Logic driven polynucleotide scanning for mapping features in a nanopore device
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Publication Number
US-12338488-B2
Publication Date
2025-06-24
Expiration Date
Abstract
The present disclosure provides an automated method of mapping one or more features of a target polynucleotide. Also provided in the present disclosure are automated methods for sequencing a polynucleotide sequence. Also provided in the present disclosure are methods of extended recapture of a polynucleotide in a nanopore device. Also provided in the present disclosure are devices and systems for carrying out the methods of the present disclosure.
Core Innovation
The invention provides a nanopore device/system and methods for extended partially or fully recapturing and mapping a polynucleotide that is previously captured in a nanopore device. The device includes at least one nanopore connected between a chamber and a geometrically constrained fluidic enclosure, where the constrained enclosure includes an inlet and an outlet for fluidic filling and electrode access. At least one electrode is positioned within the constrained fluidic volume and at least one electrode is positioned within the chamber, and a sensor provides a voltage and detects capture and translocation through the pore via a current measurement.
The method loads the polynucleotide into the chamber, applies a first voltage to capture and translocate the polynucleotide through the first pore and into the constrained fluidic enclosure, and detects capture and translocation through the first pore using the current measurement. A second voltage equal to zero mV is applied for a time period while the polynucleotide is contained within the constrained fluidic enclosure, and then a third voltage is applied to recapture and partially or fully translocate the polynucleotide from the constrained fluidic enclosure back through the first pore and into the chamber with detection of current during recapture.
In multi-pore embodiments, the invention controls movement of a target polynucleotide through a first and second pore simultaneously using different voltages at the first pore and the second pore while separate portions remain in different regions. The method detects a set of features in the current measurement of the first pore and detects the same set of features as the features pass through the second pore, and the direction of motion is controlled and reversed so that feature detection occurs in both directions across the two pores, including within a combined fully capturing, partially recapturing, and mapping sequence.
Claims Coverage
The provided material includes three independent claims. Across these independent claims, the inventive features cover zero-mV dwell recapturing in a constrained enclosure, simultaneous first and second pore control for feature mapping, and a combined sequence of full capture, partial recapture, and mapping with direction reversal.
Zero-mV dwell recapturing through a single constrained enclosure
A method for partially or fully recapturing a polynucleotide previously captured in a nanopore device, where the device includes a first pore between a chamber and a first geometrically constrained fluidic enclosure, electrodes in the chamber and the constrained enclosure, and a sensor that provides a voltage and detects capture and translocation into and through the first pore; the method applies a first voltage to capture and translocate into the constrained enclosure, detects translocation, applies a second voltage equal to zero mV for a time period while the polynucleotide is contained within the constrained enclosure, then applies a third voltage to recapture and partially or fully translocate the polynucleotide back through the first pore and into the chamber, with detection during the recapture.
Dual-pore simultaneous motion control for feature mapping in both directions
A method for mapping one or more features of a target polynucleotide using a device that controls movement through a first and second pore simultaneously, where electrodes and a sensor provide voltages and current measurements for capture and partial or full translocation through each pore; the method applies different voltages at the first pore and second pore to route and control a first portion while a separate portion remains in the first pore, detects a set of features at the first pore and detects the same set of features at the second pore, then applies voltages to control direction of motion through the first and second pores and applies voltages to reverse the direction of motion.
Full capture, partial recapture, and dual-pore feature mapping sequence with zero-mV dwell
A method for fully capturing, partially recapturing and then mapping one or more features of a target polynucleotide using a device with a first pore and a second pore between a chamber and respective geometrically constrained fluidic enclosures, electrodes in each fluidic enclosure and in the chamber, and sensors that provide voltages and current measurements that detect capture and partial or full translocation into and through each pore; the method loads the polynucleotide into the chamber, applies a first voltage to capture and translocate into the first fluidic enclosure, detects translocation, applies a second voltage equal to zero mV for a time period while the polynucleotide is in the first fluidic enclosure, applies subsequent voltages to recapture first portions through the first pore and into the chamber, to transfer the first portion through the second pore into the second fluidic enclosure while a separate portion remains in the first pore, then controls the direction and reverses the direction of motion, and detects and matches a set of features passing through both pores.
Across the independent claims, the disclosure centers on applying a second voltage equal to zero mV for a time period while the polynucleotide is contained in a geometrically constrained fluidic enclosure, and using simultaneous first/second pore voltage control, current measurement at both pores, and direction reversal to detect the same set of features through both pores in different motion directions, including within a combined fully capturing, partially recapturing, and mapping sequence.
Stated Advantages
Enables partially or fully recapturing a previously captured polynucleotide by applying a zero-mV dwell while the polynucleotide is contained in a geometrically constrained enclosure, followed by recapture and translocation back through the nanopore with current detection.
Enables mapping one or more features by detecting the same set of features as the polynucleotide passes through the first pore and the second pore during controlled motion and reversal.
Supports a combined process of fully capturing, partially recapturing, and then mapping one or more features of a target polynucleotide using sensor current measurements during capture/translocation events.
Documented Applications
Mapping one or more features of a target polynucleotide by controlling movement through a first and second pore simultaneously and detecting the same set of features as each feature passes through the first pore and then the second pore.
Partially or fully recapturing a polynucleotide previously captured in a nanopore device using a zero-mV dwell in a geometrically constrained enclosure followed by recapture and translocation back into the chamber.
Fully capturing, partially recapturing, and then mapping one or more features of a target polynucleotide using sequential capture/recapture and direction reversal through first and second pores.
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