EP-4735626-A1 - USING APERTURES TO CAPTURE POLYNUCLEOTIDES ON PARTICLES

EP4735626A1EP 4735626 A1EP4735626 A1EP 4735626A1EP-4735626-A1

Abstract

In some examples, a method of capturing a polynucleotide on a particle that includes a capture primer includes transporting the particle to a first aperture between a first fluidic compartment and a second fluidic compartment. The particle may be located in the first fluidic compartment, and the polynucleotide may be at least partially located in the second fluidic compartment. The method may include transporting the polynucleotide from the second fluidic compartment to the first fluidic compartment through the first aperture. The method may include hybridizing the polynucleotide to the capture primer.

Inventors

ARTIOLI, GIANLUCA
VON HATTEN, Xavier
RICHEZ, Alexandre
GEORGE, WAYNE
BROWN, ANDREW
KOCSIS, Istvan
WHITE, ADAM

Assignees

Illumina, Inc.

Dates

Publication Date: 20260506
Application Date: 20240627

Claims (1)

IP-2441-PCT 47CX-386101-WO WHAT IS CLAIMED IS: 1. A method of capturing a polynucleotide on a particle comprising a capture primer, the method comprising: transporting the particle to a first aperture between a first fluidic compartment and a second fluidic compartment, wherein the particle is located in the first fluidic compartment, and wherein the polynucleotide is at least partially located in the second fluidic compartment; transporting the polynucleotide from the second fluidic compartment to the first fluidic compartment through the first aperture; and hybridizing the polynucleotide to the capture primer. 2. The method of claim 1, further comprising, after hybridizing the polynucleotide to the capture primer, transporting the particle away from the first aperture. 3. The method of claim 2, wherein transporting the particle to the first aperture and away from the aperture comprises flowing a fluid, in which the particle is suspended, through the first fluidic compartment and past the aperture. 4. The method of any one of claims 1 to 3, further comprising synchronizing transport of the particle to the first aperture with transport of the polynucleotide through the first aperture. 5. The method of claim 4, wherein the synchronizing comprises: detecting transport of the polynucleotide through the first aperture; and controlling transport of the particle to the first aperture based on the detected transport of the polynucleotide through the first aperture. 6. The method of claim 4, wherein the synchronizing comprises: transporting the particle through a second aperture and into the first fluidic compartment; detecting transport of the particle through the second aperture; and controlling transport of the polynucleotide through the first aperture based on the detected transport of the particle through the second aperture. 7. The method of claim 4, wherein the synchronizing comprises: SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO electronically controlling transport of the particle through a second aperture and into the first fluidic compartment; and electronically controlling transport of the polynucleotide through the first aperture and into the first fluidic compartment. 8. The method of claim 6 or claim 7, wherein the second aperture comprises a nanopore. 9. The method of claim 6 or claim 7, wherein the second aperture has a diameter of about 50 nm to about 1000 nm. 10. The method of any one of claims 1 to 9, wherein the particle has a diameter of about 50 nm to about 1000 nm. 11. The method of any one of claims 1 to 10, wherein the first aperture comprises a nanopore. 12. The method of any one of claims 1 to 11, wherein the first aperture has a diameter of about 2 nm to about 20 nm. 13. The method of any one of claims 1 to 12, wherein transporting the polynucleotide through the first aperture comprises flowing a fluid, in which the polynucleotide is suspended, through the aperture and into the first fluidic compartment. 14. The method of claim 13, wherein a plurality of polynucleotides are suspended in the fluid at a concentration of about 1 nM to about 100 nM. 15. The method of any one of claims 1 to 14, wherein the polynucleotide is transported through the first aperture before hybridizing the polynucleotide to the capture primer. 16. The method of claim 15, wherein before being transported through the first aperture, the polynucleotide is located entirely in the second fluidic compartment. 17. The method of any one of claims 1 to 14, wherein the polynucleotide is transported through the first aperture in response to hybridizing the polynucleotide to the capture primer. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO 18. The method of claim 17, wherein prior to hybridizing the polynucleotide to the capture primer, a first portion of the polynucleotide is located in the second fluidic compartment and a second portion of the polynucleotide is located in the first fluidic compartment. 19. The method of claim 18, wherein responsive to hybridizing the polynucleotide to the capture primer, the first portion of the polynucleotide is transported from the second fluidic compartment into the first fluidic compartment through the first aperture. 20. The method of claim 19, wherein the first portion of the polynucleotide retains the first portion of the polynucleotide in the second fluidic compartment. 21. The method of claim 20, wherein the first portion of the polynucleotide comprises a DNA loop. 22. The method of claim 21, wherein when the polynucleotide is hybridized to the capture primer, force from the particle dissociates the DNA loop. 23. The method of claim 20, wherein the first portion of the polynucleotide is coupled to a structure that is at least partially located within the second fluidic compartment and retains the first portion of the polynucleotide in the second fluidic compartment. 