Evolving new molecular function
First Claim
1. a method of inducing reaction between first and second reactive units during a nucleic acid-templated chemical reaction, the method comprising the steps of:
- (a) 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 capable of annealing to said codon, wherein said codon or said anti-codon comprise first and second spaced apart regions;
(b) annealing said oligonucleotides together thereby to bring said first reactive unit and said second reaction unit into reactive proximity, wherein said codon or said anti-codon having said first and second spaced apart regions produce a loop of oligonucleotides not annealed to the corresponding anti-codon or codon; and
(c) inducing a covalent bond-forming reaction between said reactive units to produce a reaction product.
1 Assignment
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Accused Products
Abstract
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.
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Citations
131 Claims
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1. a method of inducing reaction between first and second reactive units during a nucleic acid-templated chemical reaction, 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 comprising a codon and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon capable of annealing to said codon, wherein said codon or said anti-codon comprise first and second spaced apart regions;
(b) annealing said oligonucleotides together thereby to bring said first reactive unit and said second reaction unit into reactive proximity, wherein said codon or said anti-codon having said first and second spaced apart regions produce a loop of oligonucleotides not annealed to the corresponding anti-codon or codon; and
(c) inducing a covalent bond-forming reaction between said reactive units to produce a reaction product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of inducing reaction between first and second reactive units during a nucleic acid-templated chemical reaction, 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 having a proximal end and a distal end and comprising a codon and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon capable of annealing with said codon, wherein said first reactive unit is attached to an attachment site intermediate said proximal end and said distal end of said first oligonucleotide;
(b) annealing said oligonucleotides together thereby to bring said first reactive unit and said second reactive unit into reactive proximity; and
(c) inducing a covalent bond-forming reaction between said reactive units to produce a reaction product. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis, 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 comprising a predetermined codon sequence, (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon sequence capable of annealing to said codon sequence, and (iii) a second transfer unit comprising a third reactive unit different from said second reactive unit associated with a third oligonucleotide without an anti-codon sequence capable of annealing to said codon sequence; and
(b) mixing said template, said first transfer unit and said second transfer unit under conditions to permit annealing of said second oligonucleotide of said first transfer unit to said first oligonucleotide of said template thereby to enhance covalent bond formation between said second reactive unit and said first reactive unit relative to covalent bond formation between said third reactive unit and said first reactive unit.
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25-36. -36. (canceled)
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37. A method of increasing reaction selectivity among a plurality of reactants in a nucleic acid-templated synthesis, the method comprising the steps of:
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(a) providing (i) a template comprising a first oligonucleotide comprising first and second codon sequences, (ii) a first transfer unit comprising a first reactive unit associated with a second oligonucleotide comprising a first anti-codon sequence capable of annealing to said first codon sequence, (iii) a second transfer unit comprising a second reactive unit associated with a third oligonucleotide comprising a second anti-codon sequence capable of annealing to said second codon sequence, and (iv) a third transfer unit comprising a third reactive unit associated with a fourth oligonucleotide sequence without an anti-codon sequence capable of annealing to said first codon sequence or said second codon sequence; and
(b) mixing said template, said first transfer unit, said second transfer unit and said third transfer unit under conditions to permit annealing of said first anti-codon sequence to said first codon sequence and said second anti-codon sequence to said second codon sequence thereby to enhance covalent bond formation between said first reactive unit and said second reactive unit relative to covalent bond formation between said third reactive unit and said first reactive unit or between said third reactive unit and said second reactive unit.
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38-48. -48. (canceled)
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49. A method of performing stereoselective nucleic acid-templated synthesis, the method comprising the steps of:
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(a) providing (i) a template comprising a first oligonucleotide optionally associated with a reactive unit and (ii) one or more transfer units each comprising a second oligonucleotide associated with a reactive unit;
(b) annealing said first and second oligonucleotides, thereby bringing at least two said reactive units into reactive proximity and inducing formation of a covalent bond between said reactive units to form a reaction product, wherein said reaction product comprises a chiral center and is of at least 60% stereochemical purity at said chiral center.
