WO2009005547A2 - Procédé de piégeage électrostatique de molécules dans des nanopores - Google Patents

Procédé de piégeage électrostatique de molécules dans des nanopores Download PDF

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Publication number
WO2009005547A2
WO2009005547A2 PCT/US2008/003120 US2008003120W WO2009005547A2 WO 2009005547 A2 WO2009005547 A2 WO 2009005547A2 US 2008003120 W US2008003120 W US 2008003120W WO 2009005547 A2 WO2009005547 A2 WO 2009005547A2
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WO
WIPO (PCT)
Prior art keywords
ion channel
cyclodextrin
adapter molecule
barrel
molecule
Prior art date
Application number
PCT/US2008/003120
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English (en)
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WO2009005547A3 (fr
Inventor
Henry S. White
Ryan J. White
Eric N. Ervin
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University Of Utah Research Foundation
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Publication of WO2009005547A2 publication Critical patent/WO2009005547A2/fr
Publication of WO2009005547A3 publication Critical patent/WO2009005547A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

  • the present invention pertains to the art of biotechnology and nanotechnology.
  • the invention relates to a method for altering the constriction zone within protein channel size barrel(s) or lumen(s). Such methods have use in various areas, such as chemical sensors and nucleic acid sequencing.
  • Pore-forming proteins are presently being engineered for applications in biotechnology. For instance, work has been conducted on alpha-hemolysin (alpha-HL), a bacterial toxin that forms a heptameric trans-membrane pore of known three-dimensional structure. In general, it is known to use genetic engineering and targeted chemical modification to create pores having diverse functional properties. In addition, transmembrane beta-barrels are being engineered by combining fragments from various bacterial toxins and porins, as well as polypeptide segments designed de novo. Such molecules find applications in several areas including drug delivery and the construction of biosensors.
  • alpha-HL alpha-hemolysin
  • beta-barrels are being engineered by combining fragments from various bacterial toxins and porins, as well as polypeptide segments designed de novo. Such molecules find applications in several areas including drug delivery and the construction of biosensors.
  • One type of useful biosensor is the ion channel sensor.
  • One type of ion channel sensing constitutes passing a molecule through an ion channel or protein pore present in a lipid bilayer membrane, such that each nucleotide in the molecule obstructs the pore to a different degree, causing current passing through the pore at any given moment to vary depending on the nucleotide present.
  • One application of ion channel sensors is in nucleic acid sequencing (e.g., DNA or RNA sequencing). In such an application, DNA is driven through a biological or synthetic pore by an electrical voltage.
  • a fast DNA translocation rate ( ⁇ 2 microseconds/base ( ⁇ s/base)) prevents simple electrical data acquisition
  • ⁇ s/base the diameter of the constriction zone in a pore may be too large to generate an electrical signature unique to a base as the base passes through the pore.
  • alpha-HL has a constriction zone of ⁇ 1.4 nanometers (nm).
  • Adapter molecules such as beta-cyclodextrin, have been utilized in ion channel recordings to increase detection sensitivity.
  • adapter molecules reside only briefly within the barrel or lumen of a pore.
  • the adaptor molecule heptakis (6-O-sulfo)- ⁇ -cyclodextrin (s7- ⁇ CD) resides for ⁇ 1 second within a pore when a voltage of -0.04 volts (V) is applied across the lipid bilayer membrane (trans relative to cis). The molecule then diffuses out of the barrel of the pore.
  • V -0.04 volts
  • the present invention is directed to a method for electrostatic trapping of molecules in nanopores. More specifically, the method of the present invention provides a means for altering the constriction zone within the size barrel or lumen of a protein channel or channels within an electrostatic sensing system.
  • the electrostatic sensing system includes a glass membrane with a lipid bilayer extending across a nanopore opening in the membrane.
  • An ion channel or protein channel, such as alpha-HL, is inserted into the lipid bilayer and an adaptor molecule is introduced to the system.
  • the adaptor molecule constitutes a cyclodextrin molecule.
