Selection by compartmentalised screening

• US 9,857,303 B2
• Filed: 08/12/2011
• Issued: 01/02/2018
• Est. Priority Date: 03/31/2003
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Griffiths, Andrew David, Abell, Chris, Hollfelder, Florian, Mastrobattista, Enrico
• Primary Examiner(s): Bunker, Amy M
• Application Number:
  US13/208,614
• Publication Number:
  US 20120010107A1
• Time in Patent Office: 2,335 Days
• Field of Search: 424490, 435 61, 506 9, 506 16
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules

Abstract

The invention describes a method for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, by compartmentalizing the compounds into microcapsules together with the target, such that only a subset of the repertoire is represented in multiple copies in any one microcapsules; and identifying the compound which binds to or modulates the activity of the target. The invention enables the screening of large repertoires of molecules which can serve as leads for drug development.

Claims

1. A water-in-perfluorocarbon oil emulsion library comprising:

(a) a perfluorocarbon oil comprising a fluorinated surfactant; and

(b) a plurality of individual aqueous microdroplets encapsulated by the perfluorocarbon oil, each aqueous microdroplet of the plurality of individual aqueous microdroplets comprising;

(i) a subset of non-fluorinated nucleic acids, comprising one or more non-fluorinated nucleic acids from of a repertoire of non-fluorinated nucleic acids, wherein the repertoire of non-fluorinated nucleic acids in a plurality of individual aqueous microdroplets are structurally different;

(ii) a biological target enzyme that is reactive with one or more of the nucleic acids from the repertoire of non-fluorinated nucleic acids; and

(iii) a fluorogenic substrate of the biological target enzyme;

wherein each non-fluorinated nucleic acids of the each subset of the repertoire of non-fluorinated nucleic acids is insoluble in the perfluorocarbon oil, and wherein the fluorogenic substrate of the biological target enzyme comprises an optical tag, wherein the optical tag produces a selectable change upon complexation of the fluorogenic substrate with the biological target enzyme.

2. The water-in-perfluorocarbon oil emulsion library according to claim 1, wherein the aqueous microdroplets further comprise microbeads.

3. The water-in-perfluorocarbon oil emulsion library according to claim 2, wherein the repertoire of non-fluorinated nucleic acids are releasably attached to the microbeads.

4. The water-in-perfluorocarbon emulsion library according to claim 1, wherein the biological target enzyme in each of the aqueous microdroplets is structurally different.

5. The water-in-perfluorocarbon oil emulsion library according to claim 1, wherein each aqueous microdroplet further comprises a member of a binding pair.

6. The water-in-perfluorocarbon oil emulsion library according to claim 5, wherein a member of a binding pair is an antibody.

7. A water-in-perfluorocarbon oil emulsion library comprising:

a perfluorocarbon oil comprising;

(a) a fluorosurfactant; and

(b) a plurality of individual aqueous microdroplets, wherein each aqueous microdroplet of the plurality of individual aqueous microdroplets is individually encapsulated within the perfluorinated oil and comprises;

(i) multiple copies of a single type of nucleic acid, wherein the single type of nucleic acid is insoluble in the perfluorocarbon oil; and

(ii) a target cell, wherein the target cell comprises a biological target enzyme that is reactive with one or more of the multiple copies of the single type of nucleic acid; and

a fluorogenic substrate of the biological target enzyme, wherein the fluorogenic substrate of the biological target enzyme comprises an optical tag, wherein the optical tag produces a selectable change upon complexation of the fluorogenic substrate with the biological target enzyme.

8. The water-in-perfluorocarbon oil emulsion library according to claim 7, wherein the single type of nucleic acid is attached to a microbead.

9. The water-in-perfluorocarbon oil emulsion library according to claim 8, wherein the single type of nucleic acid is releasably attached to a microbead.