(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2006/0063171 A1
`Akeson et al.
`(43) Pub. Date:
`Mar. 23, 2006
`
`US 2006OO631 71A1
`
`(54) METHODS AND APPARATUS FOR
`CHARACTERIZING POLYNUCLEOTDES
`
`(76) Inventors: Mark Akeson, Santa Cruz, CA (US);
`Daniel Branton, Lexington, MA (US);
`David W. Deamer, Santa Cruz, CA
`(US); Jeffrey R. Sampson, San
`Francisco, CA (US)
`
`Related U.S. Application Data
`(60) Provisional application No. 60/555,665, filed on Mar.
`23, 2004.
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`CI2O I/68
`(52) U.S. Cl. .................................................................. 435/6
`
`Correspondence Address:
`CLARK & ELBING LLP
`101 FEDERAL STREET
`BOSTON, MA 02110 (US)
`
`(21) Appl. No.:
`
`11/088,140
`
`(22) Filed:
`
`Mar. 23, 2005
`
`(57)
`
`ABSTRACT
`
`Systems and methods for analysis of polymers, e.g., poly
`nucleotides, are provided. The Systems are capable of ana
`lyzing a polymer at a Specified rate. One Such analysis
`System includes a Structure having a nanopore aperture and
`a molecular motor, e.g., a polymerase, adjacent the nanopore
`aperture.
`
`Oxford, Exh. 1004, p. 1
`
`

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`Patent Application Publication Mar. 23, 2006 Sheet 1 of 14
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`US 2006/0063171 A1
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`Patent Application Publication Mar. 23, 2006 Sheet 2 0f 14
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`US 2006/0063171 A1
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`Patent Application Publication Mar. 23, 2006 Sheet 3 of 14
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`Patent Application Publication Mar. 23, 2006 Sheet 5 of 14
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`Patent Application Publication Mar. 23, 2006 Sheet 8 of 14
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`Patent Application Publication Mar. 23, 2006 Sheet 14 of 14
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`US 2006/0063171 A1
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`

`

`US 2006/OO63171 A1
`
`Mar. 23, 2006
`
`METHODS AND APPARATUS FOR
`CHARACTERIZING POLYNUCLEOTDES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001. This application claims benefit of U.S. Provisional
`Application No. 60/555,665, filed Mar. 23, 2004, hereby
`incorporated by reference.
`
`STATEMENT AS TO FEDERALLY FUNDED
`RESEARCH
`0002 The invention was made with U.S. Government
`support from DARPA award number N65236-98-1-5407;
`DARPA/Air Force Office of Scientific Research award num
`ber F49620-01-1-0467; and NIH award numbers
`RO1 HGO2338 and RO1 HGO1826-04. The Government has
`certain rights in the invention.
`
`BACKGROUND OF THE INVENTION
`0003. The invention relates to the field of methods and
`apparatus for characterizing nucleic acids and other poly
`CS.
`0004 Determining the nucleotide sequence of DNA and
`RNA in a rapid manner is a major goal of researchers in
`biotechnology, especially for projects Seeking to obtain the
`Sequence of entire genomes of organisms. In addition,
`rapidly determining the sequence of a nucleic acid molecule
`is important for identifying genetic mutations and polymor
`phisms in individuals and populations of individuals.
`0005 Nanopore sequencing is one method of rapidly
`determining the Sequence of nucleic acid molecules. Nan
`opore Sequencing is based on the property of physically
`Sensing the individual nucleotides (or physical changes in
`the environment of the nucleotides (i.e., electric current))
`within an individual polynucleotide (e.g., DNA and RNA) as
`it traverses through a nanopore aperture. In principle, the
`Sequence of a polynucleotide can be determined from a
`Single molecule. However, in practice, it is preferred that a
`polynucleotide Sequence be determined from a Statistical
`average of data obtained from multiple passages of the same
`molecule or the passage of multiple molecules having the
`Same polynucleotide Sequence. The use of membrane chan
`nels to characterize polynucleotides as the molecules pass
`through the Small ion channels has been Studied by Kasian
`owicz et al. (Proc. Natl. Acad. Sci. USA. 93:13770-3, 1996,
`incorporate herein by reference) by using an electric field to
`force single stranded RNA and DNA molecules through a
`2.6 nanometer diameter nanopore aperture (i.e., ion channel)
`in a lipid bilayer membrane. The diameter of the nanopore
`aperture permitted only a Single Strand of a polynucleotide
`to traverse the nanopore aperture at any given time. AS the
`polynucleotide traversed the nanopore aperture, the poly
`nucleotide partially blocked the nanopore aperture, resulting
`in a transient decrease of ionic current. Since the length of
`the decrease in current is directly proportional to the length
`of the polynucleotide, Kasianowicz et al. were able to
`determine experimentally lengths of polynucleotides by
`measuring changes in the ionic current.
