||||I||
`
`USOO5795782A
`Patent Number:
`11
`45) Date of Patent:
`
`5,795,782
`Aug. 18, 1998
`
`United States Patent (19)
`Church et al.
`
`54 CHARACTERIZATION OF INDIVIDUAL
`POLYMERMOLECULES BASED ON
`MONOMER-INTERFACE INTERACTIONS
`
`75 Inventors: George Church, Brookline. Mass.;
`David W. Deamer, Santa Cruz, Calif.;
`Daniel Branton, Lexington; Richard
`Baldarelli, Natick, both of Mass.; John
`Kasianowicz, Darnestown, Md.
`73) Assignees: President & Fellows of Harvard
`College, Cambridge. Mass.: The
`Regents of the University of
`California, Oakland, Calif.
`
`21 Appl. No.: 405,735
`22 Filed:
`Mar 17, 1995
`(51
`int. Cl. ................................ GON 33f483
`52 U.S. Cl. .................................... 436/2: 436/151; 435/4
`58 Field of Search ............................ 435/6, 4, 5:436/2.
`436/15
`
`56
`
`References Cited
`PUBLICATIONS
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`Boulanger et al., "Characterization of Ion Channels Involved
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`Boulanger et al., “Ion Channels Are Likely to Be Involved
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`Dargent et al., “Selectivity for Maltose and Maltodextrins of
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`Dargent et al. “Effect of Point Mutations on the in-Vitro
`Pore Properties of Maltoporin, a Protein of Escherichia Coli
`Outer Membrane'. 988, J. Mo. Biol. 201:497-506.
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`cron Particles and Pores by the Resistive Pulse Technique".
`1977, J. Colloid and Interface Science, 61(2):323-35.
`Ehrmann et al., "Genetic Analysis of Membrane Protein
`Topology by a Sandwich Gene Fusion Approach". 1990,
`Proc. Natl. Acad. Sci., USA, 87:7574-78.
`Ferenci et al., "Channel Architecture in Maltoporin: Domi
`nance Studies with LamB Mutations Influencing Maltodex
`trin Binding Provide Evidence for Independent Selectivity
`Filters in Each Subunit", 1989, J. Bacteriology,
`171(2):855-61.
`(List continued on next page.)
`Primary Examiner-Charles L. Patterson, Jr.
`Attorney, Agent, or Firm-Fish & Richardson PC.
`57
`ABSTRACT
`A method is disclosed for characterizing a linear polymer
`molecule by measuring physical changes across an interface
`between two pools of media as the linear polymer traverses
`the interface and monomers of the polymer interact with the
`interface, where the physical changes are suitable to identify
`characteristics of the polymer.
`
`15 Claims, 6 Drawing Sheets
`
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`ORDO
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`Channel
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`Current
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`base
`OCEO
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`lonic Current Blocked
`by Polynucleotide in
`Channel
`
`Oxford, Exh. 1010, p. 1
`
`

`

`5,795,782
`Page 2
`
`PUBLICATIONS
`Ghadiri et al., "Artificial Transmembrane Ion Channels
`From Self-Assembling Peptide Nanotubes". 1994, Nature,
`369:301-304.
`Hall et al., “Alamethicin: A Rich Model for Channel Behav
`ior”, 1984, J. Biophys. 45:233-47.
`Hamill et al., "Improved Patch-Clamp Techniques for
`High-Resolution Current Recording from Cells and
`Cell-Free Membrane Patches", 1981, Pfligers Archiv. Eur
`J. Physiology. 391(2):85-100.
`Harrington et al., "The F Pilus of Escherichia Coli Appears
`to Support Stable DNA Transfer in the Absence of
`Wal-to-Wall Contact Between Cels', 1990, J. Bacteriod
`ogy, 172(12):7263-64.
`Heinemann et al... "Open Channel Noise IV: Estimation of
`Rapid Kinetics of Formamide Block in Gramicidin A Chan
`nels". 1988. J. Biophys. 54:757-64.
`Heinemann et al... "Open Channel Noise V: Fluctuating
`Barriers to Ion Entry in Gramicidin A Channels", 1990, J.
