Methods and compositions for delivery of molecules and complexes to reaction sites

• US 10,711,300 B2
• Filed: 07/21/2017
• Issued: 07/14/2020
• Est. Priority Date: 07/22/2016
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Kamtekar, Satwik, Bjornson, Keith, Chern, Leewin, Lin, Steven
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US15/656,622
• Publication Number:
  US 20180023134A1
• Time in Patent Office: 1,089 Days
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 2030/8827 involving nucleic acids
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The present invention provides methods, compositions, and systems for distributing molecules and complexes into reaction sites. In particular, the methods, compositions, and systems of the present invention result in an active loading of molecules and complexes into reaction sites with improved efficiency over loading by passive diffusion methods alone.

Claims

1. A method of delivering polymerase enzyme complexes to nanoscale wells, the method comprising:

(a) providing a substrate comprising an array of nanoscale wells;

(b) coating the top surface of the substrate between the nanoscale wells with a positively charged molecule to form a coated surface;

(c) applying a composition comprising polymerase enzyme complexes to the coated surface such that at least a portion of the polymerase enzyme complexes attach to the positively charged molecules, wherein the polymerase enzyme complexes comprise template nucleic acids complexed with polymerase enzymes; and

(d) after step c), releasing the polymerase enzyme complexes from the positively charged molecules to allow the polymerase enzyme complexes to passively diffuse into the nanoscale wells,

thereby delivering polymerase enzyme complexes to the nanoscale wells.

2. The method of claim 1, wherein releasing the polymerase enzyme complexes from the positively charged molecules comprises releasing the polymerase enzyme complexes from the positively charged molecules by applying a solution containing high salt to the coated surface.

3. The method of claim 1, wherein the releasing step comprises applying a size exclusion resin to the coated surface.

4. The method of claim 1, wherein the template nucleic acid molecules are further hybridized to a primer.

5. The method of claim 1, wherein the template nucleic acid molecules are at least 5 kb long.

6. The method of claim 1, wherein the template nucleic acid molecules are about 3 kb to about 20 kb in length.

7. The method of claim 1, wherein the delivering of the polymerase enzyme complexes is accomplished about 4 to about 100 times faster than seen when the surface is not coated with the positively charged molecule.

8. The method of claim 1, wherein the delivering of the polymerase enzyme complexes is accomplished at least 30, 40, 50, 60, 70 or 80 times faster than seen when the surface is not coated with the positively charged molecule.

9. The method of claim 1, wherein the delivering of the polymerase enzyme complexes is accomplished about 30 to about 150 times faster than seen when the surface is not coated with the positively charged molecule.

10. The method of claim 1, wherein the delivering of the polymerase enzyme complexes is accomplished about 20 to about 80 times faster than seen when the surface is not coated with the positively charged molecule.

11. The method of claim 1, wherein the positively charged molecule is a positively charged peptide or polypeptide.

12. The method of claim 11, wherein releasing the polymerase enzyme complexes from the positively charged molecules comprises releasing the polymerase enzyme complexes from the positively charged molecules by applying a protease to the coated surface.

13. The method of claim 12, wherein the protease comprises trypsin.

14. The method of claim 11, wherein the positively charged peptide or polypeptide is a member selected from the group consisting of Tat, poly-lysine, poly-arginine, and histone.

15. The method of claim 11, wherein the positively charged peptide or polypeptide comprises a Tat peptide.

16. The method of claim 1, wherein the positively charged molecule is a cationic polymer.

17. The method of claim 1, wherein the base of the nanoscale well comprises biotin and wherein the polymerase enzyme comprises a biotin-binding moiety, the method comprising, after the releasing step, localizing the polymerase enzyme complexes to the base of the nanoscale wells through binding of the biotin-binding moiety on the polymerase to the biotin on the base of the nanoscale well.

18. The method of claim 1, wherein the base of the nanoscale well comprises a biotin-binding moiety and wherein the polymerase enzyme comprises biotin, the method comprising, after the releasing step, localizing the polymerase enzyme complexes to the base of the nanoscale wells through binding of the biotin on the polymerase to the biotin-binding moiety on the base of the nanoscale well.

19. The method of claim 1, wherein the substrate comprises a metal or metal oxide layer on a silica-based layer, wherein the nanoscale wells are disposed through the metal or metal oxide layer to or into the silica-based layer.

20. The method of claim 1, wherein the top surface of the substrate between the nanoscale wells comprises aluminum or titanium nitride.