`0006 Baldarelli et al. (U.S. Pat. No. 6,015,714) and
`Church et al. (U.S. Pat. No. 5,795,782) describe the use of
`nanopores to characterize polynucleotides including DNA
`
`and RNA molecules on a monomer by monomer basis. In
`particular, Baldarelli et al. characterized and Sequenced the
`polynucleotides by passing a polynucleotide through the
`nanopore aperture. The nanopore aperture is imbedded in a
`Structure or an interface, which Separates two media. AS the
`polynucleotide passes through the nanopore aperture, the
`polynucleotide alters an ionic current by blocking the nan
`opore aperture. AS the individual nucleotides pass through
`the nanopore aperture, each base/nucleotide alters the ionic
`current in a manner that allows the identification of the
`nucleotide transiently blocking the nanopore aperture,
`thereby allowing one to characterize the nucleotide compo
`Sition of the polynucleotide and perhaps determine the
`nucleotide Sequence of the polynucleotide.
`0007 One disadvantage of previous nanopore analysis
`techniques is controlling the rate at which the target poly
`nucleotide is analyzed. AS described by Kasianowicz, et al.
`(Proc. Natl. Acad. Sci., USA, 93:13770-3, (1996)), nanopore
`analysis is a useful method for performing length determi
`nations of polynucleotides. However, the translocation rate
`is nucleotide composition dependent and can range between
`10 to 107 nucleotides per second under the measurement
`conditions outlined by Kasianowicz et al. Therefore, the
`correlation between any given polynucleotide's length and
`its translocation time is not Straightforward. It is also antici
`pated that a higher degree of resolution with regard to both
`the composition and Spatial relationship between nucleotide
`units within a polynucleotide can be obtained if the trans
`location rate is Substantially reduced.
`
`SUMMARY OF THE INVENTION
`0008. The invention features apparatus for characterizing
`a polynucleotide, e.g., at a specified rate, and methods of its
`use and manufacture. Typically, an apparatus includes a
`nanopore aperture and a molecular motor that is capable of
`moving a target polynucleotide with respect to the nanopore,
`e.g., at a specified rate.
`0009. In one aspect, the invention features a method for
`analyzing a target polynucleotide including introducing the
`target polynucleotide to a nanopore analysis System includ
`ing a nanopore aperture; allowing the target polynucleotide
`to move with respect to the nanopore aperture to produce a
`signal at a rate of 75-2000 Hz, e.g., 350-2000 Hz; and
`monitoring the Signal corresponding to the movement of the
`target polynucleotide with respect to the nanopore aperture,
`e.g., to measure a monomer-dependent characteristic of the
`target polynucleotide. Examples of monomer-dependent
`characteristics include the identity of a nucleotide or the
`number of nucleotides in the polynucleotide. The nanopore
`analysis System may further include a molecular motor that
`moves the polynucleotide with respect to the nanopore
`aperture. The molecular motor may also be Substantially
`immobilized inline with the nanopore aperture, e.g., by a gel
`matrix. The target polynucleotide may or may not move
`through the nanopore aperture. The method may also include
`applying a Voltage gradient to the nanopore analysis System
`to draw the target polynucleotide adjacent the nanopore
`aperture. In another embodiment, the method includes alter
`ing the rate of movement of the polynucleotide before,
`during, or after the monitoring Step. The movement may be
`increased, decreased, initiated, or Stopped, e.g., at least in
`part, from a change in Voltage, pH, temperature, Viscosity, or
`concentration of a chemical species (e.g., ions, cofactors,
`
`Oxford, Exh. 1004, p. 16
`
`

`

`US 2006/OO63171 A1
`
`Mar. 23, 2006
`
`energy Sources, or inhibitors). In certain embodiments, the
`molecular motor is a DNA polymerase, an exonuclease, or
`a helicase, and the rate of movement is 75-2000 Hz.
