Evolving new molecular function
First Claim
1. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product that is not a nucleic acid, the method comprising the steps of:
- (a) providing, in a single solution, (i) a template comprising a first reactive unit covalently attached to a first oligonucleotide defining a first codon sequence and a second, different codon sequence, wherein the codon sequences comprise at least eight bases, and (ii) a transfer unit comprising a second reactive unit covalently attached to a second oligonucleotide defining a single anti-codon sequence, wherein the anti-codon sequence is a first anti-codon sequence complementary to the first codon sequence of the template; and
(b) permitting the first codon and the first anti-codon sequences to anneal to one another to bring the first reactive unit and the second reactive unit into reactive proximity whereupon the first reactive unit reacts with the second reactive unit, without the assistance of a ribosome, to produce the reaction product that is covalently attached to the template.
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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.
146 Citations
51 Claims
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1. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product that is not a nucleic acid, the method comprising the steps of:
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(a) providing, in a single solution, (i) a template comprising a first reactive unit covalently attached to a first oligonucleotide defining a first codon sequence and a second, different codon sequence, wherein the codon sequences comprise at least eight bases, and (ii) a transfer unit comprising a second reactive unit covalently attached to a second oligonucleotide defining a single anti-codon sequence, wherein the anti-codon sequence is a first anti-codon sequence complementary to the first codon sequence of the template; and (b) permitting the first codon and the first anti-codon sequences to anneal to one another to bring the first reactive unit and the second reactive unit into reactive proximity whereupon the first reactive unit reacts with the second reactive unit, without the assistance of a ribosome, to produce the reaction product that is covalently attached to the template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. An in vitro method of performing synthesis from a nucleic acid template to produce a reaction product that is not a nucleic acid, the method comprising the steps of:
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(a) providing, in a single solution, (i) a template comprising a first reactive unit covalently attached to a first oligonucleotide defining a first codon sequence and a second, different codon sequence, (ii) a first transfer unit comprising a second reactive unit covalently attached to a second oligonucleotide defining a first anti-codon sequence capable of annealing to the first codon sequence, and (iii) a second, different transfer unit comprising a second anticodon sequence capable of annealing to the second, different codon sequence; and (b) permitting the first codon and the first anti-codon sequences to anneal to one another to bring the first reactive unit and the second reactive unit into reactive proximity whereupon the first reactive unit reacts with the second reactive unit, without the assistance of a ribosome, to produce the reaction product that is covalently attached to the template. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. 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, in a single solution, (i) a plurality of different templates comprising a plurality of different first reactive units covalently attached to a corresponding plurality of first oligonucleotides, wherein each first oligonucleotide defines at least one first codon sequence and (ii) a plurality of transfer units comprising a plurality of second reactive units covalently attached to a corresponding plurality of second oligonucleotides, wherein the second oligonucleotides each define an anti-codon sequence complementary to a first codon sequence of at least one of the different templates; and (b) annealing oligonucleotides having complementary codon and anti-codon sequences to bring the first and second reactive units into reactive proximity thereby to induce covalent bond forming reactions between the reactive units, without the assistance of a ribosome, to produce a plurality of different reaction products covalently attached to a corresponding plurality of first oligonucleotides, wherein the reaction products are not nucleic acids. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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43. An in vitro method of performing nucleic acid templated multi-step synthesis, the method comprising the steps of:
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(a) providing, in a single solution, (i) a template comprising a first reactive unit covalently associated with a first oligonucleotide comprising a plurality of different codon sequences and defining a first codon sequence and, (ii) a plurality of different transfer units each comprising a different anti-codon sequence and a corresponding reactive unit, wherein the plurality of different transfer units includes a first transfer unit comprising a second reactive unit covalently associated with a second oligonucleotide defining a first anti-codon sequence complementary to the first codon sequence of the template; and (b) annealing the first codon and first anti-codon sequences to bring the first reactive unit and the second reactive unit into reactive proximity, thereby inducing a reaction between the first and second reactive units to produce, without the assistance of a ribosome, a reaction product covalently attached to the template, wherein the reaction product is not a nucleic acid. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51)
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Specification