Source: http://www.google.com/patents/US8206914?dq=oakley+D523,461&ei=qiI4T-CjGqXf0QHz_PSUCA
Timestamp: 2015-11-30 03:07:55
Document Index: 383031706

Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60']

Patent US8206914 - Evolving new molecular function - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsNature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The power of Nature and the flexibility of organic synthesis are combined in nucleic acid-templated synthesis. The present invention provides a variety of template architectures...http://www.google.com/patents/US8206914?utm_source=gb-gplus-sharePatent US8206914 - Evolving new molecular functionAdvanced Patent SearchPublication numberUS8206914 B2Publication typeGrantApplication numberUS 12/834,072Publication dateJun 26, 2012Filing dateJul 12, 2010Priority dateAug 19, 2002Also published asCA2495881A1, CA2495881C, EP1540013A2, EP1540013A4, EP1540013B1, US7491494, US7771935, US20040180412, US20050170376, US20110190141, WO2004016767A2, WO2004016767A3Publication number12834072, 834072, US 8206914 B2, US 8206914B2, US-B2-8206914, US8206914 B2, US8206914B2InventorsDavid R. Liu, Zev J. Gartner, Jeffrey B. Doyon, Christopher T. Calderone, Matthew W. Kanan, Xiaoyu Li, Thomas M. Snyder, Daniel M. RosenbaumOriginal AssigneePresident And Fellows Of Harvard CollegeExport CitationBiBTeX, EndNote, RefManPatent Citations (112), Non-Patent Citations (295), Classifications (9), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetEvolving new molecular function
US 8206914 B2Abstract
Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The power of Nature and the flexibility of organic synthesis are combined in nucleic acid-templated synthesis. The present invention provides a variety of template architectures for performing nucleic acid-templated synthesis, methods for increasing the selectivity of nucleic acid-templated reactions, methods for performing stereoselective nucleic acid-templated reactions, methods of selecting for reaction products resulting from nucleic acid-templated synthesis, and methods of identifying new chemical reactions based on nucleic acid-templated synthesis.
Images(114) Claims(19)
1. An in vitro method of enriching a product of a nucleic acid-templated synthesis, the method comprising the steps of:
(a) providing a first library of molecules comprising a plurality of reaction products of a nucleic acid templated synthesis, which are not nucleic acids, wherein each reaction product is covalently attached to a corresponding oligonucleotide that templated the synthesis of the reaction product, and wherein each oligonucleotide comprises a nucleotide sequence indicative of the reaction product associated therewith, and wherein a portion of said reaction products are capable of binding to a preselected binding moiety;
(b) exposing said first library of molecules to said binding moiety under conditions to permit reaction product capable of binding said binding moiety to bind thereto, wherein the reaction product has a Kd for the binding moiety of no less than 0.9 nM;
(c) removing unbound reaction products; and
(d) eluting bound reaction product from said binding moiety to produce a second library of molecules enriched at least 50-fold for reaction product that binds said binding moiety relative to said first library.
2. The method of claim 1, wherein in step (b), said binding moiety is immobilized on a solid support.
3. The method of claim 1, wherein said binding moiety is a target biomolecule.
4. The method of claim 3, wherein said target biomolecule is a protein.
5. The method of claim 1, wherein in step (d), said second library is enriched at least 100-fold for reaction product that binds said binding moiety.
6. The method of claim 5, wherein in step (d), said second library is enriched at least 1,000-fold for reaction product that binds said binding moiety.
7. The method of claim 1, further comprising repeating steps (b), (c), and (d).
8. The method of claim 7, wherein repeating steps (b), (c), and (d) produces a third library enriched by at least 10,000-fold for reaction product that binds said binding moiety.
9. The method of claim 8, wherein said library is enriched by at least 100,000-fold for reaction product that binds said binding moiety.
10. The method of claim 1, wherein said oligonucleotide comprises a first sequence that identifies a first reactive unit that produced said reaction product capable of binding said preselected binding moiety.
11. The method of claim 10, wherein said oligonucleotide comprises a second sequence that identifies a second reactive unit that produced said reaction product capable of binding said preselected binding moiety.
12. The method of claim 1, comprising the additional step of amplifying oligonucleotide associated with the enriched reaction product.
13. The method of claim 1, comprising the additional step of determining the sequence of the oligonucleotide associated with the enriched reaction product.
14. The method of claim 12, comprising the additional step of determining the sequence of the amplified oligonucleotide.
15. The method of claim 13, further comprising the step of characterizing said reaction product from information in said sequence of said oligonucleotide.
16. The method of claim 15, further comprising the step of identifying a new chemical reaction that produced said reaction product.
17. The method of claim 14, further comprising the step of characterizing the reaction product from information in said sequence of said oligonucleotide.