`0010. In another aspect, the invention features an alter
`native method for analyzing a target polynucleotide includ
`ing introducing the target polynucleotide to a nanopore
`analysis System including a nanopore aperture and a molecu
`lar motor disposed adjacent the nanopore aperture, allowing
`the target polynucleotide to move with respect to the nan
`opore aperture to produce a Signal; and monitoring the Signal
`corresponding to the movement of the target polynucleotide
`with respect to the nanopore aperture, e.g., to measure a
`monomer-dependent characteristic of the target polynucle
`otide. This alternative method further includes altering the
`rate of movement of the polynucleotide before, during, or
`after the monitoring. Exemplary Schemes for altering the
`rate are described herein.
`0.011 The invention further features a nanopore analysis
`System including a structure having a nanopore aperture, and
`a molecular motor adjacent the nanopore aperture, wherein
`the molecular motor is substantially immobilized inline with
`the nanopore aperture, and the molecular motor is capable of
`moving a polynucleotide with respect to the nanopore aper
`ture a rate of 75-2000 Hz, e.g., at least 350 Hz. The rate of
`movement is controllable, e.g., by Voltage, pH, temperature,
`Viscosity, or concentration of a chemical Species. The
`molecular motor may be substantially immobilized inline
`With the nanopore aperture by a gel matrix, e.g., through a
`covalent bond. The molecular motor may be immobilized on
`the cis or trans side of the Structure. The System may also
`include a detection System operative to detect a monomer
`dependent characteristic of a polynucleotide. In certain
`embodiments, the molecular motor is a DNA polymerase, an
`exonuclease, or a helicase, and the rate of movement is
`75-2000 HZ.
`0012. In another aspect, the invention features a method
`for fabricating a nanopore analysis device including provid
`ing a structure comprising a nanopore aperture, a molecular
`motor, and a positioning polynucleotide; forming a complex
`between the positioning polynucleotide and molecular
`motor, disposing the complex adjacent the nanopore aper
`ture, and immobilizing the molecular motor adjacent the
`nanopore aperture Such that the molecular motor is Substan
`tially inline with the nanopore aperture; and removing the
`positioning polynucleotide from the complex. The disposing
`Step may include applying a voltage gradient to the nanopore
`analysis System to draw the complex to the nanopore aper
`ture. The immobilizing Step may include disposing a gel
`matrix around the complex, Such that the molecular motor is
`Substantially immobilized inline with the nanopore aperture
`by the gel matrix. In an alternative embodiment, the immo
`bilizing Step may include reacting a chemical bonding
`material disposed on the Structure with the molecular motor
`such that the molecular motor is immobilized substantially
`inline with the nanopore aperture by the chemical bonding
`material.
`0013 In various embodiments of any of the above
`aspects, the molecular motor includes a DNA polymerase, a
`RNA polymerase, a ribosome, an exonuclease, or a helicase.
`Exemplary DNA polymerases include E. coli DNA poly
`merase I, E. coli DNA polymerase I Large Fragment (Kle
`now fragment), phage T7 DNA polymerase, Phi-29 DNA
`
`polymerase, Thermus aquaticuS (Taq) DNA polymerase,
`Thermus flavus (Tfl) DNA polymerase, Thermus Thermo
`philus (Tth) DNA polymerase, Thermococcus litoralis (Tli)
`DNA polymerase, Pyrococcus furiosus (Pfu) DNA poly
`merase, VentTM DNA polymerase, Bacillus Stearothermo
`philus (Bst) DNA polymerase, AMV reverse transcriptase,
`MMLV reverse transcriptase, and HIV-1 reverse tran
`scriptase. Exemplary RNA polymerases include T7 RNA
`polymerase, T3 RNA polymerase, SP6 RNA polymerase,
`and E. coli RNA polymerase. Exemplary exonucleases
`include exonuclease Lambda, T7 Exonuclease, EXO III,
`Rec, Exonuclease, EXO I, and EXO T. Exemplary helicases
`include E-coli bacteriophage T7 gp4 and T4 gp41 gene
`proteins, E. coli protein DnaB, E. coli protein RuvB, and E.
