IP Library › Granted Patent US 12,416,625
Granted Patent B2
US 12,416,625 · App. 19/067,493 · Granted Sep 16, 2025

Deterministic control of polymer molecule translocation through a nanopore

Inventors: Daniel Branton (Lexington, MA); Stephen Jordan Fleming (Cincinnati, OH); Jene A. Golovchenko (Lexington, MA)
Assignee: President and Fellows of Harvard College
G01N33/48721B01D71/0211B01D71/74C12Q1/6869G01N27/40B82Y35/00C12Y306/01039
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Quick Facts
Patent No.
US 12,416,625
App. No.
19/067,493
Granted
Sep 16, 2025
Kind
B2
Abstract

In a method for controlling translocation of a target polymer molecule through a nanopore, a clamp is reversibly bound at a site along the target polymer molecule length; the clamp and the target polymer molecule are disposed in an ionic solution that is in fluidic communication with a nanopore having an aperture diameter less than an outer diameter of the clamp. A constant translocation force is applied across the nanopore to induce travel of the target polymer molecule into the nanopore such that the reversibly bound clamp abuts the nanopore. A voltage pulse is applied across the nanopore that advances the target polymer molecule into the nanopore by one nucleotide, without either of chemical fuel and biochemical fuel provided to the clamp. The voltage pulse is repeatedly applied to cause a plurality of nucleotides to translocate through the nanopore. An indication of each nucleotide can be acquired during nucleotide translocation.

Claims (37)

1. A method for controlling translocation of a target polymer molecule through a nanopore, the target polymer molecule having a target polymer molecule length comprising a plurality of nucleotides, comprising:

reversibly binding a clamp to the target polymer molecule at a site along the target polymer molecule length;

disposing the target polymer molecule and the clamp in an ionic solution that is in fluidic communication with the nanopore, the nanopore having an aperture diameter less than an outer diameter of the clamp;

applying a constant translocation force across the nanopore to induce travel of the target polymer molecule in the ionic solution into the nanopore such that the reversibly bound clamp abuts the nanopore;

applying across the nanopore a voltage pulse that advances the target polymer molecule into the nanopore by one nucleotide, without providing either of chemical fuel and biochemical fuel to the clamp; and

repeatedly applying the voltage pulse to cause a plurality of nucleotides to translocate through the nanopore.

2. The method of claim 1 wherein applying a constant translocation force across the nanopore comprises applying a voltage bias across the nanopore.

3. The method of claim 2 wherein applying a voltage pulse comprises applying a voltage pulse having a voltage pulse magnitude greater than a voltage bias magnitude of the voltage bias applied across the nanopore.

4. The method of claim 1 further comprising measuring current through the nanopore while a plurality of nucleotides of the target polymer molecule translocates through the nanopore.

5. The method of claim 1 further comprising acquiring a representative indication of a nucleotide as the nucleotide translocates through the nanopore.

6. The method of claim 5 wherein repeatedly applying the voltage pulse comprises conducting one repetition of voltage pulse application after acquisition of a representative indication of a nucleotide.

7. The method of claim 1 further comprising acquiring a representative indication of a sequence of nucleotides as the plurality of nucleotides translocates through the nanopore.

8. The method of claim 1 wherein reversibly binding a clamp to the target polymer molecule comprises reversibly binding a clamp selected from biological molecule clamp, biochemical clamp, protein clamp, and enzyme clamp to a plurality of nucleotides.

9. The method of claim 1 wherein reversibly binding a clamp to the target polymer molecule comprises reversibly binding a helicase enzyme to a plurality of nucleotides.

10. The method of claim 9 wherein reversibly binding a helicase enzyme to a plurality of nucleotides comprises reversibly binding a helicase enzyme selected from a SF1 family helicase and a T4 Dda helicase to a plurality of nucleotides.

11. The method of claim 1 wherein reversibly binding a clamp to the target polymer molecule comprises reversibly binding a polymerase enzyme to a plurality of nucleotides.

12. The method of claim 1 wherein applying a constant translocation force comprises applying a translocation force selected from electrophoretic force, hydrostatic force, optical force, and magnetic force.

13. The method of claim 1 wherein applying the pulse of force comprises applying a pulse of electrical voltage having an electrical voltage pulse duration less than a length of time required for the constant translocation force to induce the target polymer molecule to travel into the nanopore by one nucleotide.

14. The method of claim 1 wherein applying a voltage pulse comprises applying a voltage pulse having an electrical voltage pulse duration no greater than about one millisecond.