18. The method of claim 17, further comprising the step of identifying a new chemical reaction that produced said reaction product.
19. An in vitro method of enriching a product of a nucleic acid-templated synthesis, the method comprising the steps of:
(a) providing a first library of molecules comprising a plurality of reaction products of a nucleic acid templated synthesis, which are not nucleic acids, wherein each reaction product is covalently attached to a corresponding oligonucleotide that templated the synthesis of the reaction product, wherein each oligonucleotide comprises a nucleotide sequence indicative of the reaction product associated therewith, wherein no oligonucleotide is linked by a direct or indirect covalent or non-covalent interaction to a capturable moiety selected from the group consisting of biotin, avidin and streptavidin; and wherein a portion of said reaction products are capable of binding to a preselected binding moiety;
(b) exposing said first library of molecules to said binding moiety under conditions to permit reaction product capable of binding said binding moiety to bind thereto;
This application is a continuation of U.S. patent application Ser. No. 10/950,367, filed Sep. 24, 2004, which is a continuation of U.S. patent application Ser. No. 10/643,752, filed Aug. 19, 2003, which claims the benefit of (i) U.S. Provisional Patent Application No. 60/404,395, filed Aug. 19, 2002, (ii) U.S. Provisional Patent Application No. 60/419,667, filed Oct. 18, 2002, (iii) U.S. Provisional Patent Application No. 60/432,812, filed Dec. 11, 2002, (iv) U.S. Provisional Patent Application No. 60/444,770, filed Feb. 4, 2003, (v) U.S. Provisional Patent Application No. 60/457,789, filed Mar. 26, 2003, (vi) U.S. Provisional Patent Application No. 60/469,866, filed May 12, 2003, and (vii) U.S. Provisional Patent Application No. 60/479,494, filed Jun. 18, 2003, the disclosures of each of which are incorporated by reference herein. The application is also related to U.S. Provisional Patent Application Nos. 60/277,081 (filed Mar. 19, 2001), 60/277,094 (filed Mar. 19, 2001), 60/306,691 (filed Jul. 20, 2001), and 60/353,565 (filed Feb. 1, 2002), as well as to U.S. patent application Ser. Nos. 10/101,030 (filed Mar. 19, 2002) and 10/102,056 (filed Mar. 19, 2002), and to International Patent Application serial number US02/08546 (filed Mar. 19, 2002).
This invention was made with Government support under the Office for Naval Research under Contract No. N00014-00-1-0596 and Grant No. 00014-03-1-0749. The United States Government has certain rights in the invention.
The classic “chemical approach” to generating molecules with new functions has been used extensively over the last century in applications ranging from drug discovery to synthetic methodology to materials science. In this approach, researchers synthesize or isolate candidate molecules, assay these candidates for desired properties, determine the structures of active compounds if unknown, formulate structure-activity relationships based on available assay and structural data, and then synthesize a new generation of molecules designed to possess improved properties. While combinatorial chemistry methods (see, for example, Eliseev et al. (1999) COMBINATORIAL CHEMISTRY IN BIOLOGY 243: 159-172; Kuntz et al. (1999) CURRENT OPINION IN CHEMICAL BIOLOGY 3: 313-319; Liu et al. (1999) ANGEW. CHEM. INTL. ED. ENG. 38: 36) have increased the throughput of this approach, its fundamental limitations remain unchanged. Several factors limit the effectiveness of the chemical approach to generating molecular function. First, the ability to accurately predict the structural changes that will lead to new function is often inadequate due to subtle conformational rearrangements of molecules, unforeseen solvent interactions, or unknown stereochemical requirements of binding or reaction events. The resulting complexity of structure-activity relationships frequently limits the success of rational ligand or catalyst design, including those efforts conducted in a high-throughput manner. Second, the need to assay or screen, rather than select, each member of a collection of candidates limits the number of molecules that can be searched in each experiment. Finally, the lack of a way to amplify synthetic molecules places requirements on the minimum amount of material that must be produced for characterization, screening, and structure elucidation. As a result, it can be difficult to generate libraries of more than roughly 106 different synthetic compounds.
In contrast, Nature generates proteins with new functions using a fundamentally different method that overcomes many of these limitations. In this approach, a protein with desired properties induces the survival and amplification of the information encoding that protein. This information is diversified through spontaneous mutation and DNA recombination, and then translated into a new generation of candidate proteins using the ribosome. Unlike the linear chemical approach described above, the steps used by Nature form a cycle of molecular evolution. Proteins emerging from this process have been directly selected, rather than simply screened, for desired activities. Because the biomolecules that encode evolving proteins (e.g., DNA) can be amplified, a single protein molecule with desired activity can in theory lead to the survival and propagation of the DNA encoding its structure.
