Methods for detecting and identifying single molecules

• US 6,287,765 B1
• Filed: 05/20/1998
• Issued: 09/11/2001
• Est. Priority Date: 05/20/1998
• Status: Expired due to Term

• Current Assignee: Molecular Machines, Inc.
• Original Assignee: Molecular Machines, Inc.
• Inventors: Cubicciotti, Roger S.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/081,930
• Time in Patent Office: 1,210 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.5
• US Class Current: 435/6.11
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: Y10S 977/853 Biological sample

Abstract

Multimolecular devices and drug delivery systems prepared from synthetic heteropolymers, heteropolymeric discrete structures, multivalent heteropolymeric hybrid structures, aptameric multimolecular devices, multivalent imprints, tethered specific recognition devices, paired specific recognition devices, nonaptameric multimolecular devices and immobilized multimolecular structures are provided, including molecular adsorbents and multimolecular adherents, adhesives, transducers, switches, sensors and delivery systems. Methods for selecting single synthetic nucleotides, shape-specific probes and specifically attractive surfaces for use in these multimolecular devices are also provided. In addition, paired nucleotide-nonnucleotide mapping libraries for transposition of selected populations of selected nonoligonucleotide molecules into selected populations of replicatable nucleotide sequences are described.

Claims

1. A method of detecting a single nonnaturally occurring oligonucleotide molecule capable of specifically binding to a selected nonoligonucleotide molecule comprising:

(a) contacting a mixture comprising an assortment of nonnaturally occurring oligonucleotides with a selected nonoligonucleotide molecule; and

(b) detecting a single nonnaturally occurring oligonucleotide molecule specifically bound to said selected nonoligonucleotide molecule by at least one of scanning probe microscopy, optical trapping or flow cytometry.

2. The method of claim 1 wherein said selected nonoligonucleotide molecule is not a DNA-binding protein.

3. The method of claim 1 wherein said mixture comprises a random-sequence assortment of oligonucleotides.

4. The method of claim 1 wherein said mixture comprises at least one of a nonrandom assortment of oligonucleotides, a nucleic acid library, a combinatorial library, or a nucleotide-encoded library.

5. The method of claim 1 wherein said mixture comprises an oligonucleotide produced by at least one of amplification, replication, transcription, or in vitro evolution.

6. The method of claim 1 wherein said single nonnaturally occurring oligonucleotide molecule further comprises a nonnucleotide molecule.

7. The method of claim 6 wherein the identity of said nonnucleotide molecule is encoded by a nucleotide sequence comprising said single nonnaturally occurring oligonucleotide molecule.

8. The method of claim 1 wherein said selected nonoligonucleotide molecule is immobilized on a solid support.

9. The method of claim 1 further comprising isolating said single nonnaturally occurring oligonucleotide molecule by at least one of removing said single nonnaturally occurring oligonucleotide molecule from said mixture, removing said single nonnaturally occurring oligonucleotide molecule from said selected nonoligonucleotide molecule, transporting said single nonnaturally occurring oligonucleotide molecule to a sequencing apparatus, or transporting said single nonnaturally occurring oligonucleotide molecule to an amplification vessel.

10. The method of claim 1 further comprising at least one of determining, amplifying, replicating, or transcribing a nucleotide sequence comprising said single nonnaturally occurring oligonucleotide molecule.

11. The method of claim 1 further comprising identifying said single nonnaturally occurring oligonucleotide molecule.

12. A method of detecting a single nonnaturally occurring oligonucleotide molecule capable of specifically binding to a selected nonoligonucleotide molecule comprising:

(a) preparing a reaction mixture comprising a selected nonoligonucleotide molecule and a nonnaturally occurring oligonucleotide comprising an unknown nucleotide sequence; and

(b) detecting a single nonnaturally occurring oligonucleotide molecule specifically bound to said selected nonoligonucleotide molecule by at least one of scanning probe microscopy, optical trapping or flow cytometry.

