecting for binding activity, selected library members can be directly tested for a desired functional effect, such as an effect on cell signaling. This can, for example, be performed via FACS methodologies.
  • the binding molecules of the invention can be selected for other properties in addition to binding. For example, to select for stability of binding interactions in a desired working environment. If stability in the presence of a certain protease is desired, that protease can be part of the buffer medium used during selection. Similarly, the selection can be performed in serum or cell extracts or in any type of medium, aqueous or organic. Conditions that disrupt or degrade the template should however be avoided to allow subsequent amplification.
  • selections for other desired properties can also be performed.
  • the selection should be designed such that library members with the desired activity are isolatable on that basis from other library members.
  • library members can be screened for the ability to fold or otherwise significantly change conformation in the presence of a target molecule, such as a metal ion, or under particular pH or salinity conditions.
  • the folded library members can be isolated by performing non- denaturing gel electrophoresis under the conditions of interest. The folded library members migrate to a different position in the gel and can subsequently be extracted from the gel and isolated.
  • reaction products that fluoresce in the presence of specific ligands may be selected by FACS based sorting of translated polymers linked through their DNA templates to beads. Those beads that fluoresce in the presence, but not in the absence, of the target ligand are isolated and characterized.
  • Useful beads with a homogenous population of nucleic acid- templates on any bead can be prepared using the split-pool synthesis technique on the bead, such that each bead is exposed to only a single nucleotide sequence.
  • a different anti-template (each complementary to only a single, different template) can be synthesized on beads using a split-pool technique, and then can anneal to capture a solution-phase library.
  • Biotin-terminated biopolymers can be selected for the actual catalysis of bond- breaking reactions by passing these biopolymers over a resin linked through a substrate to avidin. Those biopolymers that catalyze substrate cleavage self-elute from a column charged with this resin. Similarly, biotin-terminated biopolymers can be selected for the catalysis of bond-forming reactions. One substrate is linked to resin and the second substrate is linked to avidin. Biopolymers that catalyze bond formation between the substrates are selected by their ability to react the substrates together, resulting in attachment of the biopolymer to the resin.
  • Library members can also be selected for their catalytic effects on synthesis of a polymer to which the template is or becomes attached.
  • the library member may influence the selection of monomer units to be polymerized as well as how the polymerization reaction takes place (e.g., stereochemistry, tacticity, activity).
  • the synthesized polymers can be selected for specific properties, such as, molecular weight, density, hydrophobicity, tacticity, stereoselectivity, using standard techniques, such as, electrophoresis, gel filtration, centrifugal sedimentation, or partitioning into solvents of different hydrophobicities.
  • the attached template that directed the synthesis of the polymer can then be identified.
  • Library members that catalyze virtually any reaction causing bond formation between two substrate molecules or resulting in bond breakage into two product molecules can be selected using the schemes proposed herein.
  • bond forming catalysts for example, hetero Diels-Alder, Heck coupling, aldol reaction, or olefin metathesis catalysts
  • library members are covalently linked to one substrate through their 5' amino or thiol termini.
  • the other substrate of the reaction is synthesized as a derivative linked to biotin.
  • those library members that catalyze bond formation cause the biotin group to become covalently attached to themselves.
  • Active bond forming catalysts can then be separated from inactive library members by capturing the former with immobilized streptavidin and washing away inactive library members
  • library members that catalyze bond cleavage reactions such as retro-aldol reactions, amide hydrolysis, elimination reactions, or olefin dihydroxylation followed by periodate cleavage can be selected.
  • library members are covalently linked to biotinylated substrates such that the bond breakage reaction causes the disconnection of the biotin moiety from the library members.
  • active catalysts but not inactive library members, induce the loss of their biotin groups.
  • Streptavidin-linked beads can then be used to capture inactive polymers, while active catalysts are able to be eluted from the beads.