Acknowledging the power and efficiency of Nature's approach, researchers have used molecular evolution to generate many proteins and nucleic acids with novel binding or catalytic properties (see, for example, Minshull et al. (1999) CURR. OPIN. CHEM. BIOL. 3: 284-90; Schmidt-Dannert et al. (1999) TRENDS BIOTECHNOL. 17: 135-6; Wilson et al. (1999) ANNU. REV. BIOCHEM. 68: 611-47). Proteins and nucleic acids evolved by researchers have demonstrated value as research tools, diagnostics, industrial reagents, and therapeutics, and have greatly expanded the understanding of the molecular interactions that endow proteins and nucleic acids with binding or catalytic properties (see, Famulok et al. (1998) CURR. OPIN. CHEM. BIOL. 2: 320-7).
Despite Nature's efficient approach to generating function, Nature's molecular evolution is limited to two types of “natural” molecules (proteins and nucleic acids) because thus far the information in nucleic acids can only be translated into proteins or into other nucleic acids. Unfortunately, many synthetic molecules of interest do not in general have nucleic acid or protein backbones. An ideal approach to generating functional molecules merges the most powerful aspects of molecular evolution with the flexibility of synthetic chemistry. Clearly, enabling the evolution of non-natural synthetic small molecules and polymers, much as Nature evolves biomolecules, would lead to much more effective methods of discovering new synthetic ligands, receptors, and catalysts difficult or impossible to generate using rational design.
Although these concepts have been brought together to permit nucleic acid-templated synthesis of small molecules (see, for example, Gartner & Liu (2001) J. AM. CHEM. SOC. 123: 6961-6963) there is still an ongoing need for improvements in these core technologies to permit the more efficient synthesis, selection, amplification, and evolution of molecules of interest.
The invention provides a variety of methods and compositions that expand the scope of template-directed synthesis, selection, amplification and evolution of molecules of interest. During nucleic acid-templated synthesis, the information encoded within a nucleic acid template is used to bring two or more reactants together into reactive proximity. These methods permit the creation of, for example, small molecule and polymer libraries that have not been possible to create to date using conventional combinational chemistries.
In one aspect, the invention provides a method of performing nucleic acid-templated synthesis using a template having an “omega” or “Ω” type architecture. This type of template permits distance-dependent nucleic acid-templated reactions to be encoded by bases far removed from the associated reactive unit. The method involves providing (i) a template comprising a first reactive unit associated with a first oligonucleotide comprising a codon and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon that is capable of annealing to the codon. The codon and/or the anti-codon include first and second regions spaced apart from one another. The oligonucleotides then are annealed together to bring the reactive units into reactive proximity. When the oligonucleotides anneal to one another, the codon (or anti-codon) with the spaced-apart regions produce a loop of oligonucleotides not annealed to the corresponding anti-codon (or codon). A covalent bond-forming reaction then is induced between the reactive units to produce the reaction product.
In one embodiment, at least one of the reactive units are attached adjacent a terminal region of its corresponding oligonucleotide. In another embodiment, the codon or anti-codon is disposed more than one base away (for example, 10, 20, 30 bases or more) from its corresponding reactive unit. The first spaced apart region typically is disposed directly adjacent a terminus of its corresponding oligonucleotide. The first spaced apart region preferably includes, for example, three, four, or five nucleotides, although other embodiments (e.g., more than five nucleotides) are also envisioned. The second region may be disposed, for example, at least twenty or at least thirty bases away from its corresponding reactive unit. More particularly, the end of the second region closest to the reactive unit may be disposed, for example, at least ten, twenty, thirty or more bases from the end of the oligonucleotide attached to its reactive unit. The template may include additional (e.g., 2, 3, 4, or more than 4) codons, in which case a corresponding number of transfer units can be annealed to the template, optionally permitting multi-step or alternative syntheses.
In another aspect, the invention provides a method of performing a nucleic acid-templated synthesis using a template having a “T” type architecture. The T architecture permits two nucleic acid-templated reactions to take place on a single template in a single step. The method involves providing (i) a template comprising a first reactive unit (e.g., a scaffold molecule) associated with a first oligonucleotide having a codon, and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide having an anti-codon capable of annealing to the codon. The first reactive unit is attached, preferably covalently, to an attachment site intermediate the proximal and distal ends of the first oligonucleotide of the template. During synthesis, the oligonucleotides of the template and transfer unit are annealed to one another to bring the reactive units into reactive proximity, and a covalent bond-forming reaction between the reactive units is induced.