15. The method of claim 1 wherein reversibly binding a clamp to the target polymer molecule comprises first disposing the target polymer molecule in the ionic solution and then reversibly binding the clamp to the target polymer molecule.

16. The method of claim 1 wherein applying a voltage pulse that advances the target polymer molecule into the nanopore by one nucleotide comprises applying a voltage pulse across a biological nanopore selected from a CsgG bacterial porin nanopore and a Mycobacterium smegmatis porin A (MspA) nanopore to advance the target polymer molecule into the biological nanopore by one nucleotide.

17. The method of claim 16 wherein applying a voltage pulse across a biological nanopore comprises applying a voltage pulse across a biological nanopore disposed in a membrane selected from a triblock copolymer membrane, a mycolic acid membrane, a tetraether lipid membrane, and a lipid bilayer membrane.

18. A method for determining a sequence of nucleotides of a target polymer molecule having a target polymer molecule length comprising a plurality of nucleotides, comprising:

reversibly binding a clamp to the target polymer molecule at a site along the target polymer molecule length;

after binding the clamp to the target polymer molecule, disposing the target polymer molecule and the reversibly-bound clamp in an ionic solution that is in fluidic communication with a nanopore, the nanopore having an aperture diameter less than an outer diameter of the clamp;

applying a constant translocation force across the nanopore to induce travel of the target polymer molecule in the ionic solution into the nanopore such that the reversibly bound clamp abuts the nanopore;

applying across the nanopore a voltage pulse that advances the target polymer molecule into the nanopore by one nucleotide, without providing either of chemical fuel and biochemical fuel to the clamp;

acquiring a representative indication of a nucleotide as the nucleotide translocates through the nanopore; and

repeatedly applying the voltage pulse to cause a plurality of nucleotides to translocate through the nanopore, with a repetition of voltage pulse application after each acquisition of a representative indication of a nucleotide.

19. The method of claim 18 wherein applying a constant translocation force across the nanopore comprises applying an electrical voltage bias across the nanopore, and wherein applying a voltage pulse across the nanopore comprises applying a voltage pulse having a voltage pulse amplitude greater than a voltage bias amplitude of the applied electrical voltage bias.

20. A method for determining a sequence of nucleotides of a target polymer molecule having a target polymer molecule length comprising a plurality of nucleotides, comprising:

disposing the target polymer molecule and a clamp in an ionic solution that is in fluidic communication with a nanopore, the nanopore having an aperture diameter less than an outer diameter of the clamp;

after disposing the target polymer molecule and the clamp in an ionic solution, reversibly binding the clamp to the target polymer molecule in the ionic solution at a site along the target polymer molecule length;

applying a constant translocation force across the nanopore to induce travel of the target polymer molecule in the ionic solution into the nanopore such that the reversibly bound clamp abuts the nanopore;

applying across the nanopore a voltage pulse that advances the target polymer molecule into the nanopore by one nucleotide, without providing either of chemical fuel and biochemical fuel to the clamp;

acquiring a representative indication of a nucleotide as the nucleotide translocates through the nanopore; and