  • Related bond formation and bond cleavage selections have been used successfully in catalytic RNA and DNA evolution (Jaschke et al. (2000) CURR. OPIN. CHEM. BIOL. 4: 257-62) Although these selections do not explicitly select for multiple turnover catalysis, RNAs and DNAs selected in this manner have in general proven to be multiple turnover catalysts when separated from their substrate moieties (Jaschke et al. (2000) CURR. OPIN. CHEM. BIOL. 4: 257-62; Jaeger et al. (1999) PROC. NATL. ACAD. SCI. USA 96: 14712-7; Bartel et al. (1993) SCIENCE 261 : 141 1-8; Sen et al. (1998) CURR. OPIN. CHEM. BIOL. 2: 680-7
  • Substrate specificity among catalysts can be selected by selecting for active catalysts in the presence of the desired substrate and then selecting for inactive catalysts in the presence of one or more undesired substrates. If the desired and undesired substrates differ by their configuration at one or more stereocenters, enantioselective or diastereoselective catalysts can emerge from rounds of selection.
  • metal selectivity can be evolved by selecting for active catalysts in the presence of desired metals and selecting for inactive catalysts in the presence of undesired metals.
  • catalysts with broad substrate tolerance can be evolved by varying substrate structures between successive rounds of selection.
  • in vitro selections can also select for specificity in addition to binding affinity.
  • Library screening methods for binding specificity typically require duplicating the entire screen for each target or non-target of interest.
  • selections for specificity can be performed in a single experiment by selecting for target binding as well as for the inability to bind one or more non-targets.
  • the library can be pre-depleted by removing library members that bind to a non-target.
  • selection for binding to the target molecule can be performed in the presence of an excess of one or more non-targets.
  • the non-target can be a homologous molecule.
  • the target molecule is a protein
  • appropriate non-target proteins include, for example, a generally promiscuous protein such as an albumin. If the binding assay is designed to target only a specific portion of a target molecule, the non-target can be a variation on the molecule in which that portion has been changed or removed.
  • the templates which are associated with the selected reaction product preferably are amplified using any suitable technique to facilitate sequencing or other subsequent manipulation of the templates.
  • Natural oligonucleotides can be amplified by any state of the art method. These methods include, for example, polymerase chain reaction (PCR); nucleic acid sequence-based amplification (see, for example, Compton (1991) NATURE 350: 91 -92), amplified anti-sense RNA (see, for example, van Gelder et al. (1988) PROC. NATL. ACAD. SCI.
  • Ligase-mediated amplification methods such as Ligase Chain Reaction (LCR) may also be used.
  • LCR Ligase Chain Reaction
  • any means allowing faithful, efficient amplification of selected nucleic acid sequences can be employed in the method of the present invention. It is preferable, although not necessary, that the proportionate representations of the sequences after amplification reflect the relative proportions of sequences in the mixture before amplification.
  • non-natural nucleotides the choices of efficient amplification procedures are fewer. As non-natural nucleotides can be incorporated by certain enzymes including polymerases it will be possible to perform manual polymerase chain reaction by adding the polymerase during each extension cycle. [00132] For oligonucleotides containing nucleotide analogs, fewer methods for amplification exist. One may use non-enzyme mediated amplification schemes (Schmidt et al. (1997)
  • NUCLEIC ACIDS RES. 25: 4797-4802 For backbone-modified oligonucleotides such as PNA and LNA, this amplification method may be used. Alternatively, standard PCR can be used to amplify a DNA from a PNA or LNA oligonucleotide template. Before or during amplification the templates or complementing templates may be mutagenized or recombined in order to create an evolved library for the next round of selection or screening.
  • Sequencing can be done by a standard dideoxy chain termination method, or by chemical sequencing, for example, using the Maxam-Gilbert sequencing procedure.
  • the sequence of the template (or, if a long template is used, the variable portion(s) thereof) can be determined by hybridization to a chip.