In one embodiment of the T type architecture, the template also includes a second, different codon capable of annealing to a second, different anti-codon sequence of a second, different transfer unit. In this embodiment, the first codon is located proximal to the attachment site and the second codon, if present, is located distal to the attachment site. If a second transfer unit comprising a third reactive unit associated with a third oligonucleotide having a second, different anti-codon sequence capable of annealing to the second codon is provided, the second transfer unit may bind to the template at the second codon position. Accordingly, when the first and second transfer units are combined with the template, the first anti-codon of the first transfer unit anneals to the first codon of the template and the second anti-codon of the second transfer unit anneals to the second codon of the template. This system permits two reactions to occur simultaneously or sequentially on a single template in a single step.
In another aspect, the invention provides a series of methods for increasing reaction selectivity between reactants in a templated synthesis. In one approach, the method comprises providing a template and at least two transfer units. The template comprises a first reactive unit associated with a first oligonucleotide comprising a predetermined codon sequence. The first transfer unit comprises a second reactive unit associated with a second oligonucleotide comprising an anti-codon sequence capable of annealing to the codon sequence. The second transfer unit comprises a third reactive unit, different from the second reactive unit. The third reactive unit, however, is associated with a third oligonucleotide that lacks an anti-codon sequence capable of annealing to the codon sequence. The template and transfer units are mixed under conditions to permit annealing of the second oligonucleotide to the first oligonucleotide, thereby to enhance covalent bond formation between the second and first reactive units relative to covalent bond formation between the third and first reactive units.
This method may be particularly helpful when the second and third reactive units are each capable of reacting independently with the first reactive unit. Furthermore, the method may also be helpful when the second and third reactive units are capable of reacting with one another, for example, to modify or inactivate one another. Accordingly, this type of method permits a series of otherwise incompatible reactions to occur in the same solution, for example, where a reaction between the second and third reactive units is incompatible with a reaction between the second reactive unit and the first reactive unit. The method may enhance covalent bond formation between the first and second reactive units by at least 2-fold, at least 5-fold, at least 10-fold, or at least 50-fold relative to covalent bond formation between the first and third reactive units. Collectively, these advantages permit a one-pot ordered multi-step synthesis, in which a sequence of reactions is programmed by the sequence of a template oligonucleotide. Thus, a sequence of at least 2, 3, 4, 5, 6, or more reactions can take place in an ordered manner in a single solution, even when the reactants would interfere with each other using conventional, non-templated chemistries.
In one embodiment, the template, the first transfer unit, and/or the second transfer unit are associated with a capturable moiety, for example, biotin, avidin, or streptavidin. If a capturable moiety is present, the method may include capturing the capturable moiety as a way to enrich a reaction product from a reaction mixture.
In another approach, the method comprises providing (i) a template comprising a first oligonucleotide having first and second codon sequences (ii) a first transfer unit, (iii) a second transfer unit, and (iv) a third transfer unit. The first transfer unit comprises a first reactive unit associated with a second oligonucleotide comprising a first anti-codon sequence capable of annealing to the first codon sequence. The second transfer unit comprises a second reactive unit associated with a third oligonucleotide comprising a second anti-codon sequence capable of annealing to the second codon sequence. The third transfer unit comprises a third reactive unit associated with a fourth oligonucleotide sequence that lacks an anti-codon sequence capable of annealing to the first or second codon sequences. The template, first transfer unit, second transfer unit, and third transfer unit then are mixed under conditions to permit (i) annealing of the first anti-codon sequence to the first codon sequence and (ii) annealing of the second anti-codon sequence to the second codon sequence thereby to enhance covalent bond formation between the first and second reactive units relative to covalent bond formation between the third reactive unit and the first reactive unit and/or between the third reactive unit the second reactive unit. This type of method may be particularly useful for producing non-natural polymers by nucleic acid-templated synthesis.
In one embodiment, the template is associated with a capturable moiety, for example, biotin, avidin, or streptavidin. The capturable moiety may also be a reaction product resulting from a reaction between the first and second reactive units when the first and second reactive units are annealed to a template. If a capturable moiety is present, the method may include capturing the capturable moiety as a way to enrich a reaction production from the reaction mixture.
This type of method is also helpful when the third reactive unit is capable of reacting with the first and/or second reactive units. In other words, the reaction between the first and third reactive units and/or between the second and third reactive units may be incompatible with the reaction between the first and second reactive units. The method may enhance covalent bond formation between the first and second reactive units by at least 2-fold, at least 5-fold, at least 10-fold, or at least 50-fold relative to covalent bond formation between the first and third reactive units.
In another aspect, the invention provides a series of methods for performing stereoselective nucleic acid-templated synthesis. The stereoselectivity of the synthesis may result from the choice of a particular template, transfer unit, reactive unit, hybridized template and transfer unit, stereoselective catalyst, or any combination of the above. The resulting product may be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% stereochemically pure.