`coli protein rho. In certain embodiments, the molecular
`motor includes a DNA polymerase, a ribosome, an exonu
`clease, or a helicase, e.g., exhibiting a rate of movement of
`a polynucleotide of 75-2000 Hz.
`0014. By “cis” is meant the side of a nanopore aperture
`through which a polymer enters the pore or across the face
`of which the polymer moves.
`0015. By “trans” is meant the side of a nanopore aperture
`through which a polymer (or fragments thereof) exits the
`pore or acroSS the face of which the polymer does not move.
`0016 By “molecular motor” is meant a molecule (e.g., an
`enzyme) that physically interacts with a polymer, e.g., a
`polynucleotide, and is capable of physically moving the
`polymer with respect to a fixed location. Although not
`intending to be bound by theory, molecular motorS utilize
`chemical energy to generate mechanical force. The molecu
`lar motor may interact with each monomer of a polymer in
`a Sequential manner.
`0017. By “polynucleotide" is meant DNA or RNA,
`including any naturally occurring, Synthetic, or modified
`nucleotide. Nucleotides include, but are not limited to, ATP,
`dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl
`CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-adenosine-TP,
`2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate,
`pyrrolo-pyrimidine triphosphate, 2-thiocytidine as well as
`the alphathiotriphosphates for all of the above, and 2'-O-
`methyl-ribonucleotide triphosphates for all the above bases.
`Modified bases include, but are not limited to, 5-Br-UTP,
`5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and
`5-propynyl-dUTP.
`0018. By “transport property” is meant a property mea
`Surable during polymer movement with respect to a nanop
`ore. The transport property may be, for example, a function
`of the Solvent, the polymer, a label on the polymer, other
`Solutes (e.g., ions), or an interaction between the nanopore
`and the Solvent or polymer.
`0019. One advantage of using molecule motors in the
`apparatus and methods described herein is that they provide
`a mechanism for controlling the rate (e.g., from 0 to 2000
`nucleotides per Second) of movement of the polynucleotide
`of interest with respect to a nanopore aperture. Another
`advantage of using molecular motorS is that they can Selec
`tively interact and act upon either Single or double Stranded
`polynucleotides. A further advantage of using molecular
`motorS is that Some molecular motorS decrease the prob
`ability of backward movement of the polynucleotide through
`the nanopore aperture, thus ensuring a defined directional
`analysis of a polynucleotide Sequence.
`
`Oxford, Exh. 1004, p. 17
`
`

`

`US 2006/OO63171 A1
`
`Mar. 23, 2006
`
`0020. Other features and advantages of the invention will
`be apparent from the following drawings, detailed descrip
`tion, and the claims.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`0021
`FIG. 1 is a schematic of an embodiment of a
`nanopore analysis System.
`0022 FIGS. 2A through 2D are diagrams of represen
`tative nanopore devices that can be used in the nanopore
`analysis system of FIG. 1.
`0023 FIG. 3 is a flow diagram of a representative
`proceSS for fabricating a nanopore device.
`0024 FIG. 4A through 4D are diagrams of a represen
`tative proceSS for fabricating a representative nanopore
`device having a molecular motor disposed on the trans side
`of the nanopore device.
`0025 FIG. 5A through 5C are diagrams of a represen
`tative proceSS for fabricating a representative nanopore
`device having a molecular motor disposed on the cis Side of
`the nanopore device.
`0.026
`FIG. 6A through 6D are diagrams of a represen
`tative process for fabricating another representative nanop
`ore device having a molecular motor disposed on the trans
`Side of the nanopore device.
`0027 FIG. 7A through 7C are diagrams of a represen
`tative process for fabricating another representative nanop
`ore device having a molecular motor disposed on the cis Side
`of the nanopore device.
`0028 FIG. 8 is a flow diagram of a representative
`proceSS for using a nanopore device.
`0029 FIG. 9A through 9D are diagrams of a represen
`tative process for using a representative nanopore device
`having a molecular motor disposed on the trans Side of the
`nanopore device.