`Biophys. 57:499-514.
`Henry et al. "Blockade of a Mitochondrial Cationic Channel
`by an Addressing Peptide: An Electrophysiological Study",
`1989, J. Menbranee Biol. 12:139-47.
`Hoshi et al., "Biophysical and Molecular Mechanisms of
`Shaker Potassium Channel Inactivation", 1990, Science,
`250:533-38.
`Hoshi et al., "Two Types of Inactivation in Shaker K”
`Channels: Effects of Alterations in the Carboxy-Terminal
`Region", 1991. Neuron. 7:547-56.
`Kubitschek, "Electronic Counting and Sizing of Bacteria",
`Nature, 1958, 182:234-35.
`Lakey et al., "The Voltage-Dependent Activity of Escheri
`chia Coli Porins in Different Planar Bilayer Reconstitu
`tions' 1989, Eur: J. Biochen. 186:303-308.
`Lopez et al., “Hydrophobic Substitution Mutations in the S4
`Sequence Alter Voltage-Dependent Gating in Shaker K"
`Channels', 1991 Netrora, 7:327-36.
`Moellerfeld et al., "Improved Stability of Black Lipid Mem
`branes by Coating with Polysaccharide Derivatives Bearing
`Hydrophibic Anchor Groups", 1986, Biochinica et Bio
`physica Acta, 857:265–70.
`Nath et al., “Transcription by T7 RNA Polymerase Using
`benzo(a)pyrene-modified templates", 1991, Carcinogen
`esis, 12(6):973-76.
`
`Neher et al. "Single-Channel Currents Recorded from
`Membrane of Denervated Frog Muscle Fibres". 1976,
`Nature. 260:799-80.
`Nowicket al., "Fluorescence Measurement of the Kinetics of
`DNA Injection by Bacteriophage I into Liposomes", 1988.
`Biochemistry, 27:7919–24.
`Ollis et al., "Domain of E. Coli DNA Polymerase I Showing
`Sequence Homology to T7 DNA Polymerase". 1985,
`Nature, 313:88-19.
`Ollis et al. "Structure of Large Fragment of Escherichia
`Coli DNA Polymerase I Complexed with dTMP", 1985.
`Nature, 313:762-66.
`Ovchinnikov et al., 3. The Cyclic Peptides: Structure. Con
`formation, and Function: P. Gramicidin S. (851). Its Analogs
`and Tyrocidines A-C (904-906), 1982, The Proteins, Third
`Edition, 5:547-55.
`Ovchinnikov et al. 3. The Cyclic Peptides: Structure. Con
`formation, and Function: T. Valinomycin (913), 1982. The
`Proteins, Third Edition, 5:563-73.
`Patton et al. "Amino Acid Residues Required for Fast
`Na-channel Inactivation: Charge Neutralizations and Dele
`tions in the I-IV Linker', 1992, Proc. Natl. Acad. Sci.
`USA, 89:10905-909.
`Shiver et al., "On the Explanation of the Acidic pH Require
`ment for In Vitro Activity of Colicin E1", 1987.J. Biological
`Chen... 262(29): 14273-281.
`Sigworth et al. "Open Channel Noise: III. High Resolution
`Recordings Show Rapid Current Fluctations in Gramicidin
`A and Four Chemical Analogues". 1987. J. Biophys.
`52:1055-64.
`Simon et al., “A Protein Conducting Channel in the Endo
`plasmic Reticulum", Cell, 65:371-80, (1991).
`Taylor et al., “Reversed' Alamethicin Conductance in Lipid
`Bilayers", 1991, J. Biophys. 59:873-79.
`Weiss et al., "Molecular Architecture and Electrostatic Prop
`erties of a Bacterial Poin', 1991, Science, 254:1627-30.
`West et al., “A Cluster of Hydrophobic Amino Acid Resi
`dues Required for Fast Na-channel Inactivation", 1992.
`Proc. Natl. Acad. Sci. USA. 89:10910-14.
`Wonderlin et al... "Optimizing Planar Lipid Bilayer Sin
`gle-Channel Recordings for High Resolution with Rapid
`Voltage Steps", 1990, J. Biophy.s., 58:289–97.