Generally, the method involves providing (i) a template comprising a first oligonucleotide that optionally is associated with a reactive unit and (ii) one or more transfer units, each comprising a second oligonucleotide associated with a reactive unit. Annealing of the first and second oligonucleotides brings at least two reactive units into reactive proximity and to react to produce a reaction product where the reaction product contains a chiral center and is of at least 60%, more preferably at least 80%, and more preferably at least 95% stereochemically pure at the chiral center. It is contemplated that this method can be accomplished when one reactive unit is associated with the template and the other reactive unit is associated with the transfer unit. Also, it is contemplated that this method can be accomplished when the template does not provide a reactive unit and two transfer units when they anneal to the template provide the two reactive units that come into reactive proximity to produce the reaction product.
In one approach, the method involves providing at least two templates and at least one transfer unit. One template includes a first oligonucleotide associated with a first reactive unit comprising a first stereochemical configuration, and the other template includes another first oligonucleotide associated with another first reactive unit having a second, different stereochemical configuration. The transfer unit comprises a second reactive unit associated with a second oligonucleotide including a sequence complementary to a sequence of the first oligonucleotide of the template. The first and second oligonucleotides then are annealed under conditions to permit the second reactive unit of the transfer unit to react preferentially with either the first reactive unit of the first stereochemical configuration or the first reactive unit of the second stereochemical configuration to produce a reaction product.
The resulting reaction product may have a particular stereochemical configuration. In one embodiment, a stereochemical configuration or macromolecular conformation of the first oligonucleotide of the template determines which one of the first reactive units reacts with the second reactive unit.
In a second approach, the method involves providing at least one template and at least two transfer units. The template includes a first oligonucleotide associated with a first reactive unit. One transfer unit comprises a second oligonucleotide associated with a second reactive unit having a first stereochemical configuration, and the other transfer unit comprises another second oligonucleotide associated with a second reactive unit having a second, different stereochemical configuration. A sequence of the second oligonucleotides is complementary to a sequence of the first oligonucleotide. The first and second oligonucleotides then are annealed under conditions to permit the first reactive unit of the template to react preferentially with either the second reactive unit having the first stereochemical configuration or with the second reactive unit having the second stereochemical configuration to produce a reaction product.
The resulting reaction product may have a particular stereochemical configuration. In one embodiment, a stereochemical configuration or macromolecular conformation of the second oligonucleotide determines which of the second reactive units reacts with the first reactive unit.
In a third approach, the method involves providing at least one template and at least two transfer units, wherein one or optionally both of the transfer units comprise a pair of reactive units with one reactive unit of the pair having a first stereochemical configuration and the other reactive unit of the pair having a second, different stereochemical configuration. The template comprises a first oligonucleotide comprising a first codon sequence and a second codon sequence. One transfer unit of a first pair of transfer units includes a second oligonucleotide with a first anti-codon sequence associated with a first reactive unit having a first stereochemical configuration. The other transfer unit of the first pair of transfer units includes another second oligonucleotide associated with a second stereochemical configuration of the first reactive unit. The second transfer unit includes a third oligonucleotide with a second anti-codon sequence associated with a second reactive unit. The template, the first pair of transfer units, and the second transfer unit are annealed to permit a member of the first pair of transfer units to react preferentially with the second transfer unit to produce a reaction product. The resulting reaction product may have a particular stereochemical configuration.
In one embodiment, a stereochemical configuration or macromolecular conformation of the second oligonucleotide determines which member of the first pair of transfer units reacts preferentially to produce the reaction product.
In one embodiment, the method involves providing a template and at least two pairs of transfer units. The template comprises a first oligonucleotide comprising first and second codon sequences. One transfer unit of the first pair comprises a second oligonucleotide with a first anti-codon sequence associated with a first reactive unit having a first stereochemical configuration. The other transfer unit of the first pair comprises the second oligonucleotide with the first anti-codon sequence associated with a first reactive unit having a second, different stereochemical configuration. One transfer unit of the second pair of transfer units comprises a third oligonucleotide having a second, different anti-codon sequence associated with a second reactive unit having a first stereochemical configuration. The other transfer unit of the second pair comprises the third oligonucleotide with the second anti-codon sequence associated with the second reactive unit having a second, different stereochemical configuration. The template, the first pair of transfer units and the second pair of transfer units are annealed to permit a member of the first pair of transfer units to react preferentially with a member of the second pair of transfer units to produce a reaction product.
In one embodiment, a stereochemical configuration or macromolecular conformation of the second oligonucleotide determines which member of the first pair of transfer units reacts preferentially to produce the reaction product. In addition, a stereochemical configuration or macromolecular conformation of the third oligonucleotide determines which member of the second pair of transfer units reacts preferentially to produce the reaction product.