  • a single- stranded template molecule associated with a detectable moiety such as a fluorescent moiety is exposed to a chip bearing a large number of clonal populations of single-stranded nucleic acids or nucleic acid analogs of known sequence, each clonal population being present at a particular addressable location on the chip.
  • the template sequences are permitted to anneal to the chip sequences.
  • the position of the detectable moieties on the chip then is determined. Based upon the location of the detectable moiety and the immobilized sequence at that location, the sequence of the template can be determined.
  • next-generation sequencing techniques are used, where during DNA sequencing, the bases of a small fragment of DNA are sequentially identified from signals emitted as each fragment is re-synthesized from a DNA template strand. This sequencing method is based on reversible dye-terminators that enable the identification of single bases as they are introduced into complementary DNA strands.
  • [00135] Small molecule compound libraries have been made using nucleic acid template synthesis, also referred to herein as DNA-programmed chemistry (DPC), in which the DNA base sequence corresponds directly to the structure of the molecule made on each unique DNA template strand. Sequence-specific DNA-templated reactions have been carried out covering a range of chemical reaction types. The process requires that the template forms a duplex specifically with the oligonucleotide in the transfer unit, a partner reagent DNA strand comprising an anti-codon sequence for one of the codons in the template. Following duplex formation, the DNA-linked reactive units were brought into close proximity, and a chemical reaction was catalyzed between the building blocks on the template and reagent strands, forming a new covalent bond linking these two small molecules together.
  • DPC DNA-programmed chemistry
  • the single-stranded DNA sequence used as the template for DPC contained several distinct codon regions of predetermined length and sequence to ensure the specificity of DNA duplex formation and thus integrity of the chemical reaction.
  • DNA templates have been designed containing fixed sequence regions, tag regions and codons for DPC reactions (see FIG. 1).
  • Using the principles described herein it has been possible to create libraries of novel small molecules conjugated to DNA oligonucleotides from analogous libraries of DNA templates where the specific base sequence in the template translates directly to a specific small molecule structure that was made on the 5 '-end of the DNA.
  • Each codon and tag region within the template corresponds to a particular structural feature or building block employed in the synthesis, and the summation of all tag and codon sequences identifies the unique structure of the attached small molecule.
  • each base employed in the single-stranded DNA template sequence is part of a longer functional region with a specific pre-determined role in the execution of DPC library synthesis or is required as part of the process by which the structures of active components of the library are determined.
  • Located at both the 3'- and 5 '-ends of the DNA template are located fixed regions of ten bases length, which represent polymerase chain reaction (PCR) ligation sites. These sites permit PCR amplification of the DNA sequences when it is required to define the specific base sequence of the DNA to directly identify library components.
  • PCR polymerase chain reaction
  • oligonucleotides attached to the small molecule By determining the particular DNA base sequence of oligonucleotides attached to the small molecule it is possible to identify compounds that have affinity for target proteins. For example, incubating the library of DNA-conjugated small molecules in the presence of a solid-phase resin-immobilized protein target acts in an affinity-based selection format to sequester compounds with protein affinity. Washing the solid-supported protein free of non- binding compounds, and then elution of binders following protein denaturation will yield active conjugates. PCR amplification of the attached DNA strands and sequencing will reveal the specific base sequence of the DNA and by extension the unique structures of the small molecule protein ligands. The success and efficiency of the protein binding hit discovery process is greatly enhanced by enlarging the number and diversity of the small molecule collection, and the library design and synthesis process has been refined to increase the productivity and efficiency of hit discovery.
  • each of 12 bases adjacent to the fixed ligation site are three independent codon regions each of 12 bases. Each of these sites in turn can form a duplex with complementary reagent DNA sequences during the DPC reaction steps.
  • the codons are designed to ensure specific interactions with the predetermined DNA sequences of the reagent 'anti-codon' sequences.