`
`Oxford, Exh. 1010, p. 2
`
`

`

`U.S. Patent
`
`Aug. 18, 1998
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`Sheet 1 of 6
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`5,795,782
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`U.S. Patent
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`Aug. 18, 1998
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`U.S. Patent
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`Aug. 18, 1998
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`U.S. Patent
`
`Aug. 18, 1998
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`Sheet 4 of 6
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`Aug. 18, 1998
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`U.S. Patent
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`Aug. 18, 1998
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`

`

`5,795,782
`
`1
`CARACTERIZATION OF INDVDUAL
`POLYMERMOLECULES BASED ON
`MONOMER-NTERFACE INTERACTIONS
`
`2
`such as the number and composition of monomers that make
`up each individual molecule, preferably in sequential order
`from any starting point within the polymer or its beginning
`or end. A heterogenous population of polymers may be
`characterized, providing a distribution of characteristics
`(such as size) within the population. Where the monomers
`within a given polymer molecule are heterogenous, the
`method can be used to determine their sequence.
`The pools of medium used in the invention may be any
`fluid that permits adequate polymer mobility for interface
`interaction. Typically, the pools will be liquids, usually
`aqueous solutions or other liquids or solutions in which the
`polymers can be distributed.
`The interface between the pools is designed to interact
`sequentially with the monomers of one polymer molecule at
`a time. As described in greater detail below, the useful
`portion of the interface may be a passage in or through an
`otherwise impermeable barrier, or it may be an interface
`between immiscible liquids. It is preferable that only one
`passage is present or functional in the impermeable barrier.
`The interface-dependent measurements made according
`to the invention can be any measurement, e.g., physical or
`electrical, that varies with polymer-interface interaction. For
`example, physical changes the monomers cause as they
`interact sequentially with the interface may be measured.
`Current changes resulting from the polymer's interference
`with ion flow at the interface may be measured. The mea
`surements may reflect the sequential interaction of the
`monomers with the interface, so as to permit evaluation of
`monomer-dependent characteristics of the polymer mol
`ecule (e.g. size or mass of individual monomers or of the
`entire polymer, or the sequence or identity of individual
`monomers which make up the polymer).
`In one embodiment, the pools include electrically con
`ductive medium which can be of the same or different
`compositions. The pools with conducting media are sepa
`rated by an impermeable barrier containing an ion
`permeable passage, and measurements of the interface char
`acteristics include establishing an electrical potential
`between the two pools such that ionic current can flow
`across the ion permeable passage. When the polymer inter
`acts sequentially with the interface at the ion permeable
`passage, the ionic conductance of the passage will change
`(e.g. decrease or increase) as each monomer interacts, thus
`indicating characteristics of the monomers (e.g., size,
`identity) and/or the polymer as a whole (e.g., size).
`In a different embodiment, the concentration of polymers
`in a solution can be determined, using the conducting
`medium and ion-permeable passage described above. As a
`voltage differential is applied across the pools, the polymer
`molecules interact with the ion-permeable passage. The
`number of interactions (conductance change events) per unit
`time is proportional to the number of polymer molecules in
`the solution. This measurement is preferably made under
`relatively low resolution recording conditions, e.g., below
`the level of resolution of individual monomer?pore interac
`tions.
`The conducting medium used can be any medium, pref
`erably a solution, more preferably an aqueous solution,
`which is able to carry electrical current. Such solutions
`generally contain ions as the current conducting agents, e.g.,
`sodium, potassium, chloride, calcium, cesium, barium,
`sulfate, phosphate. Conductance (g) across the pore or
`channel is determined by measuring the flow of current
`across the pore or channel via the conducting medium. A
`voltage difference can be imposed across the barrier between
`
`STATEMENT AS TO FEDERALLY SPONSORED
`RESEARCH
`This invention was made with Government support under
`NIH grant 1R21HG00811-01 (George Church) awarded by
`the Public Health Service and grant NSF #MCB-9421831
`(Daniel Branton) awarded by the National Science Founda
`tion. The Government has certain rights in the invention.