  • a trans-membrane voltage of between about -40 and -800 millivolts (mV) is applied via system electrodes and current through the ion channel is measured by a sensor.
  • mV millivolts
  • an adaptor molecule is sensed within the barrel of the ion channel, the voltage is increased to above -100 mV, and the adaptor molecule is trapped within the barrel for an indefinite period of time.
  • the ion channel/adaptor molecule complex may then be utilized in the analyses of other molecules, such as in nucleic acid sequencing of DNA.
  • the adaptor molecule may then be released from the ion channel by lowering the voltage to below -100 mV.
  • the present method provides significant advantages in application of single ion channels for chemical sensing and nucleic acid (e.g. DNA or RNA) sequencing. Additional objects, features and advantages of the present invention will become more readily apparent from the following detailed description of a preferred embodiment when taken in conjunction with the drawings wherein like reference numerals refer to corresponding parts
  • Figure 1 is a schematic depiction of a sensing system utilized in the present invention, including a glass nanopore membrane;
  • Figure 2A depicts a glass nanopore membrane manufacturing process
  • Figure 2B depicts a glass nanopore membrane produced by the manufacturing process of Figure 2A;
  • Figure 3 is an enlarged view of the nanopore of the system of
  • Figure 4 is a simplified cross-sectional view of a protein channel/adaptor molecule complex
  • Figure 5 is a graph of illustrating current versus time for trapping of a charged adapter molecule as a function of pressure; and Figure 6 is a chart depicting current sensed over time at -40, -100, -120 and -200 mV utilizing the system of Figure 1.
  • an electrostatic sensing system utilized in accordance with the present invention is indicated at 10.
  • the system comprises a membrane 14 including a nanopore 16 located therein, electrodes 18 and 19, an internal solution indicated at 22, an external solution indicated at 26, a sensor 28, a voltage source 30 and a controller 31.
  • system 10 also includes a pressure inducing apparatus 32 including a seal 34 for sealing an open end 35 of glass nanopore membrane 14.
  • Electrodes 18 and 19, which are preferably silver/silver chloride (Ag/AgCl) electrodes, are utilized to bias a potential across nanopore membrane 14, with the potential being referenced to the internal solution 22.
  • controller controller
  • membrane 14 is a glass nanopore membrane constituted by a conical shaped nanopore 16 located in a thin glass membrane 17.
  • a membrane 14 is manufactured utilizing a platinum (Pt) disk electrode 36 embedded at the bottom of a conical shaped pore 37 as shown in Figure 2 A. Pore 37 is then polished, and electrode 36 is etched out and removed, leaving a nanometer-sized opening or nanopore 16.
  • the circular orifice of nanopore 16 has a diameter of between approximately 5 and 5000 nm, and a glass membrane portion 17 has a thickness of approximately 20-75 ⁇ m.
  • the steady-state flux of ions and molecules across glass nanopore membrane 14 is limited by transport through nanopore 16, aiding in the study of transport in nanometer-diameter pores.
  • lipid bilayer 40 is deposited across nanopore 16 by painting techniques or other suitable methods, and a trans-membrane ion channel 44 is inserted through lipid bilayer 40.
  • ion channel 44 constitutes alpha-hemolysin (alpha-HL)
  • lipid bilayer 40 is composed of l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Dilute solutions of monomer alpha-HL or heptamer alpha-HL may be utilized to avoid multiple insertions of protein ion channels in lipid bilayer 40.
  • system 10 is set up such that ion channel 44 extends through lipid bilayer 40 as depicted in Figure 3.
  • seal 34 is applied over open end 35 of glass nanopore membrane 14 and a positive trans-membrane pressure is applied through pressure inducing apparatus 32 to ensure stable ion channel insertion.
  • Stable ion channel insertion occurs over a wide pressure range.
  • a pressure of between 40-300 mm Hg is applied as illustrated in Figure 5.
  • Adapter molecule(s), indicated at 50 is introduced into external solution 26.
  • Adapter molecule 50 can be synthesized by standard chemical methods to produce a structure with desirable functional groups, electrical charge, and electrical dipoles.
  • adapter molecule 50 may have a cyclic structure with an inner diameter d that better matches the diameter of a nucleic acid to be sequenced, than the diameter D of an ion channel barrel 46.
