Methods and compositions for high throughput identification of protein/nucleic acid binding pairs

• US 6,713,262 B2
• Filed: 06/25/2002
• Issued: 03/30/2004
• Est. Priority Date: 06/25/2002
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Leproust, Eric M., Gellibolian, Robert
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): KIM, YOUNG J
• Application Number:
  US10/180,437
• Publication Number:
  US 20030235828A1
• Time in Patent Office: 644 Days
• Field of Search: 435/6, 435/5, 435/91.2, 536/24.3, 536/24.33, 536/26.6, 530/300, 530/333, 530/350
• US Class Current: 506/4
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00729 Peptide nucleic acids [PNA]
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/531 : characterised by the capture moiety being a protein for target oligonucleotides
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

Methods and compositions for high-throughput identification of protein/nucleic acid binding pairs are provided. In the subject methods, a nucleic acid probe array, e.g., a molecular beacon probe array, is contacted with a target nucleic acid population to produce a hybridized array. The resultant hybridized array is then contacted with a population of proteins to produce a protein bound array. Any resultant array surface bound target nucleic acid/protein complexes are then detected to identify protein/nucleic acid binding pairs. In certain embodiments, the protein and/or nucleic acid members of the identified protein/nucleic acid binding pairs are further characterized. Also provided are systems and kits for use in practicing the subject methods. The subject invention finds use in a variety of different applications.

Claims

1. A method of identifying protein/nucleic acid binding pairs, said method comprising:

(a) contacting a molecular beacon array comprising a plurality of distinct molecular beacon probes with a population of target nucleic acids to produce a hybridized molecular beacon array, wherein each distinct probe of said plurality comprises a different probe sequence and all of said probes of said plurality share a common first fluorescent label;

(b) contacting said hybridized molecular beacon array with a population of fluorescently labeled proteins to produce a protein bound array, where each member of said population of fluorescently labeled proteins is labeled with a second fluorescent label that makes up a FRET pair with said first fluorescent label; and

(c) detecting any FRET generated signals from said array to identify protein/nucleic acid binding pairs on said array.

2. The method according to claim 1, wherein said method further comprises detecting any fluorescent signal from said hybridized array prior to step (b) to identify target nucleic acids hybridized to said hybridized array.

3. The method according to claim 1, wherein said method further comprises characterizing the protein of a protein/nucleic acid binding pair identified by said method.

4. The method according to claim 1, wherein said method further comprises characterizing the protein binding sequence of a nucleic acid of a protein/nucleic acid binding pair identified by said method.

5. The method according to claim 1, wherein said target nucleic acid and fluorescently labeled protein populations are generated from the same tissue/cellular source.

6. The method according to claim 1, wherein said target nucleic acid and fluorescently labeled protein populations are generated from different tissue/cellular sources.

7. The method according to claim 1, wherein said hybridized array is contacted with two differentially labeled protein populations.

8. The method according to claim 7, wherein said two differentially labeled protein populations make up a test/control pair.

9. The method according to claim 8, wherein said two differentially labeled protein populations make up a normal/disease pair.

10. The method according to claim 1, wherein said molecular beacon array comprises two or more distinct molecular beacon probes that hybridize to the same target nucleic acid, wherein said two or more distinct probes differ from each other by hybridizing to different locations of said target nucleic acid.

11. the method according to claim 1, wherein said method minter comprises a data transmission step in which a result from a reading of the array is transmitted from a first location to a second location.

12. The method according to claim 11, wherein said second location is a remote location.

13. A method comprising receiving data representing a result of a reading obtained by the method of claim 1.

14. A system for use in identifying protein/nucleic acid binding pairs, said system comprising:

(a) a molecular beacon array comprising a plurality of distinct molecular beacon probes, wherein each distinct probe of said plurality comprises a different probe sequence and all of said probes of said plurality share a common first fluorescent label;

(b) a labeling reagent for labeling a protein population with a second fluorescent label, wherein said first and second labels make up a FRET pair;

(c) reagents for generating a population of target nucleic adds, and

(d) a fluorescence detector device.

15. The system according to claim 14, wherein said molecular beacon array comprises two or more distinct molecular beacon probes that hybridize to the same target nucleic acid, wherein said two or more distinct probes differ from each other by hybridizing to different locations of said target nucleic acid.