`BACKGROUND OF THE INVENTION
`The general field of the invention is polymer character
`ization.
`Rapid, reliable, and inexpensive characterization of
`polymers, particularly nucleic acids, has become increas
`ingly important. One notable project, known as the Human
`Genome Project, has as its goal sequencing the entire human
`genome, over three billion nucleotides.
`Typical current nucleic acid sequencing methods depend
`either on chemical reactions that yield multiple length DNA
`strands cleaved at specific bases, or on enzymatic reactions
`that yield multiple length DNA strands terminated at specific
`bases. In each of these methods, the resulting DNA strands
`of differing length are then separated from each other and
`identified in strand length order. The chemical or enzymatic
`reactions, as well as the technology for separating and
`identifying the different length strands, usually involve
`tedious, repetitive work. A method that reduces the time and
`effort required would represent a highly significant advance
`in biotechnology.
`SUMMARY OF THE INVENTION
`We have discovered a method for rapid, easy character
`ization of individual polymer molecules, for example poly
`mer size or sequence determination. Individual molecules in
`a population may be characterized in rapid succession.
`Stated generally, the invention features a method for
`evaluating a polymer molecule which includes linearly
`connected (sequential) monomer residues. Two separate
`pools of liquid-containing medium and an interface between
`the pools are provided. The interface between the pools is
`capable of interacting sequentially with the individual
`monomer residues of a single polymer present in one of the
`pools. Interface-dependent measurements are continued
`over time, as individual monomer residues of a single
`polymer interact sequentially with the interface, yielding
`data suitable to infer a monomer-dependent characteristic of
`the polymer. Several individual polymers, e.g., in a heter
`ogenous mixture, can be characterized or evaluated in rapid
`succession, one polymer at a time, leading to characteriza
`tion of the polymers in the mixture.
`The method is broadly useful for characterizing polymers
`that are strands of monomers which, in general (if not
`entirely), are arranged in linear strands. Any polymer whose
`monomer units interact with the interface can be character
`ized. The method is particularly useful for characterizing
`biological polymers such as deoxyribonucleic acids, ribo
`nucleic acids, polypeptides, and oligosaccharides, although
`other polymers may be evaluated. In some embodiments, a
`polymer which carries one or more charges (e.g., nucleic
`acids, polypeptides) will facilitate implementation of the
`invention.
`The monomer-dependent characterization achieved by the
`invention may include identifying physical characteristics
`
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`5,795.782
`
`3
`the pools by conventional means, e.g., via a voltage source
`which injects or administers current to at least one of the
`pools to establish a potential difference. Alternatively, an
`electrochemical gradient may be established by a difference
`in the ionic composition of the two pools, either with
`different ions in each pool, or different concentrations of at
`least one of the ions in the solutions or media of the pools.
`In this embodiment of the invention, conductance changes
`are measured and are indicative of monomer-dependent
`characteristics.
`The term "ion permeable passages" used in this embodi
`ment of the invention includes ion channels, ion-permeable
`pores, and other ion-permeable passages, and all are used
`herein to include any local site of transport through an
`otherwise impermeable barrier. For example, the term
`includes naturally occurring. recombinant, or mutant pro
`teins which permit the passage of ions under conditions
`where ions are present in the medium contacting the channel
`or pore. Synthetic pores are also included in the definition.
`Examples of such pores can include, but are not limited to,
`chemical pores formed, e.g., by nystatin, ionophores (e.g.,
`A23 187: Pressman et al., Ann. Rev. Biochem. 45:501, 1976),
`or mechanical perforations of a membranous material. Pro
`teinaceous ion channels can be voltage-gated or voltage
`independent, including mechanically gated channels (e.g.,
`stretch-activated K channels), or recombinantly engineered
`or mutated voltage dependent channels (e.g., Na' or K
`channels constructed as is known in the art).
`Another preferred type of passage is a protein which
`includes a portion of a bacteriophage receptor which is
`capable of binding all or part of a bacteriophage ligand
`(either a natural or functional ligand) and transporting
`bacteriophage DNA from one side of the interface to the
`other. The polymer to be characterized includes a portion
`which acts as a specific ligand for the bacteriophage
`receptor, so that it may be injected across the barrierd
`interface from one pool to the other.
