Evolving new molecular function

• US 20050281819A9
• Filed: 12/23/2003
• Published: 12/22/2005
• Est. Priority Date: 03/19/2001
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Liu, David R., Kanan, Matthew W., Gartner, Zev
• Granted Patent: US 7,557,068 B2
• Time in Patent Office: Days
• US Class Current: 424/145.1
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12P 1/00 Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00

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.

Claims

1-46. -46. (canceled)

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.

48. The method of claim 47, wherein the template is attached to a solid or resinous support.

49. The method of claim 47, wherein the template comprises or essentially consists of nucleotides selected from the group consisting of deoxyribonucleic acids (DNA), and ribonucleic acids (RNA), including any analog or derivative thereof.

50. The method of claim 47, wherein the template is amplifiable.

51. The method of claim 47, wherein the template further comprises a priming site downstream of the coding nucleotides and capable of binding an oligonucleotide.

52. The method of claim 47, wherein the templated molecule is linked to the template and/or to the complementary nucleotide by means of a first linker, wherein a linker separating a reactive unit from a complementary nucleotide is a second, cleavable linker, the method further comprising cleaving the second, cleavable linker but not the first linker.

53. The method of claim 47, wherein the templated molecule comprises a scaffold modified by one or more reactive units.

54. The method of claim 53, wherein the scaffold is linked covalently to the template.

55. The method of claim 47, wherein a plurality of templates having different coding nucleotides and/or a different order of coding nucleotides is used.

56. The method according to claim 55, wherein the plurality of different templates results in a library of different templated molecules, each of said templated molecules being connected to the specific template or strand complementary to the template that templated the molecule.

57. The method of claim 47 comprising the further step of releasing the template or strand complementary to the template from the templated molecule, and obtaining a templated molecule that is not linked to the template or strand complementary to the template that templated the synthesis of the templated molecule.

58. A templated molecule obtainable in accordance with claim 47, a plurality of covalently linked functional groups each comprising a residue, wherein the covalently linked residues are capable of generating a polymer comprising, exclusively or in combination with additional portions, at least one portion selected from the group of polymer portions consisting 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, polynucleotides, PNAs, polyimines, polyacetals, polyacetates, polyvinyl, polycyclic compounds, and wherein the plurality of residues is preferably at least 2, for example from 2 to 4;

from 2 to 5;

from 2 to 6;

from 4 to 6;

from 4 to 5;

from 5 to 6;

4;

5;

6;

at least 10;

at least 20;

at least 30;

at least 50;

at least 100;

at least 150;

at least 200;

or at least 300.

59. The templated molecule according to claim 58, wherein the templated molecule is a polymer.

65. A method for screening a composition of molecules having a predetermined activity comprising:

i) establishing a first composition of templated molecules, said molecules being produced as defined in claim 47,

ii) exposing the first composition to conditions enriching said first composition with templated molecules having the predetermined activity, and

iii) optionally amplifying the templated molecules of the enriched composition to obtain a second composition,

iv) further optionally repeating step ii) to iii), and

v) obtaining a further composition having a higher ratio of templated molecules having the specific predetermined activity.

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.

61. A complex comprising a templated molecule and the template that templated the synthesis of the templated molecule.

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.

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.

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.

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.

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 optionally

iii) templating by means of said second template(s) or complementary strand(s) a second templated molecule, or a plurality of such second templated molecules.

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.