  • the length of the codon region ensures sufficient base-pairing to give high affinity duplex formation with a suitably high melting temperature such that the duplex will form and be maintained at ambient temperature.
  • each codon region contains a number of possible base sequences chosen to present specific building blocks in one diversity location in the final library compound. For example in the preparation of a triazole-linked library, each codon position (Rl through R3) contains one of 24 variants, and each of the codon sets in each of the positions Rl, R2 and R3 had its own unique set of 24 codons, making a total of 72 codon sequences used (see FIG. 2).
  • the oligonucleotide synthesis was based on a mix and split process to ensure that all permutations were generated in approximately equal amounts.
  • DNA synthesis was carried out in 24 parallel vessels, and within each the DNA was synthesized from the 3 '-end to produce specific DNA sequence comprising the fixed ligation sequence of 10 bases, two tag sequences totaling 14 bases and then a unique R3 codon sequence of 12 bases.
  • the controlled-pore glass (CPG) solid support from all 24 vessels was removed and thoroughly mixed before redistribution into 24 new vessels for the addition of the bases constituting 24 unique and distinctive R2 codon sequences.
  • CPG controlled-pore glass
  • the same process carried out for the introduction of the 24 Rl codon sequences provided the 13,824 different nascent DNA templates.
  • Subsequent oligonucleotide synthesis introduced the final fixed ligation sequence at the 5 'end of the DNA.
  • chemical modification of the 5'-hydroxyl introduced an amino group that provided a handle for addition of the first synthetic building block (linker residue).
  • FIGS. 1 and 3 show an exemplary template containing two tags, Tag 1 and Tag 2, each of which is 7 bases long. These DNA sequences are essential in the identification of the small molecule covalently attached to the template DNA at the end of the library synthesis, but they do not engage in DPC-catalyzed reactions. Instead they are used to 'hard code' for subsets of the library that might differ in the identity of the linker or spacer building blocks.
  • the Tag 2 position is kept fixed with a unique base sequence, and the mix and split process of introducing the Rl through R3 codon regions proceeds to give every codon variant (see FIG. 3).
  • the resulting product is a mixture of templates all containing a fixed Tag 2 base sequence that defines one linker building block. Without further mixing, the individual 'linker' building blocks are chemically attached to the 5 '-amino terminus maintaining a direct relationship between the Tag 2 sequence and the linker building block structure.
  • the DNA template-linker conjugates are then mixed and used in DPC reactions directly.
  • the template oligonucleotides will have been synthesized in 16 mixtures each comprising 13,824 different sequences. Combining the 16 different mixtures gives a total template library complexity of 221,184 sequences (see FIG. 4).
  • the final library products that are synthesized can be identified by sequencing the DNA, revealing the structure of the small molecule by considering both (1) the codon regions representing building blocks Rl through R3 and (2) the tag region defining the linker building block.
  • Example 3 Tag Sequence in the DNA Template Defines a Spacer Group
  • the other tag sequence in the DNA template sequence (see FIGS. 1 and 2) is used to define the spacer building block.
  • the Tag 1 position base sequence is held constant, but any library might comprise multiple library mixtures which differ only in the DNA base sequence of Tag 1.
  • Each library mixture will independently be chemically derivatized in a non-DPC step with a spacer building block. The mixtures are kept separate so that the spacer that is attached is defined by the Tag 1 base sequence ensuring that there is fidelity between the Tag 1 DNA sequence and the spacer structure (see FIG. 5).
  • the DPC library synthesis process is a method for converting the combinatorial set of DNA template sequences into a combinatorial set of small molecules that are constructed on the 5 '-end of the DNA.
  • the total library size is the mathematical product of the building block diversity at every variable position in the molecule. For example in one library, diversity was introduced through the linker position (16 variants) and each of the Rl through R3 building block positions (each 24 variants). This affords a total mixture complexity of 221,184 different library small molecule products (see FIG. 4).