repeatedly applying the voltage pulse to cause a plurality of nucleotides to translocate through the nanopore, with one repetition of voltage pulse application after each acquisition of a representative indication of a nucleotide.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 10, 2025
From: BRANTON, DANIEL; FLEMING, STEPHEN; GOLOVCHENKO, JENE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 071374/0977 →
Continuity (5)
Continuation 18912894 · Oct 11, 2024
Continuation 17319254 · May 13, 2021
Continuation 16022594 · Jun 28, 2018
Provisional Application 62526823 · Jun 29, 2017
Related Publication 20250189510A1 · Jun 12, 2025
References Cited (104)
US 5795782A · Church et al. · 1998 [cited by applicant]
US 6627067B1 · Branton et al. · 2003 [cited by applicant]
US 6673615B2 · Denison et al. · 2004 [cited by applicant]
US 7189503B2 · Akeson et al. · 2007 [cited by applicant]
US 7238485B2 · Akeson et al. · 2007 [cited by applicant]
US 7468271B2 · Golovchenko et al. · 2008 [cited by applicant]
US 7625706B2 · Akeson et al. · 2009 [cited by applicant]
US 7744816B2 · Su et al. · 2010 [cited by applicant]
US 7947454B2 · Akeson et al. · 2011 [cited by applicant]
US 8273532B2 · Gershow et al. · 2012 [cited by applicant]
US 8673556B2 · Akeson et al. · 2014 [cited by applicant]
US 8698481B2 · Lieber et al. · 2014 [cited by applicant]
US 9274097B2 · Golovchenko et al. · 2016 [cited by applicant]
US 9611140B2 · Russo et al. · 2017 [cited by applicant]
US 9617591B2 · Moysey et al. · 2017 [cited by applicant]
US 9702849B2 · Lieber et al. · 2017 [cited by applicant]
US 9815082B2 · Golovchenko et al. · 2017 [cited by applicant]
US 10221450B2 · Heron et al. · 2019 [cited by applicant]
US 11035847B2 · Branton · 2021 [cited by examiner]
US 11634763B2 · Moysey et al. · 2023 [cited by applicant]
US 12140587B2 · Branton · 2024 [cited by examiner]
US 20040055875A1 · Siwy et al. · 2004 [cited by applicant]
US 20090136958A1 · Gershow et al. · 2009 [cited by applicant]
US 20100188109A1 · Edel et al. · 2010 [cited by applicant]
US 20110226623A1 · Timp et al. · 2011 [cited by applicant]
US 20120234679A1 · Garaj et al. · 2012 [cited by applicant]
US 20130244340A1 · Davis et al. · 2013 [cited by applicant]
US 20140051068A1 · Cherf et al. · 2014 [cited by applicant]
US 20140335512A1 · Moysey et al. · 2014 [cited by applicant]
US 20150031020A1 · Jayasinghe et al. · 2015 [cited by applicant]
US 20150060276A1 · Golovchenko et al. · 2015 [cited by applicant]
US 20150060277A1 · Golovchenko et al. · 2015 [cited by applicant]
US 20150153309A1 · Luan et al. · 2015 [cited by applicant]
US 20150191709A1 · Heron et al. · 2015 [cited by applicant]
US 20160231307A1 · Xie · 2016 [cited by applicant]
US 20170199149A1 · Gundlach et al. · 2017 [cited by applicant]
US 20180148481A2 · Howorka et al. · 2018 [cited by applicant]
US 20190345550A1 · Bowen et al. · 2019 [cited by applicant]
CN 102445480 · 2012 [cited by applicant]
CN 102590314 · 2012 [cited by applicant]
EP 1441213 · 2004 [cited by applicant]
EP 1443318 · 2004 [cited by applicant]
EP 2875152B1 · 2019 [cited by applicant]
WO 2004028673 · 2004 [cited by applicant]
WO 2008071982 · 2008 [cited by applicant]
WO 2013098562A2 · 2013 [cited by applicant]
WO 2013123379 · 2013 [cited by applicant]
WO 2013123379A2 · 2013 [cited by applicant]
WO 2015175789A1 · 2015 [cited by applicant]
PCT/US2018/040152, International Search Report: Form PCT/ISA/210 first sheet, second sheet, continuation of second sheet, and patent family annex sheet, Jan. 2019. [cited by applicant]
PCT/US2018/040152, Written Opinion of the International Searching Authority: Form PCT/ISA/237 cover sheet, sheets Box No. I-Box No. VIII, and Separate Sheet sheets 1-2, Jan. 2019. [cited by applicant]
Deamer et al., “Three decades of nanopore sequencing,” Nature Biotechnology, vol. 34, No. 5, pp. 518-524, May 2016. [cited by applicant]
Albertorio et al., “Base dependent DNA-carbon nanotube interactions: activation enthalpies and assembly-disassembly control,” Nanotechnology, vol. 20, pp. 395101(1-9), Sep. 2009. [cited by applicant]