In another aspect, the invention provides a method for enriching a product of a templated synthesis reaction. The method comprises providing a first library of molecules comprising a plurality of reaction products associated with a corresponding plurality of oligonucleotides, wherein each oligonucleotide comprises a nucleotide sequence indicative of the associated reaction product. A portion of the reaction products in the first library are capable of binding to a preselected moiety. The first library then is exposed to the binding moiety under conditions to permit reaction product capable of binding the binding moiety to do so. Unbound reaction products are removed, and bound reaction product then is eluted from the binding moiety to produce a second library of molecules enriched at least 10-fold, more preferably at least 50-fold, relative to the first library, for reaction products that bind the binding moiety.
In one embodiment, the binding moiety, for example, a target biomolecule, for example, a protein, is immobilized on a solid support. In another embodiment, the second library is enriched at least 100-fold or at least 1,000-fold for reaction products that bind to the binding moiety. Furthermore, it is contemplated that the steps of exposing the library to the binding moiety, removing unbound reaction products, and eluting bound reaction products can be repeated (e.g., repeated one, two, three or more times). Repetition of these steps preferably yields a second library enriched at least 1,000-fold, more preferably, at least 10,000-fold, or, more preferably, at least 100,000-fold, for reaction products that bind to the binding moiety.
In one embodiment, the oligonucleotide attached to the selected library member includes a first sequence that identifies a first reactive unit that produced the reaction product bindable by the preselected binding moiety. Preferably, the oligonucleotide also includes a second sequence that identifies a second reactive unit that produced the reaction product bindable by the preselected binding moiety. By sequencing the oligonucleotide attached to the selected library member it is possible to determine what reactants reacted with one another to produce the reaction product. Accordingly, using this approach it is possible to deduce the structure of the selected library member from the reaction history.
The method may further comprise the step of amplifying the oligonucleotide associated with the enriched reaction product and, preferably, determining the sequence of the amplified oligonucleotide. Furthermore, the reaction product can be further characterized by using information encoded within the sequence of the oligonucleotide. For example, the sequence of the oligonucleotide may be determined and then from the sequence it is possible to determine what reactive units reacted to produce the reaction product. Using a similar approach, it is possible to identify the existence of new chemical reactions that produced the reaction product.
In another aspect, the invention provides a variety of methods for identifying the existence of new chemical reactions. One approach involves, providing a library of molecules comprising a plurality of reaction products associated with a corresponding plurality of oligonucleotides, wherein each oligonucleotide includes a nucleotide sequence indicative of an associated reaction product. A particular reaction product associated with its corresponding oligonucleotide then is selected, and characterized. Following characterization of the reaction product and identification of the reactive units that reacted to create the reaction product, it is possible to identify one or more new chemical reactions necessary to produce the reaction product.
In one embodiment, the method further includes, after selecting the reaction product, amplifying its corresponding oligonucleotide. The amplified oligonucleotide can then be sequenced to identify what reactive units reacted to produce the reaction product. The oligonucleotide may also be amplified for use in preparing more of the selected reaction product. In other embodiments, the oligonucleotide may be mutated, and the resulting mutated oligonucleotide may be used in the creation of a second generation library.
A second approach involves providing (i) a template and (ii) a first transfer unit. The template comprises a first reactive unit associated with a first oligonucleotide comprising a codon. The transfer unit comprises a second reactive unit associated with a second oligonucleotide comprising an anti-codon capable of annealing to the codon. The oligonucleotides are annealed to bring the first and second reactive units into reactive proximity. A covalent bond-forming reaction is induced between the reactive units to produce a reaction product. The reaction product then is characterized, and a new chemical reaction necessary to make the reaction product is identified using information encoded by the template to identify the first and second reactive units that reacted to produce the reaction product. The method may also include the step of selecting the reaction product prior to its characterization.
In a third approach, the invention involves providing at least (i) a template, (ii) a first transfer unit and (iii) a second transfer unit. The first transfer unit comprises a first reactive unit associated with a first oligonucleotide. The second transfer unit comprises a second reactive unit associated with a second oligonucleotide. The template includes sequences capable of annealing to the first and second oligonucleotides. During the method, the oligonucleotides are annealed to the template to bring the reactive units into reactive proximity and a covalent bond-forming reaction is induced between the reactive units to produce a reaction product. The reaction product then is characterized, for example, by using information encoded by the template to identify the first and second reactive units that reacted with one another to produce the reaction product. Based on the characterization, it is then possible to identify one or more new chemical reactions that were necessary to make the reaction product. The method may also include the step of selecting the reaction product prior to its characterization.
Although the methods of the invention are useful with small numbers of templates and transfer units, use of larger numbers of templates (e.g., 10, 50, 100, 1000, or more) and of transfer units for each codon (e.g., 10, 20, 30, 50, or more) permits the synthesis of large libraries of molecules that can be screened simultaneously using the sensitivity afforded by amplification.