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50-53. -53. (canceled)
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54. A method of performing stereoselective nucleic acid-templated synthesis, the method comprising the steps of:
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(a) providing (i) at least two templates, one template comprising a first oligonucleotide associated with a first reactive unit having a first stereochemical configuration and the other template comprising a said first oligonucleotide associated with a said first reactive unit having a second, different stereochemical configuration and (ii) at least one transfer unit comprising a second reactive unit associated with a second oligonucleotide, wherein a sequence of said second oligonucleotide is complementary to a sequence of said first oligonucleotide; and
(b) annealing said first and second oligonucleotides together under conditions to permit said second reactive unit of said transfer unit to react preferentially with either said first reactive unit having said first stereochemical configuration or said first reactive unit having said second stereochemical configuration to produce a reaction product.
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55. A method of performing stereoselective nucleic acid-templated synthesis, the method comprising the steps of:
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(a) providing (i) template comprising a first oligonucleotide associated with a first reactive unit and (ii) at least two transfer units, one transfer unit comprising a second oligonucleotide associated with a second reactive unit having a first stereochemical configuration and the other transfer unit comprising a said second oligonucleotide associated with a said second reactive unit having a second, different stereochemical configuration, wherein a sequence of said second oligonucleotide is complementary to a sequence of said first oligonucleotide; and
(b) annealing said first and second oligonucleotides together under conditions to permit said first reactive unit of said template to react preferentially with either said second reactive unit having said first stereochemical configuration or said second reactive unit having said second stereochemical configuration to produce a reaction product.
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56-59. -59. (canceled)
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60. A method of performing stereoselective nucleic acid-templated synthesis, the method comprising the steps of:
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(a) providing (i) a template comprising a first oligonucleotide comprising a first codon sequence and a second codon sequence, (ii) a first pair of transfer units, wherein one transfer unit of said first pair comprises a second oligonucleotide with a first anti-codon sequence associated with a first reactive unit having a first stereochemical configuration and the other transfer unit of said first pair comprises a said second oligonucleotide associated with a said first reactive unit having a second stereochemical configuration, and (iii) a second pair of transfer units, wherein one transfer unit of the second pair comprises a third oligonucleotide with a second anti-codon sequence associated with a second reactive unit having a first stereochemical configuration and the other transfer unit of said second pair comprises a said third oligonucleotide associated with a second reactive unit having a second stereochemical configuration; and
(b) annealing said template, said first pair of transfer units, and said second pair of transfer units under conditions to permit a member of said first pair of transfer units to react preferentially with a member of said second pair of transfer units to produce a reaction product.
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61-64. -64. (canceled)
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65. A method of enriching a product of a nucleic acid-templated synthesis, the method comprising the steps of:
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(a) 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 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;
(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.
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66-83. -83. (canceled)
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84. A method of identifying a new chemical reaction, the method comprising the steps of:
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(a) providing a 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 reaction product associated therewith;
(b) selecting a particular reaction product associated with its corresponding oligonucleotide;
(c) characterizing the reaction product; and
(d) identifying a new chemical reaction that made the reaction product using information encoded by said corresponding oligonucleotide.
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85-87. -87. (canceled)
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88. A method of identifying a new chemical reaction, 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 comprising a codon and (ii) a transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon, wherein said codon and said anti-codon are capable of annealing together;
(b) annealing the oligonucleotides together thereby to bring said first reactive unit and said second reactive unit into reactive proximity;
(c) inducing a covalent bond-forming reaction between said reactive units to produce a reaction product;
(d) characterizing the reaction product; and
(e) identifying a new chemical reaction to make the reaction product using information encoded by the template to identify the first reactive unit and the second reactive unit that reacted to produce the reaction product.
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89-95. -95. (canceled)
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96. A method of identifying a new chemical reaction, the method comprising:
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(a) providing (i) a first transfer unit comprising a first reactive unit associated with a first oligonucleotide, (ii) a second transfer unit comprising a second reactive unit associated with a second oligonucleotide, and (iii) a template comprising sequences capable of annealing to said first oligonucleotide and to said second oligonucleotide;
(b) annealing said oligonucleotides to said template thereby to bring said first and second reactive units into reactive proximity;
(c) inducing a covalent bond-forming reaction between said reactive units to produce a reaction product;
(d) characterizing said reaction product; and
(e) identifying a new chemical reaction to make said reaction product using information encoded by said template to identify said first reactive unit and said second reactive unit that reacted to produce the reaction product.
