Evolving new molecular function

• US 7,557,068 B2
• Filed: 12/23/2003
• Issued: 07/07/2009
• 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., Gartner, Zev J., Kanan, Matthew W.
• Primary Examiner(s): Kim; Young J
• Assistant Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US10/744,605
• Publication Number:
  US 20050025766A1
• Time in Patent Office: 2,023 Days
• Field of Search: None
• US Class Current: 506/23
• 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. An in vitro method for synthesizing a templated molecule comprising a plurality of functional groups, the method comprising the steps of

i) providing at least one template comprising a sequence of n codons,

wherein each codon comprises at least one base capable of recognizing a predetermined complementary anti-codon, 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 anti-codon comprising at least one base capable of recognizing a predetermined codon of the at least one template,

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 anti-codon,

iii) contacting the at least one template with the plurality of transfer units, and

iv) 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 of at least one transfer unit to a functional group of a reactive unit of at least one other transfer unit,

wherein the templated molecule is linked by means of a covalent linker 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.

2. The method of claim 1, wherein the template is attached to a solid or resinous support.

3. The method of claim 1, wherein the template comprises or essentially consists of nucleotides selected from the group consisting of:

i) deoxyribonucleic acids (DNA), ii) ribonucleic acids (RNA), and iii) an analog or derivative of i) or ii).

4. The method of claim 1, wherein the template is amplifiable.

5. The method of claim 1, wherein the template further comprises a priming site downstream of the codon and capable of binding an oligonucleotide.

6. The method of claim 1, wherein the templated molecule comprises a scaffold modified by one or more reactive units.

7. The method of claim 6, wherein the scaffold is linked covalently to the template.

8. The method of claim 1, wherein, in step i), a plurality of templates having different codons and/or a different order of codons is provided.

9. The method according to claim 8, wherein the plurality of different templates results in a library of different templated molecules, each of the templated molecules being connected to the specific template that templated the molecule.

10. The method of claim 1 comprising the further step of releasing the template from the templated molecule, and obtaining a templated molecule that is not linked to the template that templated the synthesis of the templated molecule.

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

i) establishing a first composition of templated molecules, the templated molecules being produced by the method of claim 1,

ii) exposing the first composition to conditions enriching the 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.