  • An adapter molecule 50 having the precise ideal structure for electrostatic trapping can be designed and synthesized, for the above applications.
  • adapter molecule 50 examples include alpha-cyclodextrin (4.7-5.3 A), ⁇ -cyclodextrin (6.0-6.5 A), and ⁇ -cyclodextrin (7.5-8.3 A), each having a well defined pore diameter.
  • These molecules can be modified to include charge groups and other functionality as desired. More specifically, the structure, size and charge of adapter molecule 50 may be optimized for different applications, e.g., DNA sequencing. Furthermore, adapter molecule 50 may be modified or chosen for optimal temporal translocation control of a molecule through barrel 46. In the preferred method of the present invention, adapter molecule 50 is a cyclodextrin.
  • adapter molecule 50 is heptakis(6-O- sulfo)- ⁇ -cyclodextrin (s7- ⁇ CD).
  • the preferred internal solution 22 is 1 ⁇ M alpha-HL, 1 M potassium chloride (KCl) and 10 mM phosphate buffer (PBS - pH 7.4), while the external solution 26 is 1 M KCl and 10 mM PBS (pH 7.4) containing 50 ⁇ M s7- ⁇ CD.
  • a DC voltage ranging between approximately -40 to -800 mV (trans relative to cis) is then applied via electrodes 18 and 19.
  • An electrical parameter, such as current through ion channel 44, is measured by sensor 28.
  • a characteristic drop in current is sensed by sensor 28 as a consequence of the increased resistance through ion channel 44.
  • a potential such as between -120 and -800 mV, is applied and electrostatic trapping of adapter molecule 50 within barrel 46 occurs.
  • a potential in the order of -150 mV is utilized to insert adapter molecule 50 into barrel 46, and a disassociation voltage of less than -150 mV is utilized to remove adapter molecule 50 from barrel 46.
  • Adaptor molecule 50 can be trapped for an indefinite period of time (e.g., minutes, hours, days, etc.) inside ion channel 44.
  • trapping creates a long-lived adapter molecule/ion channel structure, which can be utilized in nucleic acid sequencing.
  • molecules of interest 60 such as nucleic acids, may be introduced to system 10 such that translocation of molecule 60 can occur through the reduced lumen diameter d of adapter molecule 50, thus increasing sensitivity of sensing system 10.
  • electrostatic trapping of s7- ⁇ CD and other adapter molecules 50 in the lumen or barrel 46 of ion channel 44 reduces the size of the channel through which molecule 60 passes, thereby reducing the translocation rate and reducing the size of the constriction zone in which a base signature is generated.
  • the voltage may be reduced to release adapter molecule 50 from barrel 46 of ion channel 44.
  • the conductivity of ion channel 44 is the same before and after electrostatic trapping of adapter molecule 50, demonstrating that the method of the present invention does not damage or alter ion channel 44.
  • the following experimental methods and materials where used to demonstrate electrostatic trapping of a -7 charged molecule s7- ⁇ CD within the internal cavity of an alpha-HL trans-membrane protein ion channel.
  • the experiment was performed using a glass nanopore membrane modified with 3-cyanopropyldimethylchlorosilane.
  • the internal nanopore solution was 1 ⁇ M alpha-HL, 1 M KCl and 10 mM phosphate buffer (PBS - pH 7.4).
  • the external solution was 1 M KCl 10 mM PBS (pH 7.4) containing 50 ⁇ M s7- ⁇ CD.
  • Two Ag/AgCl electrodes were used to bias a potential across the nanopore membrane, with potential referenced to the internal solution.
  • a bilayer composed of DPhPC was painted across the nanopore (450nm) and a single alpha-HL was inserted into the bilayer membrane.
  • a DC voltage ranging between -40 to -200 mV (trans relative to cis) was applied to monitor the effects of s7- ⁇ CD binding events inside the alpha-HL channel as a function of the applied voltage.
  • a positive trans-membrane pressure from 40-300 mm Hg was applied to ensure stable protein insertion.
  • Important to the present invention is the use of high voltages (>-0.1V) to extend the lifetime of s7- ⁇ CD within the lumen of the alpha-HL channel.
  • FIG. 6A-D illustrates the current sensed over time respectively utilizing -40, -100, -120 and -200 mV of applied potential.
  • a charged adapter molecule such as s7- ⁇ CD
  • a protein ion channel e.g., alpha-HL
  • electrostatic voltage across the lipid bilayer membrane in which the ion channel is inserted.
  • cyclodextrins e.g., both positively and negatively charged different functional groups, and alpha and gamma cyclodextrin
  • proteins and nucleic acids e.g., RNA

