Optical sorting method

• US 20020119459A1
• Filed: 06/29/2001
• Published: 08/29/2002
• Est. Priority Date: 01/07/1999
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Griffiths, Andrew
• Granted Patent: US 6,808,882 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; (b) expressing the genetic elements to produce their respective gene products within the microcapsules; (c) sorting the genetic elements which produce the gene product having the desired activity using a change in the optical properties of the genetic elements. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

Claims

1. A method for isolating one or more genetic elements encoding a gene product having a desired activity the expression of which results, directly or indirectly, in the modification of an optical property of a genetic element encoding the gene product, comprising the steps of:

(a) compartmentalising genetic elements into microcapsules;

(b) expressing the genetic elements to produce their respective gene products within the microcapsules;

(c) sorting the genetic elements which produce the gene product(s) having the desired activity according to a change in the optical properties of the genetic elements, whereby a genetic element encoding a gene product having said desired activity is isolated.

2. The method according to claim 1, wherein in step (b) the activity of the desired gene product within the microcapsule results, directly or indirectly, in the modification of the genetic element encoding the gene product, thereby enabling the isolation of that genetic element.

3. The method according to claim 2, wherein the modification of the genetic element encoding the gene product occurs within the microcapsule, and where said modification induces a change in an optical property of said genetic element.

4. The method according to claim 2, wherein the modification of the genetic element enables said genetic element to be further modified outside the microcapsule so as to induce a change in the optical properties of said genetic element.

5. The method according to claim 2, wherein said genetic element comprises a ligand and the desired gene product within the microcapsule binds, directly or indirectly, to said ligand, thereby enabling the isolation of the genetic element.

6. The method according to claim 5, wherein said ligand is encoded by said genetic element.

7. The method according to claim 2, wherein said genetic element comprises a substrate and the activity of the desired gene product within the microcapsule results, directly or indirectly, in the conversion of said substrate into a product which remains linked to said genetic element thereby enabling the isolation of said genetic element.

8. The method according to claim 2, wherein the activity of the desired gene product within the microcapsule results, directly or indirectly, in the generation of a product that forms a complex with the genetic element encoding the gene product, thereby enabling the isolation of that genetic element.

9. The method of claim 2 wherein the activity of the desired gene product within the microcapsule results, directly or indirectly, in the alteration of the expression of a second gene within the microcapsule and the activity of the product of said second gene modifies the optical properties of the genetic element, thereby enabling the isolation of that genetic element.

10. The method according to claim 1, wherein step (b) comprises expressing the genetic elements to produce their respective gene products within the microcapsules, linking the gene products to the genetic elements encoding them to form complexes and isolating the complexes thereby formed.

11. The method according to claim 10, wherein in step (c) the complexes formed in step (b) are directly sorted, based on their changed optical properties, thereby isolating a genetic element encoding a gene product having the desired activity.

12. The method according to claim 10, wherein in step (c) the complexes formed in step (b) are further reacted to induce a change in optical properties of the genetic element, said change being conditionally dependent on the presence in the complex of a gene product with the desired activity.

13. The method according to claim 10, wherein the complexes are subjected to a further compartmentalisation step in order to isolate a genetic element encoding a gene product having the desired activity.

14. The method according to claim 1, wherein the change in optical properties of the genetic element results from the binding of a gene product to said genetic element, wherein said gene product has distinctive optical properties.

15. The method according to claim 1, wherein the change in optical properties of the genetic element results from the binding of a ligand that has distinctive optical properties by the gene product.

16. The method according to claim 1, wherein the change in optical properties of the genetic element is due to a change in the optical properties of the gene product when bound to ligand.

17. The method according to claim 1, wherein the change in optical properties of the genetic element is due to a change in the optical properties of the ligand when bound by the gene product.

18. The method according to claim 1, wherein the change in optical properties of the genetic element is due to a change in the optical properties of both ligand and gene product on binding.

19. The method according to claim 1, wherein the change in optical properties of the genetic element results from a detectable difference in the optical properties of the substrate and product of a reaction catalyzed by a gene product of said genetic element.