Bates et al., “Dynamics of DNA Molecules in a Membrane Channel Probed by Active Control Techniques,” Biophysical Journal, vol. 84, pp. 2366-2372, Apr. 2003. [cited by applicant]
Fleming et al., Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore, Biophysical Journal, vol. 112, pp. 368-375, Supplemental Information Cover sheet and pp. 1-7, Jan. 2017. [cited by applicant]
Lu et al., “Thermal Motion of DNA in an MspA Pore,” Biophysical Journal, vol. 109, pp. 1439-1445, Oct. 2015. [cited by applicant]
Branton et al., “The potential and challenges of nanopore sequencing,” Nat. Biotechnol. vol. 26, No. 10, pp. 1146-1153, Oct. 2008. [cited by applicant]
Cherf et al., “Automated forward and reverse ratcheting of DNA in a nanopore at 5-A precision,” Nature Biotechnology, vol. 30, No. 4, pp. 344-348, Apr. 2012. [cited by applicant]
Chu et al., “Real-Time Monitoring of DNA Polymerase Function and Stepwise Single-Nucleotide DNA Strand Translocation through a Protein Nanpore,” Angew. Chem., vol. 122, pp. 10304-10307, Nov. 2010. [cited by applicant]
Fologea et al., “DNA conformation and base number simultaneously determined in a nanopore,” Electrophoresis, vol. 28, pp. 3186-3192, 2007. [cited by applicant]
Meller, “Dynamics of polynucleotide transport through nanometre-scale pores,” J. Phys.: Condens. Matter, vol. 15, pp. R581-R607, Apr. 2003. [cited by applicant]
Nakane et al., “A Nanosensor for Transmembrane Capture and Identification of Single Nucleic Acid Molecules,” Biophysical Journal, vol. 87, pp. 615-621, Jul. 2004. [cited by applicant]
Zwolak et al., “Colloquium: Physical approaches to DNA sequencing and detection,” Revs. of Modern Physics, vol. 80, pp. 141-165, Jan. 2008. [cited by applicant]
Baker et al., “ClpXP, an ATP-powered unfolding and protein-degradation machine,” Biochimica et Biophysica Acta, vol. 1823, pp. 15-28, Jun. 2011. [cited by applicant]
Nivala et al., “Unfoldase-mediated protein translocation through an a-hemolysin nanopore,” Nat. Biotechnol. vol. 31, No. 3, pp. 247-250, Mar. 2013. [cited by applicant]
Lieberman et al., “Processive Replication of Single DNA Molecules in a Nanopore Catalyzed by phi29 DNA Polymerase,” J. Am. Chem. Soc., vol. 132, No. 50, pp. 17961-17972, Dec. 2010. [cited by applicant]
Lu et al., Control and Thermal Motion of DNA in an MspA Pore, NHGRI Advanced DNA Sequencing Technology Development Meeting, San Diego, CA, p. 1, May 2015. [cited by applicant]
Mulkidjanian et al., “Inventing the dynamo machine: the evolution of the F-type and V-type ATPases,” Nature Reviews Microbiology, vol. 5, pp. 892-899, Nov. 2007. [cited by applicant]
Itoh et al., “Mechanically driven ATP synthesis by F1-ATPase,” Nature, vol. 427, pp. 465-468, Jan. 2004. [cited by applicant]
Von Hippel et al., “A General Model for Nucleic Acid Helicases and Their ‘Coupling’ within Macromolecular Machines,” Cell, vol. 104, No. 2, pp. 177-190, Jan. 2001. [cited by applicant]
Saikrishnan et al., “Mechanistic Basis of 5′-3′ Translocation in SF1B Helicases,” Cell, vol. 137, pp. 849-859, May 2009. [cited by applicant]
Langford et al., “Unsupported planar lipid membranes formed from mycolic acids of [cited by applicant]
Zhao et al., “Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores,” Science, vol. 279, pp. 548-552, Jan. 1998. [cited by applicant]
Chinese Patent Application No. 201880056523.4, CNIPA Communication: First Office Action pp. 1-6, and Search Report, 2 pages, Apr. 2021. [cited by applicant]
Manrao et al., “Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase,” Nature Biotechnology, vol. 30, No. 4, pp. 349-354, Mar. 2012. [cited by applicant]
Laszlo et al., “Decoding long nanopore sequencing reads of natural DNA,” Nature Biotechnology, vol. 32, No. 8, pp. 829-833, Jun. 2014. [cited by applicant]
Schreiber et al., “Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands,” PNAS Early Edition, downloaded at http://pnas.org/cgi/doi/10.1073/pnas.… [cited by applicant]
Chinese Patent Application No. 201880056523.4, Response to CNIPA First Office Action: Response discussion pp. 1-4, claim amendments pp. 1-6, Aug. 2021. [cited by applicant]
