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.
56 Citations
68 Claims
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1-46. -46. (canceled)
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47. A method for synthesizing a templated molecule comprising a plurality of functional groups, said method comprising the steps of
i) providing at least one template comprising a sequence of n coding nucleotides, wherein each coding nucleotide comprises at least one base capable of recognizing a predetermined complementary nucleotide, and wherein n is an integer of more than 1, ii) providing a plurality of transfer units, wherein each transfer unit comprises a) at least one complementary nucleotide comprising at least one base capable of recognizing a predetermined coding nucleotide, b) at least one reactive unit comprising at least one functional group and at least one reactive group, and c) at least one linker separating the at least one reactive unit from the at least one complementary nucleotide, iii) contacting each of said coding nucleotides with a complementary nucleotide capable of recognizing said coding nucleotide, iv) optionally, obtaining a strand complementary to the template, and v) obtaining a templated molecule comprising covalently linked, functional groups by linking, by means of a reaction involving reactive groups, a functional group of at least one reactive unit to a functional group of another reactive unit, - wherein the templated molecule is capable of being linked by means of a linker to the strand complementary to the template or to the template that templated the synthesis of the templated molecule, and
wherein the synthesis of the templated molecule does not involve ribosome mediated translation of an mRNA. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 65)
- wherein the templated molecule is capable of being linked by means of a linker to the strand complementary to the template or to the template that templated the synthesis of the templated molecule, and
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60. A templated molecule comprising a sequence of covalently linked, functional groups, wherein the templated molecule does not comprise or consist of a peptide or a polynucleotide.
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61. A complex comprising a templated molecule and the template that templated the synthesis of the templated molecule.
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62. A templated molecule comprising a sequence of covalently linked, functional groups, wherein the templated molecule is linked by means of a linker to the template or to the strand complementary to the template that templated the synthesis of the templated molecule, wherein the templated molecule does not comprise or consist of an α
- -peptide or a polynucleotide.
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63. A molecule comprising a sequence of covalently linked building blocks, wherein the sequence of covalently linked building blocks comprises a sequence of complementary nucleotides complementary to the template that templated the synthesis of the templated molecule, and wherein the templated molecule is linked to the complementary nucleotides or to the template that templated its synthesis.
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64. A method for selecting complexes or templated molecules having a predetermined activity, said method comprising the step of performing a selection procedure and selecting templated molecules based on predetermined selection criteria.
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66. A method for amplifying a strand complementary to the template or the template that templated the synthesis of the templated molecule having, or potentially having a predetermined activity, said method comprising the step of contacting the template with amplification means, and amplifying the template.
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67. A method for altering the sequence of a templated molecule, including generating a templated molecule comprising a novel or altered sequence of functional groups, wherein said method preferably comprises the steps of
i) providing a first strand complementary to a template or a first template capable of templating the first templated molecule, or a plurality of such first strands complementary to templates or first templates capable of templating a plurality of first templated molecules, ii) mutating or modifying the sequence of the first complementary strand or the first template, or the plurality of first complementary strands or first templates, and generating a second template or a second complementary strand, or a plurality of second templates or second complementary strands, - wherein said second template(s) or complementary strand(s) is capable of templating the synthesis of a second templated molecule, or a plurality of second templated molecules,
wherein said second templated molecule(s) comprises a sequence of covalently linked, functional groups that is not identical to the sequence of functional groups of the first templated molecule(s), and optionallyiii) templating by means of said second template(s) or complementary strand(s) a second templated molecule, or a plurality of such second templated molecules.
- wherein said second template(s) or complementary strand(s) is capable of templating the synthesis of a second templated molecule, or a plurality of second templated molecules,
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68. A transfer unit comprising
i) a complementary nucleotide capable of specifically recognizing a coding nucleotide having a base, ii) at least one functional entity selected from a precursor of peptides wherein the amino acid residues are in the L-form or in the D-form, polyamides, polyesters, polycarbamates, polycarbonates, polyureas, polyethers, poly-thioethers, polyethylenes, PNAs, polyimines, polyacetals, polyacetates, polyvinyl, and polycyclic compounds, and iii) a linker separating the functional entity from the complementary nucleotide.
Specification