20. The method according to claim 1, wherein said gene product is an enzyme and both the substrate and product of the reaction catalyzed by said enzyme have similar optical properties, but only the product, and not the substrate of the reaction binds to, or reacts with, the genetic element, thereby changing the optical properties of the genetic element.

21. The method according to claim 1, wherein one or more further reagents specifically bind to, or specifically react with, the gene product attached to the genetic element, thereby altering the optical properties of the genetic element.

22. The method according to claim 1, wherein a non-desired activity of a gene product results in a change in the optical properties of the genetic element which is distinct from that change resulting from the desired activity.

23. The method according to claim 22, wherein the change in optical properties resulting from the non-desired activity is used to negatively select the genetic elements.

24. The method according to claim 22, wherein negative selection is combined with positive selection, thereby improving reaction specificity.

25. The method according to claim 24, wherein the reaction specificity is improved by an increase in binding specificity.

26. The method according to claim 24, wherein the reaction specificity is improved by an increase in regio- and/or stereo-selectivity for substrate and/or product.

27. The method according to claim 1, wherein the genetic elements are isolated from a library of genetic elements encoding a repertoire of gene products.

28. The method according to claim 1, wherein each genetic element encodes two or more gene products, and wherein, in order for the optical properties of a genetic element to be modified, each of said gene products has a desired activity.

29. The method according to claim 1, wherein each genetic element encodes two or more products and the gene products must bind to each other in the microcapsule in order for the optical properties of the genetic element to be modified and the genetic elements sorted.

31. The method according to claim 1, said method further comprising iteratively repeating one or more of steps (a) to (d).

32. The method according to claim 1, said method further comprising amplifying the genetic elements.

33. The method according to claim 1, wherein said compartmentalizing comprises microencapsulation achieved by forming a water-in-oil emulsion of an aqueous solution in an oil-based medium.

34. The method according to claim 1, wherein said one or more the genetic elements comprise a gene attached to a microbead.

35. The method according to claim 34, wherein the microbead is nonmagnetic, magnetic or paramagnetic.

36. The method according to claim 1, wherein said sorting is based on a change in fluorescence of a genetic element encoding a gene product having said desired activity.

37. The method according to claim 36, wherein said sorting is performed using a fluorescence activated cell sorter (FACS) or its equivalent.

38. The method according to claim 36, wherein said change in fluorescence is due to fluorescence resonance energy transfer (FRET).

39. The method according to claim 1, wherein the internal environment of said microcapsules is modified by the addition of one or more reagents to the oil phase.

40. The method according to claim 1, wherein genetic elements modified directly or indirectly by the activity of the desired gene product are further modified by Tyramide Signal Amplification (TSA™

;

NEN), resulting directly or indirectly in a change in the optical properties of said genetic elements thereby enabling their separation.

41. A gene product produced by a genetic element isolated according to the method of claim 1.

30. The method according to claim I further comprising the additional step, after step (c), of:

(d) introducing one or more mutations into the genetic element(s) isolated in step (c).

42. A method for preparing a gene product, comprising the steps of:

(a) preparing plurality of a genetic elements encoding a plurality of gene products;

(b) compartmentalising said genetic elements into microcapsules;

(c) expressing the genetic elements to produce their respective gene products within the microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and

(e) expressing the gene product having the desired activity.

43. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(a) preparing a repertoire of genetic elements encoding gene product;

(b) compartmentalising the genetic elements into microcapsules;

(c) expressing the genetic elements to produce their respective gene products within the microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and

(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds.

44. A method for preparing a compound or compounds comprising the steps of:

(a) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;

(b) preparing genetic elements encoding variants of the polypeptide which facilitates said at least one step;

(c) compartmentalising the genetic elements into microcapsules;

(d) expressing the genetic elements to produce their respective gene products within the microcapsules;

(e) sorting the genetic elements which produce polypeptide gene product(s) having the desired activity using a change in their optical properties; and

(f) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate said at least one step of the synthesis.