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97-103. -103. (canceled)
- 104. A reagent of the general formula
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108. (canceled)
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112. 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 associated with a first oligonucleotide comprising a predetermined codon sequence, (ii) a first transfer unit comprising a second reactive unit associated with a second oligonucleotide comprising an anti-codon sequence capable of annealing to said codon sequence, and (iii) a second transfer unit comprising a third reactive unit different from said second reactive unit associated with a third oligonucleotide without an anti-codon sequence capable of annealing to said codon sequence; and
(b) mixing said template, said first transfer unit and said second transfer unit under conditions to permit annealing of said second oligonucleotide of said first transfer unit to said first oligonucleotide of said template thereby to enhance covalent bond formation between said second reactive unit and said first reactive unit relative to covalent bond formation between said third reactive unit and said first reactive unit wherein the covalent bond between said second reactive unit and said first reactive unit occurs via a chemical reaction selected from the group consisting of a substitution reaction, a carbon-carbon bond forming reaction, an elimination reaction, an acylation reaction, an addition reaction, a rearrangement reaction, an oxidative reaction and a reductive reaction.
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113. A 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, without the assistance of a ribosome, a reaction selected from the group consisting of a substitution reaction, a carbon-carbon bond forming reaction, an elimination reaction, an acylation reaction, an addition reaction, a rearrangement reaction, an oxidative reaction and a reductive reaction 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.
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114-117. -117. (canceled)
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118. A reagent of the general formula
oligonucleotide−- linker−
reactive unit,wherein the oligonucleotide identifies the reactive unit, and wherein the reactive unit has the formula —
R1—
R2(O)—
R3 wherein R1 is an aryl group or an alkyl chain;
R2 is C or S(O);
R3 is a hydrogen atom, an alkyl group or an alkenyl group, and wherein the linker is a chemical moiety that covalently connects the oligonucleotide and the reactive unit. - View Dependent Claims (119)
- linker−
- 120. A microarray comprising a plurality of single stranded oligonucleotides each of which is immobilized at a separate addressable location of a solid support, the immobilized oligonucleotides being hybridized to a plurality of products of nucleic acid templated synthesis, wherein each product is associated with a template comprising a plurality of codons that encode reactive groups that reacted to form at least part of the product of nucleic acid templated synthesis.
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122. A method for synthesizing a templated reaction product, comprising the steps of:
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(a) providing one or more transfer units, each comprising an anti-codon, a first reactive unit, and a linker connecting the anti-codon and the first reactive unit;
(b) providing a second reactive unit attached to a first region of a codon and capable of a covalent bond-forming reaction with the first reactive unit, the first region of the codon being complementary to a first portion of the anti-codon;
(c) providing a second region of a codon, the second region of the codon being complementary to a second portion of the anti-codon;
(d) annealing the first and second portions of the anti-codon to the first and second regions of the codon under conditions allowing sequence-specific nucleic acid hybridization; and
(e) allowing the first and second reactive units to form a covalent bond, thereby synthesizing the reaction product. - View Dependent Claims (123, 124, 125, 126, 127, 128, 129, 130)
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131. A method for synthesizing a complex comprising a reaction product and a template for the synthesis thereof, the method comprising the steps of:
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(a) providing a template comprising two or more codons and a first reactive group, wherein at least one codon comprises first and second spaced apart regions, wherein the first spaced apart region of the codon is in close proximity with the first reactive group and the second spaced apart region of the codon is in a distal portion of the template, (b) providing two or more transfer units, each transfer unit comprising (i) an anti-codon capable of recognizing a codon of the template, (ii) a second reactive unit, and (iii) a linker connecting the anti-codon and the second reactive unit, wherein the anti-codon of one of the transfer units recognizes the first and second spaced apart regions, (c) contacting the template with a transfer unit under conditions allowing sequence-specific nucleic acid hybridization, (d) allowing the first and second reactive units to form a covalent bond, (e) cleaving a linker, and (f) repeating, for a transfer unit having an anti-codon capable of hybridizing to a new codon, steps (c) to (f) one or more times.
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Specification