13. The method of claim 12 wherein said selected nonoligonucleotide molecule is not a DNA-binding protein.

14. The method of claim 12 further comprising isolating said single nonnaturally occurring oligonucleotide molecule by at least one of removing said single nonnaturally occurring oligonucleotide molecule from said mixture, removing said single nonnaturally occurring oligonucleotide molecule from said selected nonoligonucleotide molecule, transporting said single nonnaturally occurring oligonucleotide molecule to a sequencing apparatus, or transporting said single nonnaturally occurring oligonucleotide molecule to an amplification vessel.

15. The method of claim 12 further comprising at least one of determining, amplifying, replicating, or transcribing a nucleotide sequence comprising said single nonnaturally occurring oligonucleotide molecule.

16. A method of detecting a single nonnaturally occurring oligonucleotide molecule capable of specifically binding to a selected target that is not a DNA-binding protein comprising:

(a) selecting a target that is not a DNA-binding protein;

(b) contacting the selected target with a mixture comprising an assortment of nonnaturally occurring oligonucleotides; and

(c) detecting a single nonnaturally occurring oligonucleotide molecule specifically bound to said selected target by at least one of scanning probe microscopy, optical trapping or flow cytometry.

17. The method of claim 16 wherein said mixture contains an oligonucleotide comprising an unknown nucleotide sequence.

18. The method of claim 16 further comprising isolating said single nonnaturally occurring oligonucleotide molecule by at least one of removing said single nonnaturally occurring oligonucleotide molecule from said mixture, removing said single nonnaturally occurring oligonucleotide molecule from said selected target, transporting said single nonnaturally occurring oligonucleotide molecule to a sequencing apparatus, or transporting said single nonnaturally occurring oligonucleotide molecule to an amplification vessel.

19. The method of claim 16 further comprising at least one of determining, amplifying, replicating, or transcribing a nucleotide sequence comprising said single nonnaturally occurring oligonucleotide molecule.

20. A method of detecting a single nonnaturally occurring oligonucleotide conjugate molecule capable of specifically binding to a selected nonoligonucleotide molecule, said method comprising:

(a) preparing a reaction mixture comprising a selected nonoligonucleotide molecule and a plurality of different nonnaturally occurring oligonucleotide conjugates, each different nonnaturally occurring oligonucleotide conjugate comprising a different nonnucleotide moiety attached to an oligonucleotide; and

(b) detecting a single nonnaturally occurring oligonucleotide conjugate molecule specifically bound to said selected nonoligonucleotide molecule by at least one of scanning probe microscopy, optical trapping or flow cytometry.

21. The method of claim 20 wherein said selected nonoligonucleotide molecule is not a DNA-binding protein.

22. The method of claim 20 wherein the identity of said nonnucleotide moiety is encoded by a nucleotide sequence comprising said single oligonucleotide conjugate.

23. The method of claim 20 further comprising isolating said single nonnaturally occurring oligonucleotide conjugate by at least one of removing said single nonnaturally occurring oligonucleotide conjugate from said mixture, removing said single nonnaturally occurring oligonucleotide conjugate from said selected nonoligonucleotide molecule, transporting said single nonnaturally occurring oligonucleotide conjugate to a sequencing apparatus, or transporting said single nonnaturally occurring oligonucleotide conjugate to an amplification vessel.

24. The method of claim 20 further comprising at least one of determining, amplifying, replicating, or transcribing a nucleotide sequence comprising said single nonnaturally occurring oligonucleotide conjugate.

25. A method of detecting a single nonoligonucleotide molecule comprising:

(a) selecting a nonnaturally occurring oligonucleotide capable of specifically binding to a selected nonoligonucleotide molecule;

(b) preparing a reaction mixture comprising said nonnaturally occurring oligonucleotide and said selected nonoligonucleotide molecule; and

(c) detecting a single complex comprising said nonnaturally occurring oligonucleotide specifically bound to said selected nonoligonucleotide molecule by at least one of scanning probe microscopy, optical trapping or flow cytometry.

26. The method of claim 25 wherein said selected nonoligonucleotide molecule is not a DNA-binding protein.

27. The method of claim 25 wherein said nonnaturally occurring oligonucleotide further comprises a nonnucleotide molecule.