24. The method of claim 23, wherein the structure comprises a DNA loop, a DNA hairpin, a cruciform folded double strand, or a dendrimer. 25. The method of claim 23, wherein when the polynucleotide is hybridized to the capture primer, force from the particle dissociates the first portion of the polynucleotide from the structure, and the structure remains within the second fluidic compartment. 26. The method of any one of claims 1 to 14, wherein the polynucleotide is transported through the first aperture after hybridizing the polynucleotide to the capture primer. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO 27. The method of claim 26, wherein the capture primer extends through the aperture and hybridizes to the polynucleotide in the second fluidic compartment. 28. The method of claim 27, wherein when the polynucleotide is hybridized to the capture primer, force from the particle pulls the polynucleotide through the first aperture and into the first fluidic compartment. 29. The method of any one of claims 1 to 28, wherein the polynucleotide comprises an adapter that is complementary to the capture primer. 30. The method of any one of claims 1 to 29, wherein the capture primer comprises an amplification primer. 31. The method of any one of claims 1 to 30, wherein the particle comprises a plurality of capture primers. 32. The method of any one of claims 1 to 31, wherein the polynucleotide is single- stranded. 33. The method of any one of claims 1 to 31, wherein the polynucleotide is double- stranded. 34. A method of capturing a polynucleotide on a particle comprising a capture primer, the method comprising: transporting the particle to an aperture between a first fluidic compartment and a second fluidic compartment, wherein the particle is located in the first fluidic compartment and the polynucleotide is coupled to a structure located in the second compartment; hybridizing the polynucleotide to the capture primer; and dissociating the polynucleotide from the structure using a force applied by the particle. 35. A method of capturing a polynucleotide on a particle comprising a capture primer, the method comprising: SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO transporting the particle to an aperture between a first fluidic compartment and a second fluidic compartment, wherein the particle is located in the first fluidic compartment and the polynucleotide is located in the second compartment; extending the capture primer through the aperture; hybridizing the polynucleotide to the capture primer; generating an amplicon of the polynucleotide using the capture primer; and transporting the amplicon from the second fluidic compartment to the first fluidic compartment through the aperture. 36. A method of generating a clonal cluster of a polynucleotide on a particle, the method comprising: capturing the polynucleotide on the particle using the method of any one of claims 1 to 35; and using a plurality of amplification primers on the particle to amplify the polynucleotide. 37. A device for capturing a polynucleotide on a particle comprising a capture primer, the device comprising: a first fluidic compartment; a second fluidic compartment; a first aperture defined through a wall between the first fluidic compartment and the second fluidic compartment; and a controller to: transport the particle within the first fluidic compartment to the first aperture; and transport the polynucleotide from the second fluidic compartment to the first fluidic compartment through the first aperture so as to hybridize the polynucleotide to the capture primer. 38. The device of claim 37, wherein the controller further is to transport the particle away from the first aperture after the polynucleotide is hybridized to the capture primer. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO 39. The device of claim 38, wherein the controller is to transport the particle to the first aperture and away from the aperture by flowing a fluid, in which the particle is suspended, through the first fluidic compartment and past the aperture. 40. The device of any one of claims 36 to 39, wherein the controller further is to synchronize transport of the particle to the first aperture with transport of the polynucleotide through the first aperture. 41. The device of claim 40, wherein the controller is to synchronize transport using operations comprising: detecting transport of the polynucleotide through the first aperture; and controlling transport of the particle to the first aperture based on the detected transport of the polynucleotide through the first aperture. 42. The device of claim 40, wherein the controller is to synchronize transport using operations comprising: transporting the particle through a second aperture and into the first fluidic compartment; detecting transport of the particle through the second aperture; and controlling transport of the polynucleotide through the first aperture based on the detected transport of the particle through the second aperture. 43. The device of claim 40, wherein the controller is to synchronize transport using operations comprising: electronically controlling transport of the particle through a second aperture and into the first fluidic compartment; and electronically controlling transport of the polynucleotide through the first aperture and into the first fluidic compartment. 44. The device of any one of claims 40 to 43, wherein the second aperture comprises a nanopore. 45. The device of any one of claims 40 to 44, wherein the second aperture has a diameter of about 50 nm to about 1000 nm. