Nanoreporters and methods of manufacturing and use thereof

• US 9,371,563 B2
• Filed: 03/11/2013
• Issued: 06/21/2016
• Est. Priority Date: 12/23/2005
• Status: Active Grant

• Current Assignee: Institute for System Biology, Nanostring Technologies, Inc.
• Original Assignee: Institute for System Biology, Nanostring Technologies, Inc.
• Inventors: Geiss, Gary K., Ferree, Sean M., Webster, Philippa Jane, Dimitrov, Krassen M.
• Primary Examiner(s): Crow, Robert T
• Assistant Examiner(s): Dauner, Joseph G
• Application Number:
  US13/794,424
• Publication Number:
  US 20130230851A1
• Time in Patent Office: 1,198 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to coded, labeled probes that are capable of binding to and identifying target molecules based on the probes'"'"' label codes. Methods of making and using such probes are also provided. The probes can be used in diagnostic, prognostic, quality control and screening applications.

Claims

1. A method comprising

(a) providing a first nucleic acid molecule comprising

(i) a first label attachment region, comprising a first DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a first RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the first label attachment region, to the first RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a first signal;

(ii) a second label attachment region, which is non-overlapping with the first label attachment region, comprising a second DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a second RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the second label attachment region, to the second RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a second signal;

(iii) a third label attachment region, which is non-overlapping with the first label attachment region and is non-overlapping with the second label attachment region, comprising a third DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a third RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the third label attachment region,

wherein the third RNA molecule is not attached to a label monomer and lacks a signal;

(iv) a first target-specific sequence capable of binding to a target nucleic acid; and

(v) a first moiety that is capable of selectively binding to a substrate or to a first binding moiety on the substrate;

(b) providing a second nucleic acid molecule comprising;

(i) a second target-specific sequence capable of binding to the target nucleic acid, wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the target nucleic acid; and

(ii) a second moiety that is capable of selectively binding to the substrate or to a second binding moiety on the substrate;

(c) forming a nucleic acid complex by binding the first nucleic acid molecule and the second nucleic acid molecule to the target nucleic acid;

(d) forming an immobilized nucleic acid complex by immobilizing a first portion of the nucleic acid complex on the substrate by selectively binding one of the first moiety or the second moiety to the substrate;

(e) forming an extended nucleic acid complex by applying to immobilized nucleic acid complex a force sufficient to extend the nucleic acid complex;

(f) selectively immobilizing a second portion of the extended nucleic acid complex by selectively binding to the substrate the moiety that is not selectively bound to the substrate in step (d), wherein the substrate is selected from the group consisting of a membrane, a bead, a filter, a porous material, and a glass surface, and

(g) removing the force, wherein the nucleic acid complex remains selectively immobilized in an extended state.

2. The method of claim 1, wherein said force is gravity, hydrodynamic force, electromagnetic force, flow-stretching, a receding meniscus technique or combinations thereof.

3. The method of claim 1, wherein said force is a direct current electrical field.

4. The method of claim 1, wherein the first or second moiety selectively binds to the substrate via one or more non-covalent bonds.

5. The method of claim 1, wherein the first or second moiety selectively binds to the substrate via one or more covalent bonds.

6. The method of claim 1, wherein the extended nucleic acid complex is immobilized in an oriented state.

7. The method of claim 1, wherein the force is sufficient to orient the nucleic acid complex.

8. The method of claim 1, wherein the extended nucleic acid complex is extended between the first portion and the second portion.

9. The method of claim 1, wherein the first portion is a terminus of the extended nucleic acid complex.

10. The method of claim 1, wherein the second portion is a terminus of the extended nucleic acid complex.

11. The method of claim 1, wherein the first portion and the second portion are termini of the extended nucleic acid complex.

12. The method of claim 1, wherein the first moiety and the second moiety are, respectively, non-covalently linked to the first nucleic acid molecule and to the second nucleic add molecule.

13. The method of claim 1, wherein the first moiety and the second moiety are, respectively, covalently linked to the first nucleic acid molecule and to the second nucleic acid molecule.

14. The method of claim 1, wherein the substrate comprises the first binding moiety on the substrate for binding to the first moiety and the second binding moiety on the substrate for binding to the second moiety.

15. The method of claim 1, wherein the first moiety and the second moiety are independently selected from the group consisting of ligands, antigens, carbohydrates, nucleic acids, receptors, lectins, antibodies, succinamides, amines, aldehydes, epoxies, thiols, biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin and anti-FITC.

16. The method of claim 1, wherein the first binding moiety on the substrate and the second binding moiety on the substrate are independently selected from the group consisting of ligands, antigens, carbohydrates, nucleic acids, receptors, lectins, antibodies, succinamides, amines, aldehydes, epoxies, thiols, biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin and anti-FITC.

17. The method of claim 1, wherein the membrane is comprised of nitrocellulose, nylon, glass, a polymer, a gel, dextran, cellulose, or latex.

18. The method of claim 1, wherein the membrane or filter is comprised of nitrocellulose or nylon.

19. The method of claim 1, wherein the porous material is comprised of an acrylic, styrene methyl methacrylate copolymer, or ethylene/acrylic acid.

20. The method of claim 1, wherein the substrate comprises a form selected from the group consisting of a disk, a slab, a strip, a bead, a submicron particle, a coated magnetic bead, a gel pad, a microtiter well, a slide, a membrane, and a frit.

21. The method of claim 1, wherein the substrate is coated with streptavidin, biotinylated BSA, aldehyde, or epoxy.

22. The method of claim 1, wherein the first label attachment region and the second label attachment region, and the third label attachment region each comprises an optimized transcription start sequence.