  • Increasing the numerical complexity of the library can be achieved in several different and additive ways, but the inclusion of a fourth codon is one way to increase library size (see FIG. 6).
  • the fourth codon permits the addition of a new building block position during DNA-programmed chemistry (DPC).
  • DPC DNA-programmed chemistry
  • Using 24 variants for the new building block introduced by this codon will provide 331,776 different template sequences.
  • a library of 5,308,416 unique compounds is created, which is a 24-fold increase in library diversity as compared to a 16-mixture pool of three codon templates, which provides only 221,184 compounds.
  • the length of the codon region ensures sufficient base-pairing between the template and the reagent oligonucleotides, such that there is sufficient specificity in the DNA duplex formation.
  • the choice of codons of 12 bases length ensures a high level of fidelity between the codon sequence and the identity of the building block added to the small molecule being synthesized on the 5 '-end of the DNA.
  • the length is chosen to give high affinity duplex formation with a suitably high melting temperature (above ambient temperature), and also to minimize any mismatched DNA.
  • the number of base sequence permutations that can be achieved with four bases in each of the 12 base positions is 16,777,216, providing a significant choice of alternate sequences for each building block.
  • Appropriate computer algorithms can be used to select codon sequences, to compile all possible full DNA template sequences, and to determine that there is absolute fidelity and no ambiguity in the matching of anti-codons on the reagent DNA to the template codons.
  • codons recognized only their designated anti-codons and that the reagent strands containing the anti-codons can bind only to their complementary codons, not to any other codon, nor to any other sequence of bases in any of the templates which are concurrently present in the DPC reaction mixture.
  • the vast diversity of codon sequences ensures that 24 unique codon sequences that work in every library template context can readily be selected.
  • the codon repertoire may be extended by lengthening the codon base sequence.
  • Example 6 Use of a Multivalent (Trivalent) Building Block
  • the DNA-programmed chemistry (DPC) approach permits building blocks to be added to the growing small molecule on the 5 '-end of the DNA independent of any base sequence.
  • a typical building block is a bifunctional reagent such as an amino acid, although depending on the chemistry used in small molecule synthesis, the building block is not necessarily limited to just this type of building block.
  • the amino acid is attached to the reagent DNA anticodon sequence through the amino group.
  • the carboxylic acid is activated, typically with a standard peptide coupling reagent, and the amide bond is generated between the amino acid and the free amine group on the small molecule intermediate attached to the 5 '-end of the DNA template.
  • the newly created molecule is sandwiched between the template and reagent DNA strands.
  • the scissile bond can be selectively cleaved allowing the separation from the now redundant reagent DNA.
  • the template now has a newly modified small molecule on the 5 '-end with a free exposed amine group which is available for further chemical derivitization.
  • a trifunctional building block An example might be the use of a suitably protected diamino acid.
  • Such building blocks have been attached to the DNA template through the carboxylic acid, with the two amine groups exposed for further chemistry. To prevent ambiguity in the synthesis, the two amines will be protected in different ways, so that either of both amines can be independently revealed when required.
  • one amine has been protected as the Fmoc-derivative, which can be revealed by treating with piperidine, and the other as an azide, which was converted to the free amine at a later stage of the library synthesis by a reduction or hydrogenation reaction.
  • DPC reactions have been used to add amino acids onto the free amine of the trifunctional group.
  • the amine on the trifunctional building block was revealed by suitable chemical conversion, and the two amines (that on the amino acid added by DPC, and that revealed on the trifunctional building block) can be linked by a bis-carboxylic acid spacer molecule to generate a macrocycle.
  • the terminal functional group might be a carboxy alkyne introduced as a spacer by amide coupling to the terminal amine on the third bifunctional building block.
  • Production of the final macrocyclic product has been achieved by a copper (I) salt-catalyzed Huisgen cyclization, onto the azide of the trifunctional linker molecule, to give a 1,2,3-triazole product.