`The protein channels or pores of the invention can include
`those translated from one or more natural and/or recombi
`nant DNA molecule(s) which includes a first DNA which
`encodes a channel or pore forming protein and a second
`DNA which encodes a monomer-interacting portion of a
`monomer polymerizing agent (e.g., a nucleic acid
`polymerase). The expressed protein or proteins are capable
`of non-covalent association or covalent linkage (any linkage
`herein referred to as forming an "assemblage” of "heterolo
`gous units"), and when so associated or linked, the poly
`merizing portion of the protein structure is able to polymer
`ize monomers from a template polymer, close enough to the
`channel forming portion of the protein structure to measur
`ably affect ion conductance across the channel.
`Alternatively, assemblages can be formed from unlike
`molecules, e.g., a chemical pore linked to a protein
`polymerase, but these assemblages still fall under the defi
`nition of a "heterologous" assemblage.
`The invention also includes the recombinant fusion
`protein(s) translated from the recombinant DNA molecule(s)
`described above, so that a fusion protein is formed which
`includes a channel forming protein linked as described
`above to a monomer-interacting portion of a nucleic acid
`polymerase. Preferably, the nucleic acid polymerase portion
`of the recombinant fusion protein is capable of catalyzing
`polymerization of nucleotides. Preferably, the nucleic acid
`polymerase is a DNA or RNA polymerase, more preferably
`T7 RNA polymerase.
`The polymer being characterized may remain in its origi
`nal pool, or it may cross the passage. Either way, as a given
`
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`polymer molecule moves in relation to the passage, indi
`vidual monomers interact sequentially with the elements of
`the interface to induce a change in the conductance of the
`passage. The passages can be traversed either by polymer
`transport through the central opening of the passage so that
`the polymer passes from one of the pools into the other, or
`by the polymer traversing across the opening of the passage
`without crossing into the other pool. In the latter situation,
`the polymer is close enough to the channel for its monomers
`to interact with the passage and bring about the conductance
`changes which are indicative of polymer characteristics. The
`polymer can be induced to interact with or traverse the pore.
`e.g., as described below, by a polymerase or other template
`dependent polymer replicating catalyst linked to the pore
`which draws the polymer across the surface of the pore as it
`synthesizes a new polymer from the template polymer, or by
`a polymerase in the opposite pool which pulls the polymer
`through the passage as it synthesizes a new polymer from the
`template polymer. In such an embodiment, the polymer
`replicating catalyst is physically linked to the ion-permeable
`passage, and at least one of the conducting pools contains
`monomers suitable to be catalytically linked in the presence
`of the catalyst. A "polymer replicating catalyst," "polymer
`izing agent" or "polymerizing catalyst” is an agent that can
`catalytically assemble monomers into a polymer in a tem
`plate dependent fashion-i.e., in a manner that uses the
`polymer molecule originally provided as a template for
`reproducing that molecule from a pool of suitable mono
`mers. Such agents include, but are not limited to, nucleotide
`polymerases of any type, e.g., DNA polymerases, RNA
`polymerases, tRNA and ribosomes.
`The characteristics of the polymer can be identified by the
`amplitude or duration of individual conductance changes
`across the passage. Such changes can identify the monomers
`in sequence, as each monomer will have a characteristic
`conductance change signature. For instance, the volume.
`shape, or charges on each monomer will affect conductance
`in a characteristic way. Likewise, the size of the entire
`polymer can be determined by observing the length of time
`(duration) that monomer-dependent conductance changes
`occur. Alternatively, the number of monomers in a polymer
`(also a measure of size) can be determined as a function of
`the number of monomer-dependent conductance changes for
`a given polymer traversing a passage. The number of
`monomers may not correspond exactly to the number of
`conductance changes, because there may be more than one
`conductance level change as each monomer of the polymer
`passes sequentially through the channel. However, there will
`be a proportional relationship between the two values which
`can be determined by preparing a standard with a polymer
`of known sequence.
