Evolving new molecular function
First Claim
1. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:
- (a) providing (i) a template comprising a first reactive unit covalently attached to 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;
(b) annealing the first codon and first anti-codon sequences to bring the first reactive unit and the second reactive unit into reactive proximity; and
(c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product.
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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
71 Claims
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1. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:
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(a) providing (i) a template comprising a first reactive unit covalently attached to 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; (b) annealing the first codon and first anti-codon sequences to bring the first reactive unit and the second reactive unit into reactive proximity; and (c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)
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10. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:
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(a) providing (i) an amplifiable template comprising a first reactive unit associated with a first oligonucleotide defining a first codon sequence, and (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide defining an anti-codon sequence complementary to the first codon sequence; (b) annealing the codon and anti-codon sequences to bring the first reactive unit and the second reactive unit into reactive proximity; (c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product; and (d) amplifying the template that encoded the reaction product. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product, wherein the reaction product is not a nucleic acid, the method comprising the steps of:
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(a) providing (i) a template comprising a first reactive unit associated with a first oligonucleotide defining a first codon sequence and a second, different codon sequence, and (ii) a first 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; (b) annealing the first codon sequence and the first anti-codon sequence to bring the first reactive unit and the second reactive unit into reactive proximity; (c) inducing a reaction between the first and second reactive units without assistance of a ribosome to produce the reaction product; and (d) selecting the reaction product still associated with the template. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. An in vitro method of performing synthesis from a nucleic acid template to produce a modified scaffold molecule, the method comprising the steps of:
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(a) providing (i) a template comprising a scaffold molecule associated with a first oligonucleotide defining a first codon sequence and a second, different codon sequence, the scaffold molecule comprising a plurality of sites for modification, and (ii) a first transfer unit comprising a reactive unit covalently attached to a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence; (b) annealing the first codon sequence and the first anti-codon sequence to bring the scaffold molecule and the reactive unit into reactive proximity; and (c) inducing a covalent bond-forming reaction between the reactive unit and the scaffold molecule without assistance of a ribosome to produce the modified scaffold molecule. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. An in vitro method of producing a plurality of different reaction products in a single solution, the method comprising the steps of:
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(a) providing a plurality of different templates comprising a plurality of first reactive units associated with a corresponding plurality of first oligonucleotides, wherein each first oligonucleotide defines at least one codon sequence; (b) providing a plurality of transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein the second oligonueleotides each define an anti-codon sequence complementary to a codon sequence; (c) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity; and (d) inducing covalent bond-forming reactions between the reactive units without the assistance of a ribosome to produce the plurality of different reaction products. - View Dependent Claims (50, 51, 52, 53, 54)
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Specification