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO 46. The device of any one of claims 37 to 45, wherein the particle has a diameter of about 50 nm to about 1000 nm. 47. The device of any one of claims 37 to 46, wherein the first aperture comprises a nanopore. 48. The device of any one of claims 37 to 47, wherein the first aperture has a diameter of about 20 nm to about 200 nm. 49. The device of any one of claims 37 to 48, wherein the controller is to transport the polynucleotide through the first aperture by flowing a fluid, in which the polynucleotide is suspended, through the aperture and into the first fluidic compartment. 50. The device of claim 48, wherein a plurality of polynucleotides are suspended in the fluid at a concentration of about 1nM to about 100 nM. 51. The device of any one of claims 37 to 50, wherein the controller is to transport the polynucleotide through the first aperture before the polynucleotide is hybridized to the capture primer. 52. The device of claim 51, wherein before being transported through the first aperture, the polynucleotide is located entirely in the second fluidic compartment. 53. The device of any one of claims 37 to 50, wherein the polynucleotide is transported through the first aperture in response to hybridizing the polynucleotide to the capture primer. 54. The device of claim 53, wherein prior to the polynucleotide being hybridized to the capture primer, a first portion of the polynucleotide is located in the second fluidic compartment and a second portion of the polynucleotide is located in the first fluidic compartment. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO 55. The device of claim 54, wherein responsive to the polynucleotide hybridizing to the capture primer, the first portion of the polynucleotide is transported from the second fluidic compartment into the first fluidic compartment through the first aperture. 56. The device of claim 54 or claim 55, wherein the first portion of the polynucleotide retains the first portion of the polynucleotide in the second fluidic compartment. 57. The device of claim 56, wherein the first portion of the polynucleotide comprises a DNA loop. 58. The device of claim 57, wherein when the polynucleotide is hybridized to the capture primer, force from the particle dissociates the DNA loop. 59. The device of claim 56, wherein the first portion of the polynucleotide is coupled to a structure that is at least partially located within the second fluidic compartment and retains the first portion of the polynucleotide in the second fluidic compartment. 60. The device of claim 59, wherein the structure comprises a DNA loop, a DNA hairpin, a cruciform folded double strand, or a dendrimer. 61. The device of claim 59 or claim 60, wherein when the polynucleotide is hybridized to the capture primer, force from the particle dissociates the first portion of the polynucleotide from the structure, and the structure remains within the second fluidic compartment. 62. The device of any one of claims 37 to 50, wherein the controller is to transport the polynucleotide after the polynucleotide is hybridized to the capture primer. 63. The device of claim 62, wherein the capture primer extends through the aperture and hybridizes to the polynucleotide in the second fluidic compartment. 64. The device of claim 63, wherein when the polynucleotide is hybridized to the capture primer, force from the particle pulls the polynucleotide through the first aperture and into the first fluidic compartment. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO 65. The device of any one of claims 37 to 64, wherein the polynucleotide comprises an adapter that is complementary to the capture primer. 66. The device of any one of claims 37 to 65, wherein the capture primer comprises an amplification primer. 67. The device of any one of claims 37 to 66, wherein the particle comprises a plurality of capture primers. 68. The device of any one of claims 37 to 67, wherein the polynucleotide is single- stranded. 69. The device of any one of claims 37 to 67, wherein the polynucleotide is double- stranded. 70. A device for capturing a polynucleotide on a particle comprising a capture primer, the device comprising: a first fluidic compartment; a second fluidic compartment; a first aperture defined through a wall between the first fluidic compartment and the second fluidic compartment; and a controller to: transport the particle within the first fluidic compartment to the first aperture so that the polynucleotide hybridizes with a capture primer coupled to a structure located in the second fluidic compartment, and so that the polynucleotide dissociates from the structure using a force applied by the particle. 71. A device for capturing a polynucleotide on a particle comprising a capture primer, the device comprising: a first fluidic compartment; a second fluidic compartment; a first aperture defined through a wall between the first fluidic compartment and the second fluidic compartment; and a controller to: SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO transport the particle within the first fluidic compartment to the first aperture so that the capture primer extends through the aperture to hybridize to the polynucleotide in the second fluidic compartment such that an amplicon of the polynucleotide is generated in the second compartment and the amplicon is transported from the second fluidic compartment to the first fluidic compartment through the first aperture. 72. A method of generating a clonal cluster of a polynucleotide on a particle, the method comprising: capturing the polynucleotide on the particle using the device of any one of claims 37 to 71; and using a plurality of amplification primers on the particle to amplify the polynucleotide. SMRH:4864-1468-1289.1

