Evolving new molecular function
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Abstract
Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The present invention provides methods, compositions, and systems for synthesizing, selecting, amplifying, and evolving non-natural molecules based on nucleic acid templates. The sequence of a nucleic acid template is used to direct the synthesis of non-natural molecules such as unnatural polymers and small molecules. Using this method combinatorial libraries of these molecules can be prepared and screened. Upon selection of a molecule, its encoding nucleic acid template may be amplified and/or evolved to yield the same molecule or related molecules for re-screening. The inventive methods and compositions of the present invention allow for the amplification and evolution of non-natural molecules in a manner analogous to the amplification of natural biopolymer such as polynucleotides and protein.
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Citations
57 Claims
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1-46. -46. (Cancelled)
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47. A method for the synthesis of a library of chemical compounds, wherein each of the chemical compounds is bonded to a template, the method comprising the steps of:
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(a) providing a plurality of different templates each comprising one or more reactive units and a number of unique codons;
(b) providing a plurality of different transfer units, each transfer unit comprising an anti-codon unit associated with a reactive unit through a linker moiety, wherein the anti-codon of each transfer unit complements a unique codon of the template;
(c) contacting the plurality of different templates with a subset of the plurality of different transfer units, said subset having anti-codons that complement a corresponding subset of unique codons, under conditions which allow specific hybridization of the anti-codons to the unique codons of the templates;
(d) reacting the reactive unit of the template and the reactive unit of the transfer unit to form a chemical bond, thereby forming a nascent templated molecule;
(e) contacting, under conditions allowing specific hybridization, the plurality of different templates harboring the nascent templated molecules with a further subset of the plurality of transfer units, said subset having anti-codons complementary to a corresponding subset of unique codons of the template in the vicinity of the nascent templated molecules;
(f) allowing the reactive units of the transfer units to form a chemical bond with the nascent templated molecules;
(g) optionally, cleaving one or more of the linkers, provided that at least one linker remains to connect the nascent templated molecule with the template which directed the synthesis thereof, (h) optionally repeating steps (e) through (g); and
(i) obtaining a chemical compound attached via a linker to the template which directed the synthesis thereof.
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48. A method for the synthesis of a chemical compound bonded to a template, the method comprising the steps of:
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(a) providing a template comprising one or more reactive units and a number of unique codons;
(b) providing a plurality of different transfer units, each transfer unit comprising an anti-codon unit associated with a reactive unit through a linker moiety, wherein the anti-codon of each transfer unit complements a unique codon of the template;
(c) contacting the template with a transfer unit having an anti-codon that complements a unique codon, under conditions which allow specific hybridization of the anti-codon to the unique codon of the template;
(d) reacting the reactive unit of the template and the reactive unit of the transfer unit to form a chemical bond, thereby forming a nascent templated molecule;
(e) contacting, under conditions allowing specific hybridization, the template harboring the nascent templated molecule with a further transfer unit having an anti-codon complementary to a unique codon of the template in the vicinity of the nascent templated molecule;
(f) allowing the reactive unit of the transfer unit to form a chemical bond with the nascent templated molecule;
(g) optionally, cleaving one or more of the linkers, provided that at least one linker remains to connect the nascent templated molecule with the template which directed the synthesis thereof;
(h) optionally repeating steps (e) through (g); and
(i) obtaining a chemical compound attached via a linker to the template which directed the synthesis thereof.
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49. An in vitro method of performing nucleic acid templated synthesis, the method comprising the steps of:
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(a) providing (i) a template comprising a first reactive unit covalently associated with a first oligonucleotide defining a first codon sequence, and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence of the template; and
(b) combining the template and the transfer unit under conditions such that the first codon and first anti-codon sequences anneal to one another to bring the first reactive unit and the second unit into reactive proximity whereupon the first reactive unit and the second reactive unit react with one another via a chemical reaction selected from the group consisting of a carbon-carbon bond forming reaction, an organometallic coupling reaction, a cycloaddition reaction, an amine addition reaction, an amide bond forming reaction, an imine forming reaction, a reductive amination reaction, a thiol addition reaction, a phosphoester forming reaction and a phosphonamide forming reaction. - View Dependent Claims (51, 52, 53)
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50. An in vitro method of inducing a chemical reaction without the assistance of a ribosome, the method comprising the steps of:
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(a) providing a reactive unit, a first oligonucleotide comprising a sequence indicative of the identity of the reactive unit, and a second oligonucleotide covalently attached to a small molecule scaffold comprising sites for functionalization;
(b) forming a complex comprising the first and second oligonucleotides; and
(c) inducing a reaction, without the assistance of a ribosome, between the reactive unit and the scaffold at one of the sites for functionalization such that the complex comprises a sequence identifying the reactive unit that reacted with the scaffold, wherein the reaction is selected from the group consisting of a carbon-carbon bond forming reaction, an organometallic coupling reaction, a cycloaddition reaction, an amine addition reaction, an amide bond forming reaction, an imine forming reaction, a reductive amination reaction, a thiol addition reaction, a phosphoester forming reaction and a phosphonamide forming reaction.
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54. A method of performing nucleic acid templated synthesis, the method comprising the steps of:
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(a) providing (i) a template comprising a first reactive unit covalently associated with a first oligonucleotide defining a first codon sequence, and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence of the template, wherein one of the first reactive unit and the second reactive unit comprise a reactive amino functionality and the other of the first reactive unit and the second reactive unit comprises a functionality that permits formation of a covalent bond with the reactive amino functionality; and
(b) combining the template and the transfer unit under conditions such that the first codon and anti-codon sequences anneal to one another to bring the first reactive unit and the second unit into reactive proximity whereupon the first reactive unit and the second reactive unit react with one another to form a covalent bond via the reactive amino functionality.
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55. A reagent of the formula
oligonucleotide— - linker—
reactive unit,wherein the oligonucleotide identifies the reactive unit, the reactive unit has the formula —
R1—
R2(O)—
R3wherein R1 is an aryl group;
R2 is C or S(O); and
R3 is hydrogen or an alkenyl group, andthe linker is a chemical moiety that covalently connects the oligonucleotide to the reactive unit. - View Dependent Claims (56, 57)
- linker—
Specification