The terms “small molecule scaffold” or “molecular scaffold” as used herein, refer 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 moieties 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.
FIG. 1 depicts known sequence-specific oligomerizations of complimentary oligonucleotides catalyzed by single-stranded nucleic acid templates.
FIG. 2 is a schematic representation of one embodiment of nucleic acid-templated synthesis where a reactive unit is attached to a template at the start of synthesis.
FIG. 3 is a schematic representation of a second embodiment of nucleic acid-templated synthesis where a reactive unit is not attached to the template at the start of synthesis.
FIG. 4 is a schematic representation of a third embodiment of nucleic acid-templated synthesis suitable for polymer synthesis.
FIGS. 5A-F are schematic representations of various exemplary templates useful in nucleic acid-templated synthesis.
FIGS. 6A-E are schematic representations of desirable and undesirable possible interactions between a codon of a template and an anti-codon of a transfer unit.
FIGS. 7A-G are schematic representations of various template architectures useful in nucleic acid-templated synthesis.
FIG. 8 is a schematic representation of a method for producing a template, containing, from the 5′-end to the 3′-end, a small molecule functional group, a DNA hairpin, an annealing region, a coding region, and a PCR primer binding site.
FIG. 9 is a schematic representation of a general method for making a library of reaction products.
FIG. 10 is a graph showing the relationship between the effective concentration of target protein and the fraction of ligand that binds the target.
FIGS. 11A-B are schematic representations of methods for screening a library for bond-cleavage (FIG. 11A) and bond-formation (FIG. 11B) catalysts.
FIG. 12 is a schematic representation of an in vitro selection scheme for identifying non-natural polymer catalysts of bond-forming reactions.
FIG. 13 is a schematic representation of an in vitro selection scheme for identifying non-natural polymer catalysts of bond-cleaving reactions.
FIG. 14 is a schematic representation of exemplary reagents and their use in a recombination method for diversifying a template library.
FIG. 15 depicts synthetic reactions directed by hairpin (H) and end-of-helix (E) DNA templates. Reactions were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) after the indicated reaction times. Lanes 3 and 4 contained templates quenched with excess β-mercaptoethanol prior to reaction.
FIG. 16 depicts the results of reactions between matched (M) or mismatched (X) reagents linked to thiols (S) or primary amines (N) and templates functionalized with the variety of electrophiles.
FIGS. 17A-17B depict various mismatch reactions analyzed by denaturing PAGE. FIG. 17A depicts results of reactions in which H templates linked to an iodoacetamide group were reacted with thiol reagents containing 0, 1, or 3 mismatches at 25� C. FIG. 17B depicts results of reactions in which the reactions in FIG. 17A were repeated at the indicated temperatures for 16 hours.
FIG. 18 depicts a reaction performed using a 41-base E template and a 10-base reagent designed to anneal 1-30 bases from the 5′ end of the template.
FIG. 19 depicts a repeat of the n=10 reaction in FIG. 18 in which the nine bases following the 5′-NH2-dT were replaced with various backbone analogues.
FIG. 20 depicts the n=1, n=10, and n=1 mismatched (mis) reactions described in FIG. 18 which were repeated with template and reagent concentrations of 12.5, 25, 62.5 or 125 nM.
FIGS. 21A-21B are a schematic representation of a method for translating, selecting, and amplifying a synthetic molecule that binds streptavidin from a DNA-encoded library.
FIG. 22A depicts DNA sequencing results of a PCR amplified pool of nucleic acid templates of FIGS. 21A-21B before and after selection.
FIG. 22B is a schematic representation of a method for creating and evolving libraries of non-natural molecules using nucleic acid-templated synthesis, where —R1 represents the library of product functionality transferred from reagent library 1 and —R1B represents a selected product.
FIGS. 23A-23D are schematic representations of exemplary DNA-templated reactions.
FIG. 24 depicts analysis by denaturing PAGE of representative DNA-templated reactions listed in FIGS. 23 and 25.
FIGS. 25A-25B are schematic representations of DNA-templated amide bond formation reactions mediated by EDC and sulfo-NHS or by DMT-MM for a variety of substituted carboxylic acids and amines.
FIG. 26A-26B depict an analysis of the distance independent nature of certain nucleic acid-templated reactions. FIG. 26A is a schematic representation showing a model for distance-independent nucleic acid-templated synthesis. FIG. 26B depicts the results of denaturing PAGE of a DNA-templated Wittig olefination between complementary aldehyde-linked template 11 and phosphorous ylide reagent 13 from FIG. 23B with either zero bases (lanes 1-3) or ten bases (lanes 4-6) separating annealed reactants.
FIG. 27 is a schematic representation of exemplary nucleic acid-templated complexity building reactions.