`0030 FIG. 10A through 10D are diagrams of a repre
`Sentative proceSS for using a representative nanopore device
`having a molecular motor disposed on the trans Side of the
`nanopore device.
`0.031
`FIG. 11 is a schematic depiction of regulating
`DNA delivery into a nanoscale pore using a molecular motor
`as a brake. This Schematic shows W exonuclease digesting
`dsDNA and feeding the ssDNA product into an O.-hemolysin
`pore. The applied electric field acroSS the pore is required to
`capture the DNA/enzyme complex and then drive the
`SSDNA product sequentially through the detector. The sche
`matic is to Scale.
`0.032
`FIG. 12 is a schematic depiction and experimental
`data from binding of E. coli Exonuclease I to ssDNA
`64mers. Molecules were captured by applying a 180 mV
`bias (trans side positive). The buffer used was 1M KCl, 10
`mM HEPES(KOH) at pH 8.0 and 23° C. No Mg" was
`present. Each point represents capture and translocation of
`one DNA molecule. The top graph shows results for 1 uM
`of a ssDNA 64 mer. The bottom graph shows the results
`following addition of 1 uM of Exo I.
`0033 FIG. 13 is a schematic depiction of the structure of
`exonuclease from Kovall et al. Science 277:1824 (1997).
`A) Crystal structure of the homotrimer looking down
`
`through the pore which contains the catalytic domain that
`processively hydrolyzes nucleotides from one Strand of
`dsDNA leaving one DNA strand intact. B) Schematic view
`of dsDNA entering the larger pore orifice and ssDNA exiting
`the Smaller orifice.
`0034 FIG. 14A-14C are graphs showing the capture of
`dsDNA molecules bound to
`exonuclease. A) Events
`caused by capture of ssDNA 60 mers at 5 uM. B) Events
`caused by annealing of a SSDNA complement to the original
`ssDNA 60 mer for 15 minutes. C) Events seen after exonu
`clease (2.5uM ) addition to the dsDNA formed in B).
`0035 FIG. 15 is graph showing the anticipated effect of
`load on dwell time of the exonuclease/dsDNA complex
`absent Mg.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`0036) The invention features an apparatus for character
`izing polymers, Such as polynucleotides, e.g., at a Specified
`rate. Typically, an apparatus of the invention includes a
`nanopore aperture and a molecular motor disposed adjacent
`the aperture, where the molecular motor is capable of
`moving a polymer with respect to the aperture. In alternative
`embodiments, other methods are employed to control the
`rate of movement of the polymer. By making measurements
`as the polymer is moved, the polymer may be characterized.
`The following discussion will focus on polynucleotides, but
`the invention is applicable to any other polymer (e.g.,
`proteins, polypeptides, polysaccharides, lipids, and Synthetic
`polymers) that can be moved via a molecular motor.
`0037 Apparatus
`0038 FIG. 1 illustrates a representative embodiment of a
`nanopore analysis System 10 that can be used in character
`izing polymerS Such as polynucleotides. The nanopore
`analysis System 10 includes, but is not limited to, a nanopore
`device 12 and a nanopore detection System 14. The nanopore
`device 12 and the nanopore detection System 14 are coupled
`So that data regarding the target polynucleotide can be
`measured.
`0039. A typical nanopore detection system 14 includes
`electronic equipment capable of measuring characteristics of
`the polynucleotide as it interacts with the nanopore aperture,
`a computer System capable of controlling the measurement
`of the characteristics and Storing the corresponding data,
`control equipment capable of controlling the conditions of
`the nanopore device, and one or more detectors capable of
`measuring transport properties in the device.
`0040. The nanopore detection system 14 can measure
`transport properties, Such as, but not limited to, the ampli
`tude or duration of individual conductance or electron
`tunneling current changes acroSS a nanopore aperture. Such
`changes can identify the monomers in Sequence, as each
`monomer has a characteristic conductance change Signature.
`For instance, the Volume, shape, or charges on each mono
`mer can affect conductance in a characteristic way. Like
`wise, the Size of the entire polynucleotide can be determined
`by observing the length of time (duration) that monomer
`dependent conductance changes occur. Alternatively, the
`number of nucleotides in a polynucleotide (also a measure of
`Size) can be determined as a function of the number of
`nucleotide-dependent conductance changes for a given
`
`Oxford, Exh. 1004, p. 18
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`
`nucleic acid traversing the nanopore aperture. The number
`of nucleotides may not correspond exactly to the number of
`conductance changes, because there may be more than one
`conductance level change as each nucleotide of the nucleic
`acid passes Sequentially through the nanopore aperture.