`The mixture of polymers used in the invention does not
`need to be homogenous. Even when the mixture is
`heterogenous, only one molecule interacts with a passage at
`a time, yielding a size distribution of molecules in the
`mixture, and/or sequence data for multiple polymer mol
`ecules in the mixture.
`In preferred embodiments, the passage is a natural or
`recombinant bacterial porin molecule. In other preferred
`embodiments, the passage is a natural or recombinant
`voltage-sensitive or voltage gated ion channel, preferably
`one which does not inactivate (whether naturally or through
`recombinant engineering as is known in the art). "Voltage
`sensitive" or "gated" indicates that the channel displays
`activation and/or inactivation properties when exposed to a
`particular range of voltages. Preferred channels for use in the
`invention include the ot-hemolysin toxin from S. aureus and
`maltoporin channels.
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`
`S
`In an alternate embodiment of the invention, the pools of
`medium are not necessarily conductive, but are of different
`compositions so that the liquid of one pool is not miscible in
`the liquid of the other pool and the interface is the immis
`cible interface between the pools. In order to measure the
`characteristics of the polymer, a polymer molecule is drawn
`through the interface of the liquids, resulting in an interac
`tion between each sequential monomer of the polymer and
`the interface. The sequence of interactions as the monomers
`of the polymer are drawn through the interface is measured,
`yielding information about the sequence of monomers that
`characterize the polymer. The measurement of the interac
`tions can be by a detector that measures the deflection of the
`interface (caused by each monomer passing through the
`interface) using reflected or refracted light, or a sensitive
`gauge capable of measuring intermolecular forces. Several
`methods are available for measurement of forces between
`macromolecules and interfacial assemblies, including the
`surface forces apparatus (Israelachvili. Intermolecular and
`Surface Forces, Academic Press, New York, 1992), optical
`tweezers (Ashkin et al., Oppt. Lett, 11:288, 1986; Kuo and
`Sheetz, Science, 260:232, 1993; Svoboda et al., Nature
`365:721, 1993), and atomic force microscopy (Quate. F.
`Surf Sci. 299:980, 1994; Mate et al., Phys. Rev. Lett.
`59:1942, 1987; Frisbie et al., Science 265:71, 1994; all
`hereby incorporated by reference).
`The interactions between the interface and the monomers
`in the polymer are suitable to identify the size of the
`polymer. e.g., by measuring the length of time during which
`the polymer interacts with the interface as it is drawn across
`the interface at a known rate, or by measuring some feature
`of the interaction (such as deflection of the interface, as
`described above) as each monomer of the polymer is
`sequentially drawn across the interface. The interactions can
`also be sufficient to ascertain the identity of individual
`monomers in the polymer.
`This invention offers advantages particularly in nucle
`otide sequencing, e.g., reduction in the number of sequenc
`ing steps, and increasing the speed of sequencing and the
`length of molecule capable of being sequenced. The speed of
`the method and the size of the polymers it can sequence are
`particular advantages of the invention. The linear polymer
`may be very large, and this advantage will be especially
`useful in reducing template preparation time, sequencing
`errors and analysis time currently needed to piece together
`small overlapping fragments of a large gene or stretch of
`polymer.
`Other features and advantages of the invention will be
`apparent from the following description of the preferred
`embodiments thereof, and from the claims.
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`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a schematic representation of an embodiment of
`DNA characterization by the method of the invention. The
`unobstructed ionic current (illustrated for the channel at the
`top of the diagram), is reduced as a polymeric molecule
`begins its traversal through the pore (illustrated for the
`channel at the bottom of the diagram). The monomeric units
`of the polymer (drawn as different sized ovals on the strand)
`interfere sequentially and differentially with the flow of ions
`through the channel.
`FIG. 2 is a schematic representation of an implementation
`of DNA sequencing by the method of the invention. In this
`embodiment, the polymer is drawn across the opening of the
`channel, but is not drawn through the channel. The channel.
`e.g., a porin, is inserted in the phospholipid bilayer. A
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`polymerase domain is fused by its N-terminus to the
`C-terminus of one of the porin monomers (the porin
`C-termini are on the periplasmic side of the membrane in
`both Rhodobacter capsulatus and LamB porins). Fusions on
`the other side of the membrane can also be made. Malto
`oligosaccharides can bind and block current from either side.