FIGS. 28A-28B depict strategies for DNA-templated synthesis using autocleaving linkers (FIGS. 28A and 28B), scarless linkers (FIG. 28C), and useful scar linkers (FIG. 28D).
FIG. 29 depicts results from nucleic acid-templated reactions with various linkers.
FIGS. 30A-30B are schematic representations depicting strategies for purifying products of DNA-templated synthesis using an autocleaving reagent linker (FIG. 30A) or scar and non scar linkers (FIG. 30B).
FIGS. 31A-B depict an exemplary DNA-templated multi-step tripeptide synthesis.
FIGS. 32A-B depict an exemplary DNA-templated multi-step synthesis.
FIG. 33 depicts DNA-templated amide bond formation reactions in which reagents and templates are complexed with dimethyldidodecylammonium cations.
FIG. 34 shows denaturing PAGE gels with representative DNA-templated amine acylation, Wittig olefination, 1,3-dipolar cycloaddition, and reductive amination reactions using the end-of-helix (E) and omega (Ω) architectures.
FIGS. 35A-35D are bar charts showing a comparison of end-of-helix (E), hairpin (H), and omega (Ω) architectures for mediating DNA-templated amine acylation (FIG. 35A), Wittig olefination (FIG. 35B), 1,3-dipolar cycloaddition (FIG. 35C), or reductive amination reactions (FIG. 35D).
FIG. 36 is a table showing the melting temperatures of selected template-reagent combinations using the omega (Ω) and end-of-helix (E) architectures.
FIG. 37 is a bar chart showing the efficiencies of DNA-templated reactions mediated by a template having the T architecture.
FIGS. 38A-38C depict two DNA-templated reactions on a single template in one solution mediated by templates having a T architecture.
FIG. 39A-39C are schematic illustrations showing the relative rates of product formation from (S)- and (R)-bromides in H template (FIG. 39A) or E template (FIGS. 39B and 39C) mediated stereoselective DNA-templated substitution reactions.
FIGS. 40A-40D depict results on reaction stereoselectivity when aromatic bases between the reactive groups are deleted and restored. The Figures show changes in stereoselectivity as a result of restoring aromatic DNA bases from the 5′ end (FIGS. 40A-40C) or from the 3′ end (FIG. 40D) of the 12-base intervening region.
FIGS. 41A-41B show the stereoselectivities of DNA-templated reactions mediated by right-handed helix (B-form) (FIG. 41A) or left-handed helix (Z-form) (FIGS. 41A and 41B) hairpin architectures.
FIGS. 42A-42D shows graphical representations of product yield versus time for exemplary stereoselective DNA-templated reactions used to calculate kS/kR. FIG. 42A corresponds to the reaction shown in FIG. 39A; FIG. 42B corresponds to the reaction shown in FIG. 39B; FIG. 42C corresponds to the reaction shown in FIG. 44A and FIG. 42D corresponds to the reaction shown in FIG. 44B.
FIGS. 43A-43F are a schematic representations showing template and reagent structures that incorporate achiral, flexible linkers.
FIG. 44A-44B are graphical representations of circular dichroism spectra obtained for B-form (FIG. 44A) and Z-form (FIG. 44B) template-reagent complexes.
FIG. 45 shows a representative denaturing PAGE analysis of reactions using the CG-rich sequences at low and high salt concentrations.
FIG. 46 is a schematic representation of a DNA-templated synthesis in which maleimides, aldehydes, or amines are subjected to multiple DNA-templated reaction types in a single solution.
FIG. 47 depicts templates and reagents used pairwise in 12-reactant one-pot DNA-templated reactions.
FIG. 48 depicts a “one-pot” DNA-templated reaction containing 12 reactants and at least seven possible reaction types which generates only 6 sequence-programmed products out of at least 28 possible products.
FIG. 49 is a schematic representation of a method for diversifying a DNA-templated library by sequentially exposing or creating reactive groups.
FIGS. 50A-50E are schematic representations of exemplary nucleic acid-templated deprotections useful in the practice of the invention.
FIGS. 51A-51B are schematic representations of exemplary nucleic acid-templated functional group interconversions useful in the practice of the invention.
FIG. 52 is a schematic representation showing the assembly of transfer units along a nucleic acid template.
FIG. 53 is a schematic representation showing the polymerization of dicarbamate units along a nucleic acid template to form a polycarbamate.
FIG. 54 is a schematic representation showing cleavage of a polycarbamate polymer from a nucleotide backbone.
FIG. 55 is a schematic representation showing the synthesis of a DNA-templated macrocyclic fumaramide library.
FIG. 56 is a schematic representation of the amine acylation and cyclization steps of various fumaramide library members of FIG. 55.
FIG. 57 shows exemplary amino acid building blocks for the synthesis of a DNA-templated macrocyclic fumaramide library.
FIG. 58 is a schematic representation of a method of c