Abstract

L'invention concerne un procédé de piégeage électrostatique de molécules dans des nanopores. De préférence, ledit procédé utilise une membrane de verre (14) ayant une bicouche lipidique (40) s'étendant à travers un nanopore (16) formé dans celle-ci. Un canal ionique (44), tel qu'alpha-HL, est introduit dans la bicouche lipidique (40). Une molécule adaptatrice (50), telle que la cyclodextrine, est introduite dans le système et piégée à l'intérieur du canal ionique (44) à l'aide d'une tension transmembranaire de l'ordre de -120 mV ou plus pour créer une molécule adaptatrice/structure de canal ionique ayant des applications particulières dans le séquençage d'acide nucléique. La molécule adaptatrice (50) peut être sélectivement libérée du canal ionique (44) par réduction de la tension transmembranaire.
PCT/US2008/003120 2007-03-09 2008-03-10 Procédé de piégeage électrostatique de molécules dans des nanopores WO2009005547A2 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US90599707P 2007-03-09 2007-03-09
US60/905,997 2007-03-09
US90699307P 2007-03-13 2007-03-13
US60/906,993 2007-03-13
US91969407P 2007-03-23 2007-03-23
US60/919,694 2007-03-23
US92612207P 2007-04-24 2007-04-24
US60/926,122 2007-04-24

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WO2009005547A2 true WO2009005547A2 (fr) 2009-01-08
WO2009005547A3 WO2009005547A3 (fr) 2009-02-19

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105273991A (zh) * 2010-02-08 2016-01-27 吉尼亚科技公司 用于在纳米孔中操作分子的系统和方法
US9678055B2 (en) 2010-02-08 2017-06-13 Genia Technologies, Inc. Methods for forming a nanopore in a lipid bilayer
US9759711B2 (en) 2013-02-05 2017-09-12 Genia Technologies, Inc. Nanopore arrays
US9869655B2 (en) 2011-01-24 2018-01-16 Genia Technologies, Inc. System for detecting electrical properties of a molecular complex
US10036725B2 (en) 2013-10-17 2018-07-31 Genia Technologies, Inc. Non-faradaic, capacitively coupled measurement in a nanopore cell array
US10724987B2 (en) 2012-02-27 2020-07-28 Roche Sequencing Solutions, Inc. Sensor circuit for controlling, detecting, and measuring a molecular complex

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Publication number Priority date Publication date Assignee Title
GB201619930D0 (en) * 2016-11-24 2017-01-11 Oxford Nanopore Tech Apparatus and methods for controlling insertion of a membrane channel into a membrane

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105273991B (zh) * 2010-02-08 2019-05-10 吉尼亚科技公司 用于在纳米孔中操作分子的系统和方法
US9678055B2 (en) 2010-02-08 2017-06-13 Genia Technologies, Inc. Methods for forming a nanopore in a lipid bilayer
US10371692B2 (en) 2010-02-08 2019-08-06 Genia Technologies, Inc. Systems for forming a nanopore in a lipid bilayer
CN105273991A (zh) * 2010-02-08 2016-01-27 吉尼亚科技公司 用于在纳米孔中操作分子的系统和方法
US9869655B2 (en) 2011-01-24 2018-01-16 Genia Technologies, Inc. System for detecting electrical properties of a molecular complex
US10156541B2 (en) 2011-01-24 2018-12-18 Genia Technologies, Inc. System for detecting electrical properties of a molecular complex
US10330633B2 (en) 2011-01-24 2019-06-25 Genia Technologies, Inc. System for communicating information from an array of sensors
US10724987B2 (en) 2012-02-27 2020-07-28 Roche Sequencing Solutions, Inc. Sensor circuit for controlling, detecting, and measuring a molecular complex
US11275052B2 (en) 2012-02-27 2022-03-15 Roche Sequencing Solutions, Inc. Sensor circuit for controlling, detecting, and measuring a molecular complex
US9759711B2 (en) 2013-02-05 2017-09-12 Genia Technologies, Inc. Nanopore arrays
US10809244B2 (en) 2013-02-05 2020-10-20 Roche Sequencing Solutions, Inc. Nanopore arrays
US10036725B2 (en) 2013-10-17 2018-07-31 Genia Technologies, Inc. Non-faradaic, capacitively coupled measurement in a nanopore cell array
US10393700B2 (en) 2013-10-17 2019-08-27 Roche Sequencing Solutions, Inc. Non-faradaic, capacitively coupled measurement in a nanopore cell array

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