`However, there is a proportional relationship between the
`two values that can be determined by preparing a Standard
`with a polynucleotide having a known Sequence. Other
`detection Schemes are described herein.
`0041 FIGS. 2A through 2D illustrate representative
`embodiments of a nanopore device 12a .
`.
`. 12d. The
`nanopore device 12a . . . 12d includes, but is not limited to,
`a structure 22 that Separates two independent adjacent pools
`of a medium. The two adjacent pools are located on the cis
`Side and the trans Side of the nanopore device 12a . . . 12d.
`The Structure 22 includes, but is not limited to, at least one
`nanopore aperture 24, e.g., So dimensioned as to allow
`Sequential monomer-by-monomer translocation (i.e., pas
`Sage) from one pool to another of only one polynucleotide
`at a time, and detection components that can be used to
`measure transport properties.
`0.042
`Exemplary detection components have been
`described in WO 00/792.57 and can include, but are not
`limited to, electrodes directly associated with the Structure
`22 at or near the pore aperture, and electrodes placed within
`the cis and trans pools. The electrodes may be capable of, but
`limited to, detecting ionic current differences acroSS the two
`pools or electron tunneling currents acroSS the pore aperture.
`0.043 Nanopores. The structure 22 contains one or more
`nanopore apertures 24 and may be made of any appropriate
`material, Such as, but not limited to, Silicon nitride, Silicon
`oxide, mica, polyimide, or lipids. The Structure 22 may
`further include detection electrodes and detection integrated
`circuitry. The nanopore aperture 24 may be a simple aperture
`in Structure 22 or it may be composed of other materials,
`Such as proteins, that can assemble So as to produce a
`channel through Structure 22. The nanopore aperture may be
`dimensioned So that only a single Stranded polynucleotide
`can pass through the nanopore aperture 24 at a given time,
`So that a double or Single Stranded polynucleotide can pass
`through the nanopore aperture 24, So that neither a Single nor
`a double Stranded polynucleotide can pass through the
`nanopore aperture 24, or So that more than one double
`Stranded polynucleotide can pass through the nanopore
`aperture 24. A molecular motor 26 disposed adjacent to a
`nanopore aperture 24 can be used to determine whether a
`Single or double Stranded polynucleotide is analyzed by the
`nanopore analysis System 10 and the type of polynucleotide
`(e.g., RNA or DNA and single or double stranded) that may
`pass through the nanopore aperture 24. The nanopore aper
`ture 24 may have a diameter of, e.g., 3 to 20 nanometers (for
`analysis of Single or double Stranded polynucleotides), and
`of, e.g., 1.6 to 4 nanometers (for analysis of Single Stranded
`polynucleotides). When a molecular motor is employed, the
`Size of the nanopore aperture 24 may be significantly larger
`than the radial dimension of a polynucleotide.
`0044 Any nanopore of the appropriate size may be used
`in the methods of the invention. Nanopores may be biologi
`cal, e.g., proteinaceous, or Solid-state. Suitable nanopores
`are described, for example, in U.S. Pat. Nos. 6,746,594,
`6,673,615, 6,627,067, 6,464,842, 6,362,002, 6,267,872,
`6,015,714, and 5,795,782 and U.S. Publication Nos. 2004/
`
`0121525, 2003/0104428, and 2003/0104428. An exemplary
`method for fabricating Solid-State membranes is the ion
`beam sculpting method described in Li et al. Nature 412:166
`(2001) and in Chen et al. Nano Letters 4:1333 (2004).
`0045 Molecular Motors.
`0046) Any molecular motor that is capable of moving a
`polynucleotide of interest may be employed in the apparatus
`of the invention. Desirable properties of a molecular motor
`include: Sequential action, e.g., addition or removal of one
`nucleotide per turnover; no backtracking along the target
`polynucleotide; no slippage of the motor on the target
`polynucleotide due to forces, e.g., from an electric field,
`employed to drive a polynucleotide to the motor; retention
`of catalytic function when disposed adjacent a nanopore
`aperture; high processivity, e.g., the ability to remain bound
`to target polynucleotide and perform at least 1,000 rounds of
`catalysis before dissociating.