`The polymerase is shown just prior to binding to the
`promoter. A non-glucosylated base is shown near a pore
`opening, while a penta-glucosylated cytosine is shown 10 bp
`away. The polymerase structure represented is that of DNA
`polymerase I (taken from Ollis et al.. 1985. Nature,
`313:762-66), and the general porin model is from Jap (1989.
`J. Mol. Biol, 205:407-19).
`FIG. 3 is a schematic representation of DNA sequencing
`results by the method of the invention. The schematic
`depicts, at very high resolution, one of the longer transient
`blockages such as can be seen in FIG. 4. The monomeric
`units of DNA (bases G. A.T. and C) interfere differentially
`with the flow of ions through the pore, resulting in discrete
`conductance levels that are characteristic of each base. The
`order of appearance of the conductance levels sequentially
`identifies the monomers of the DNA.
`FIG. 4 is a recording of the effect of polyadenylic acid
`(poly A) on the conductance of a single ot-hemolysin chan
`nel in a lipid bilayer between two aqueous compartments
`containing 1 M NaCl, 10 mM Tris, pH 7.4. Before addition
`of RNA, the conductance of the channel was around 850 pS.
`The cis compartment, to which poly A is added, is -120 mV
`with respect to the trans compartment. After adding poly A
`to the cis compartment, the conductance of the o-hemolysin
`channel begins to exhibit transient blockages (conductance
`decreases to about 100 pS) as individual poly A molecules
`are drawn across the channel from the cis to the trans
`compartment. When viewed at higher resolution (expanded
`time scale, at top), the duration of each transient blockage is
`seen to vary between less than 1 msec up to 10 msec. Arrows
`point to two of the longer duration blockages. See FIGS. 5A
`and 5B for histograms of blockage duration.
`FIGS. 5A and 5B are comparisons of blockage duration
`with purified RNA fragments of 320nt (FIG.5A) and 1100nt
`(FIG.5B) lengths. The absolute number of blockades plotted
`in the two histograms are not comparable because they have
`not been normalized to take into account the different
`lengths of time over which the data in the two graphs were
`collected.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`As summarized above, we have determined a new method
`for rapidly analyzing polymers such as DNA and RNA. We
`illustrate the invention with two primary embodiments. In
`one embodiment, the method involves measurements of
`ionic current modulation as the monomers (e.g., nucleotides)
`of a linear polymer (e.g. nucleic acid molecule) pass
`through or across a channel in an artificial membrane.
`During polymer passage through or across the channel, ionic
`currents are reduced in a manner that reflects the properties
`of the polymer (length, concentration of polymers in
`solution, etc.) and the identities of the monomers. In the
`second embodiment, an immiscible interface is created
`between two immiscible liquids, and, as above, polymer
`passage through the interface results in monomer interac
`tions with the interface which are sufficient to identify
`characteristics of the polymer and/or the identity of the
`OOes.
`The description of the invention will be primarily con
`cerned with sequencing nucleic acids, but this is not
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`intended to be limiting. It is feasible to size and sequence
`polymers other than nucleic acids by the method of the
`invention, including linear protein molecules which include
`monomers of amino acids. Other linear arrays of monomers,
`including chemicals (e.g., biochemicals such as
`polysaccharides), may also be sequenced and characterized
`by size.
`I. Polymer Analysis. Using Conductance Changes Across An
`Interface
`Sensitive single channel recording techniques (i.e., the
`patch clamp technique) can be used in the invention, as a
`rapid, high-resolution approach allowing differentiation of
`nucleotide bases of single DNA molecules, and thus a fast
`and efficient DNA sequencing technique or a method to
`determine polymer size or concentration (FIGS. 1 and 2).
`We will describe methods to orient DNA to a pore molecule
`in two general configurations (see FIGS. 1 and 2) and record
`conductance changes across the pore (FIG. 3). One method
`is to use a pore molecule such as the receptor for bacte
`riophage lambda (LamB) or ot-hemolysin, an