16. The system according to claim 14, wherein said system includes two different labeling reagents for producing two differentially labeled protein populations that are each labeled with a different second fluorescent label that makes up a FRET pair with said first fluorescent label.

17. The system according to claim 14, wherein said fluorescence detector device is a fluorescent scanner.

18. The system according to claim 14, wherein said system further comprises reagents necessary for identifying a protein component of an identified protein/nucleic acid binding pair.

19. A kit for use in identifying protein/nucleic acid binding pairs, said kit comprising:

(a) a molecular beacon array comprising a plurality of distinct molecular beacon probes, wherein each distinct probe of said plurality comprises a different probe sequence and all of said probes of said plurality share a common first fluorescent label;

(b) a labeling reagent for labeling a protein population with a second fluorescent label, wherein said first and second labels make up a FRET pair; and

(c) reagents for generating a population of target nucleic acids.

20. The kit according to claim 19, wherein said molecular beacon array comprises two or more distinct molecular beacon probes that hybridize to the same target nucleic acid, wherein said two or more distinct probes differ from each other by hybridizing to different locations of said target nucleic acid.

21. The kit according to claim 19, wherein said kit includes two different labeling reagents for producing two differentially labeled protein populations that are each labeled with a different second fluorescent label that makes up a FRET pair with said first fluorescent label.

22. The kit according to claim 19, wherein said kit further comprises reagents necessary for identifying a protein comprising of an identified protein/nucleic acid binding pair.

23. A substrate comprising a surface having at least one protein/nucleic acid binding pair immobilized thereon, wherein each protein/nucleic acid binding pair comprises:

(a) a molecular beacon probe comprising a first fluorescent label;

(b) an unlabeled target nucleic acid hybridized to said molecular beacon probe; and

(c) a fluorescently labeled protein labeled with a second fluorescent label and bound to said unlabeled target nucleic acid, wherein said second fluorescent label and said first fluorescent label make up a FRET pair.

24. The substrate according to claim 23, wherein said substrate comprises two or more different protein/nucleic acid binding pairs immobilized on said surface.

25. A method of identifying protein/nucleic acid binding pairs, said method comprising:

(a) contacting a nucleic acid probe array comprising a plurality of distinct probe nucleic acids with a population of target nucleic acids to produce a hybridized array, wherein each distinct probe nucleic acid of said plurality comprises a different probe sequence;

(b) contacting said hybridized array with a population of labeled proteins to produce a protein bound array; and

(c) detecting any surface bound protein/target nucleic acid complexes to identify protein/nucleic acid binding pairs on said array.

26. The method according to claim 25, wherein said labeled proteins are labeled with a first fluorescent label.

27. The method according to claim 26, wherein said target nucleic acids are labeled with a second fluorescent label, wherein said first and second fluorescent labels make up a FRET pair.

28. The method according to claim 25, wherein said labeled proteins arc labeled with an indirectly detectable label.

29. The method according to claim 25, wherein said method further comprises contacting said hybridized array with a second population or labeled proteins that arc distinguishably labeled from said first population of labeled proteins.

30. A method of identifying protein/nucleic acid binding pairs, said method comprising:

(a) contacting a molecular beacon array comprising a plurality of distinct molecular beacon probes with a population of target nucleic acids to produce a hybridized molecular beacon array, wherein each distinct probe of said plurality comprises a different probe sequence and all of said probes of said plurality share a common first fluorescent label;

(b) contacting said hybridized molecular beacon array with at least one fluorescently labeled protein to produce a protein bound array, where said at least one fluorescently labeled protein is labeled with a second fluorescent label that makes up a FRET pair with said first fluorescent label; and

(c) detecting any FRET generated signals from said array to identify protein/nucleic acid binding pairs on said array.

31. A method of identifying protein/nucleic acid binding pairs, said method comprising:

(a) contacting a nucleic acid probe array comprising a plurality of distinct probe nucleic acids with a population of target nucleic acids ro produce a hybridized array, wherein each distinct probe nucleic acid of said plurality comprises a different probe sequence;

(b) contacting said hybridized array with at least one labeled protein to produce a protein bound array; and

(c) detecting any surface bound protein/target nucleic acid complexes to identify protein/nucleic acid binding pairs on said array.