  • the trifunctional building block can thus be considered to be a diversity generating element that, by being employed at different stages of the synthesis, can result in small molecule products with highly divergent architectures (see FIG. 7). Inclusion of additional trifunctional building blocks can add further structural diversity to the library with minimal additional synthetic effort, and will result in multiple libraries of similar numerical complexity.
  • each codon position encodes for a building block collection of 24 different building blocks.
  • Each of the three codon positions has a different set of 24 building blocks associated with it.
  • the building blocks are added to the growing small molecule by sequential DPC reactions. However there does not need to be a direct relationship between the sequence of DPC catalyzed building block adding events and the relative positions of the encoding codons within the DNA template.
  • the first building block could be introduced by using the codon 1 position.
  • a set of building blocks could be introduced in the first step equally successfully by using the second codon position or indeed the third.
  • Certain library architectures can be engineered to contain reactive functional groups such as carboxylic acids and amines. These functional groups are further derivatized by reaction of the entire library mixture with soluble reagents. For example, making a library comprising multiple diversity positions might conclude the DPC steps with an exposed nucleophilic amino group. Rather than using this amine for a further DPC step, or for cyclization to yield a macrocycle, the amine is derivatized with a number of different soluble reagents. To maximize the total number of library compounds, the library mixture at the end of the DPC steps is split into multiple aliquots such that each aliquot contains every library component. Each aliquot is treated with a different soluble reagent.
  • reactive functional groups such as carboxylic acids and amines.
  • a library aliquot containing a nucleophilic amine is reacted with acid chlorides, anhydrides, sulfonyl chlorides, isocyanates, isothiocyanates or nucleophilic aromatic systems such as chloroheterocycles.
  • a library mixture with a free, terminal carboxylic acid is reacted with amines using routine amide coupling conditions to generate amide derivatives.
  • a mixture of 5,971,968 library products containing a terminal amino group is split into 50 aliquots and each aliquot reacted with a different acyl chloride, anhydride, sulfonyl chloride or other electrophilic reagent to generate a total of 298,598,400 different library products in 50 mixtures.
  • Example 9 Addition of a DNA Tag Sequence by Ligation to Define a Soluble Reagent or the Sequence of Building Block Addition
  • Tag sequences can be used to define and later identify non-DPC steps, and as a consequence of their use, it is necessary to keep pools of template sequences separate until the point the non-DPC building block is added to the growing small molecule.
  • An example of this is the addition of the linker at the start of the small molecule synthesis. This step is undertaken with individual pools of templates, each comprising a unique tag sequence and each resulting in the addition of only one linker building block.
  • the pools of templates each containing different linkers can be combined subsequently and prior to the DPC reactions.
  • An alternative method is to add the linker to a template mixture and then add a tag DNA sequence to the 3 '-end of the DNA template by a ligation reaction.
  • the tag can be just 7 bases long and would be unique for the linker that has been added.
  • the sequence of the added tag can be identified and the nature of the non-DPC building block or soluble reagent can also be unambiguously determined.

Abstract

La présente invention concerne des procédés et des compositions destinées à étendre le champ d'application de réactions chimiques qui peuvent être mises en oeuvre au cours de synthèses organiques dirigées par des matrices d'acide nucléique, et pour la production de bibliothèques de petites molécules de dimension, de champ d'application et de complexité plus importants qu'auparavant.
PCT/US2015/068308 2014-12-31 2015-12-31 Procédés et compositions pour une synthèse dirigées par matrices d'acide nucléique, de grandes bibliothèques de petites molécules complexes WO2016109808A1 (fr)

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US20090149347A1 (en) * 2005-06-07 2009-06-11 President And Fellows Of Harvard College Ordered Multi-Step Synthesis by Nucleic Acid-Mediated Chemistry
US20090035824A1 (en) * 2005-06-17 2009-02-05 Liu David R Nucleic acid-templated chemistry in organic solvents

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