`0047 A molecular motor 26 includes, e.g., polymerases
`(i.e., DNA and RNA), helicases, ribosomes, and exonu
`cleases. The molecular motor 26 that is used according to the
`methods described herein will depend, in part, on the type of
`target polynucleotide being analyzed. For example, a
`molecular motor 26 Such as a DNA polymerase or a helicase
`is useful when the target polynucleotide is DNA, and a
`molecular motor Such as RNA polymerase is useful when
`the target polynucleotide is RNA. In addition, the molecular
`motor 26 used will depend, in part, on whether the target
`polynucleotide is single-stranded or double-stranded. Those
`of ordinary skill in the art would be able to identify the
`appropriate molecular motorS 26 useful according to the
`particular application.
`0048 DNA polymerases have been demonstrated to
`function as efficient molecular motors 26. Exemplary DNA
`polymerases include E. coli DNA polymerase I, E. coli DNA
`polymerase I Large Fragment (Klenow fragment), phage T7
`DNA polymerase, Phi-29 DNA polymerase, thermophilic
`polymerases (e.g., Thermus aquaticus (Taq) DNA poly
`merase, Thermus flavus (Tfl) DNA polymerase, Thermus
`Thermophilus (Tth) DNA polymerase, Thermococcus lito
`ralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu)
`DNA polymerase, VentTM DNA polymerase, or Bacillus
`Stearothermophilus (Bst) DNA polymerase), and a reverse
`transcriptase (e.g., AMV reverse transcriptase, MMLV
`reverse transcriptase, or HIV-1 reverse transcriptase). Other
`suitable DNA polymerases are known in the art. In one
`embodiment, approximately 300 nucleotides per Second are
`threaded through the clamp of a DNA polymerase in a
`ratchet-like linear fashion, which decreases the probability
`of backward movement of the polynucleotide. In certain
`embodiments, E. coli DNA polymerase I, the Klenow frag
`ment, phage T7 DNA polymerase, Taq polymerase, and the
`Stoffel fragment are excluded from the molecular motors
`employed in the invention.
`0049 RNA polymerases, like DNA polymerases, can
`also function as efficient molecular motorS 26. Exemplary
`RNA polymerases include T7 RNA polymerase, T3 RNA
`polymerase, SP6 RNA polymerase, and E. coli RNA poly
`merases. In certain embodiments, T7 RNA polymerase is
`excluded from the molecular motors employed in the inven
`tion.
`0050. The molecular motor 26 may also include a single
`Strand Specific or double-Strand Specific exonuclease. EXO
`
`Oxford, Exh. 1004, p. 19
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`
`nuclease Lambda, which is a trimeric enzyme isolated from
`the E. coli bacteriophage Lambda, is particularly well Suited
`to be the molecular motor 26 for a number of reasons. First,
`it acts upon double-stranded DNA, which is a preferred
`Substrate for genetic analysis. Second, it is a highly proces
`Sive enzyme and acts upon only one Strand of the double
`stranded DNA, which facilitates the movement of a given
`DNA molecule with respect to a nanopore aperture 24.
`Further, the digestion rate is about 10-50 nucleotides per
`second (van Oijen et al. Science 301:1235 (2003); Perkins et
`al. Science 301:1914 (2003)). Exonuclease Lambda may
`also be excluded from the molecular motors employed in the
`invention. Additional exonucleases include, for example, T7
`Exonuclease, EXO III, Rec.J Exonuclease, EXO I, and EXO T.
`0051. Another type of molecular motor 26 is a helicase.
`Helicases are proteins, which move along polynucleotide
`backbones and unwind the polynucleotide So that the pro
`ceSSes of DNA replication, repair, recombination, transcrip
`tion, mRNA splicing, translation, and ribosomal assembly,
`can take place. Helicases include both RNA and DNA
`helicases. Helicases have previously been described in U.S.
`Pat. No. 5,888,792. Exemplary helicases include hexameric
`helicases Such as the E-coli bacteriophage T7 gp4 and T4
`gp41