Description

IP-2441-PCT 47CX-386101-WO USING APERTURES TO CAPTURE POLYNUCLEOTIDES ON PARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/511,338, filed on June 30, 2023 and entitled “Using Apertures to Capture Polynucleotides on Particles,” the entire contents of which are incorporated by reference herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The material in the accompanying sequence listing is hereby incorporated by reference into the application. The accompanying sequence listing XML file, named “IP- 2441-PCT.xml”, was created on June 17, 2024 and is 8 kB in size. FIELD [0003] This application generally relates to capturing polynucleotides. BACKGROUND [0004] Cluster amplification is an approach to amplifying polynucleotides, for example for use in genetic sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 primers) coupled to a substrate surface in a flowcell, and form “seeds” at random locations on the surface. Cycles of amplification are performed to form clusters on the surface around each seed. The clusters include copies, and complementary copies, of the seed polynucleotides. In some circumstances, the substrate is patterned so as to define regions that bound different clusters, such as wells that may be filled with respective clusters. SUMMARY [0005] Examples provided herein are related to using apertures to capture polynucleotides on particles. Devices for performing such capture also are disclosed. [0006] Some examples herein provide a method of capturing a polynucleotide on a particle including a capture primer. The method may include transporting the particle to a first aperture between a first fluidic compartment and a second fluidic compartment. The particle is located in the first fluidic compartment, and the polynucleotide is at least partially located SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO in the second fluidic compartment. The method may include transporting the polynucleotide from the second fluidic compartment to the first fluidic compartment through the first aperture. The method may include hybridizing the polynucleotide to the capture primer. [0007] In some examples, the method further includes, after hybridizing the polynucleotide to the capture primer, transporting the particle away from the first aperture. In some examples, transporting the particle to the first aperture and away from the aperture includes flowing a fluid, in which the particle is suspended, through the first fluidic compartment and past the aperture. [0008] In some examples, the method further includes synchronizing transport of the particle to the first aperture with transport of the polynucleotide through the first aperture. In some examples, the synchronizing includes: detecting transport of the polynucleotide through the first aperture; and controlling transport of the particle to the first aperture based on the detected transport of the polynucleotide through the first aperture. In some examples, the synchronizing includes: transporting the particle through a second aperture and into the first fluidic compartment; detecting transport of the particle through the second aperture; and controlling transport of the polynucleotide through the first aperture based on the detected transport of the particle through the second aperture. In some examples, the synchronizing includes: electronically controlling transport of the particle through a second aperture and into the first fluidic compartment; and electronically controlling transport of the polynucleotide through the first aperture and into the first fluidic compartment. In some examples, the second aperture includes a nanopore. In some examples, the second aperture has a diameter of about 50 nm to about 1000 nm. [0009] In some examples, the particle has a diameter of about 50 nm to about 1000 nm. [0010] In some examples, the first aperture includes a nanopore. [0011] In some examples, the first aperture has a diameter of about 2 nm to about 20 nm. [0012] In some examples, transporting the polynucleotide through the first aperture includes flowing a fluid, in which the polynucleotide is suspended, through the aperture and into the first fluidic compartment. In some examples, a plurality of polynucleotides are suspended in the fluid at a concentration of about 1 nM to about 100 nM. SMRH:4864-1468-1289.1 IP-2441-PCT 47CX-386101-WO [0013] In some examples, the polynucleotide is transported through the first aperture before hybridizing the polynucleotide to the capture primer. In some examples, before being transported through the first aperture, the polynucleotide is located entirely in the second fluidic compartment. [0014] In some examples, the polynucleotide is transported through the first aperture in response to hybridizing the polynucleotide to the capture primer. In some examples, prior to hybridizing the polynucleotide to the cap