European Patent Application No. 21189186, EPO Communication: EPO Form 1507N p. 1, EPO Form 1503 1 sheet, EPO Form P0459 Annex 1 sheet, EPO Form P04A42 1 sheet, EPO Form 1703 sheets 1-2, Nov. 2021. [cited by applicant]
European Patent Application No. 21189186, Response to EPO Communication: Response discussion pp. 1-2, claim amendments pp. 1-3, Jun. 2022. [cited by applicant]
Japanese Patent Application No. 2019-572544, JPO Notice of Reasons for Refusal, pp. 1-2, Jul. 2022. [cited by applicant]
Japanese Patent Application No. 2019-572544, Response to JPO Notice of Reasons for Refusal: Response discussion pp. 1-2, claim amendments pp. 1-5, Sep. 2022. [cited by applicant]
Fleming, “Probing nanopore-DNA interactions with MspA,” Ph.D. Thesis, pp. 1-196, Harvard University, May 2019. [cited by applicant]
Chinese Patent Application No. 202210133705.9, Preliminary Amendments to original claims, pp. 1-5, Nov. 2022. [cited by applicant]
European Patent Application No. 21189186.6-1001, Communication pursuant to Art. 94(3) EPC Letter and sheet 1, Form 2906 Sheet 1, Dec. 2022. [cited by applicant]
Korean Patent Application No. 10-2020-7002841, Remarks by Applicant, pp. 1-3, Preliminary amendments to claims, pp. 1-7, Mar. 2023. [cited by applicant]
Japanese Patent Application No. 2019-572544, Notice of Reasons for Refusal, pp. 1-2, Feb. 2023. [cited by applicant]
European Patent Application No. 21189186.6, Response to Art. 94(3) EPC communication pp. 1-3, claim amendments pp. 1-4, Apr. 2023. [cited by applicant]
Japanese Patent Application No. 2019-572544, Applicant response to Notice of Reasons for Refusal, Remarks pp. 1-2, amendments to the claims, pp. 1-5, May 2023. [cited by applicant]
Heron, “Molecular engineering DNA and RNA for nanopore sequencing,” Chapter 8, Nanopore Sequencing: An Introduction, Branton and Deamer, WSPC Publishing, Mar. 2019. [cited by applicant]
Dittrich et al., “PcrA Helicase, a Prototype ATP-Driven Molecular Motor,” Structure, vol. 14, pp. 1345-1353, Sep. 2006. [cited by applicant]
Carson et al., “Challenges in DNA motion control and sequence readout using nanopore devices,” Nanotechnology vol. 26, No. 7, HHS Public Access Author Manuscript, pp. 1-24, Jan. 2016. [cited by applicant]
Yu et al., “Structure-Based Model of the Stepping Motor of PcrA Helicase,” Biophysical Journal, vol. 91, pp. 2097-2114, Sep. 2006. [cited by applicant]
Yu et al., “How Directional Translocation is Regulated in a DNA Helicase Motor,” Biophysical Journal, vol. 93, pp. 3783-3797, Dec. 2007. [cited by applicant]
Enemark et al., “On helicases and other motor proteins,” Current Opinion in Structural Biology, vol. 18, pp. 243-287, Mar. 2008. [cited by applicant]
Caldwell et al., “Helicase SPRNTing through the nanopore,” PNAS, vol. 114, No. 5, pp. 11809-11811, Nov. 2017. [cited by applicant]
Japanese Patent Application No. 2023-164691, Notice of Reasons for Refusal pp. 1-4, Nov. 2024. [cited by applicant]
Chinese Patent Application No. 202210133715.9, First Office Action, pp. 1-4, text of first office action pp. 1-4, Search Report pp. 1-3, Oct. 2024. [cited by applicant]
Chinese Patent Application No. 202210133715.9, Response to First Office Action, Remarks pp. 1-4, listing of claims with amendments pp. 1-3, Apr. 2025. [cited by applicant]
Langford et al., “Unsupported planar lipid membranes formed from mycolic acids of [cited by applicant]
Lu et al., “Pressure-controlled motion of single polymers through solid-state nanopores,” Nano Lett. vol. 13, No. 7, pp. 3048-3052, Jul. 2013. [cited by applicant]
Kasianowicz et al., “Characterization of individual polynucleotide molecules using a membrane channel,” Proc. Natl. Aca. Sci. USA, vol. 93, pp. 13770-13773, Nov. 1996. [cited by applicant]
Keyser et al., “Direct force measurements on DNA in a solid-state nanopore,” NaturePhysics, vol. 2, pp. 473-477, Jul. 2006. [cited by applicant]
Akeson et al., “Microsecond Time-Scale Discrimination Among Polycytidylic Acid, Polyadenylic Acid, and Polyuridylic Acid as Homopolymers or as Segments Within Single RNA Molecules,” Biophysical Jnl., vol. 77, pp. 3227-3… [cited by applicant]