EP3500684A1 - Séquençage par nanopores à base optique à l'aide d'agents d'extinction - Google Patents

Séquençage par nanopores à base optique à l'aide d'agents d'extinction

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Publication number
EP3500684A1
EP3500684A1 EP17841831.5A EP17841831A EP3500684A1 EP 3500684 A1 EP3500684 A1 EP 3500684A1 EP 17841831 A EP17841831 A EP 17841831A EP 3500684 A1 EP3500684 A1 EP 3500684A1
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European Patent Office
Prior art keywords
fluorescent
polynucleotide
nanopore
labels
nucleotides
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EP17841831.5A
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German (de)
English (en)
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EP3500684A4 (fr
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Stephen C. Macevicz
Steven Menchen
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Quantapore Inc
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Quantapore Inc
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Publication of EP3500684A4 publication Critical patent/EP3500684A4/fr
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    • 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
    • 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

Definitions

  • optically based nanopore sequencing would be advanced if methods were available for eliminating or reducing optical noise from labels outside detection zones.
  • the present invention is directed to methods and compositions for optically based polynucleotide analysis using nanopores.
  • the invention is directed to the use of quenching agents to reduce or eliminate spurious optical signals generated by polynucleotide labels outside of detection zones.
  • the invention is directed to a method of analyzing a polynucleotide comprising the steps of (a) translocating a polynucleotide through a nanopore, wherein different kinds of nucleotides of the polynucleotide are labeled with different flurorescent labels that generate distinguishable fluorescent signals and wherein the nanopore constrains the nucleotides to move single file through a detection zone; (b) exciting the fluorescent labels; (c) quenching fluorescent signals from excited fluorescent labels outside of the detection zone using a non-fluorescent quenching agent; (d) detecting fluorescent signals emitted from the fluorescent labels as the fluorescent labels pass through the detection zone; and (e)
  • the invention is directed to a method of determining nucleotide sequences of polynucleotides comprising the following steps: (a) providing a nanopore array comprising a solid phase membrane having a first side, a second side, and a plurality of nanopores therethrough, the solid phase membrane separating a first chamber and a second chamber such that each nanopore provides fluid communication between the first chamber and the second chamber, and each nanopore has an exit to the second chamber and is dimensioned to pass a single stranded nucleic acid but not a double stranded nucleic acid and each nanopore constrains the nucleotides of a translocating polynucleotide to move single file through a detection zone at its exit; (b) translocating polynucleotides from the first chamber to the second chamber through the nanopores, each polynucleotide having one or more fluorescent labels attached to nucleotides thereof, such that different kinds of nucleotides are labeled with different
  • the present invention advantageously overcomes the above problems in the field of optically based nanopore sequencing by providing quenching agents to suppress or eliminate unwanted optical noise in the form of fluorescent signals generated by fluorescent labels outside of a detection zone.
  • Figs. 1A-1B illustrate polynucleotides with fluorescent labels and sources of optical noise when such polynucleotides are analyzed using a nanopore.
  • FIGs. 2A-2C illustrate embodiments of the invention employing quenching agents in a trans chamber, a cis chamber and in both cis and trans chambers.
  • FIG. 3 illustrates an embodiment of the invention using a protein nanopore and epi- illumination with a metal layer on the nanopore array to reduce background or TIR with FRET excitation.
  • Fig. 4 illustrates the basic components of a confocal epi-illumination system.
  • Fig. 5 illustrates elements of a TIRF system for excitation of optical labels in or near a nanopore array without FRET signal generation.
  • Fig. 6 is a flow chart illustrating a step for calling nucleotide sequences based on measurements of optical signals comprising light from multiple optical labels.
  • Figs. 7A-7C illustrate embodiments employing two and three fluorescent labels.
  • the invention is directed to methods and devices for analyzing polynucleotides, such as DNA, RNA, and the like, using nanopores and optical detection. More particularly, the invention is directed to determining nucleotide sequences of one or more polynucleotides using nanopores and optical detection. In one aspect, the invention is directed to, and includes, reducing optical noise in collected signals by using of non-fluorescent quenching agents to suppress and/or eliminate fluorescent signals from fluorescent labels excited outside of desired detection zones.
  • Figs. 1A and IB illustrate diagrammatically a problem addressed by this aspect of the invention.
  • a labeled polynucleotide such as illustrated by (100) is generated from a target
  • polynucleotide which may be obtained from a sample. As represented, a portion of nucleotides of labeled polynucleotide (100) has attached (for example) one of two fluorescent labels, "f ' (102) or "g" (104). Labeled polynucleotide (100) and/or similarly labeled polynucleotides are translocated through a nanopore, such as that represented by nanopore (106) in solid phase membrane (108). Nanopores may have a wide variety of formats and compositions, as described more fully below, but are employed to carry out substantially the same function, namely, to constrain nucleotides of a label polynucleotide to move single file through a detection zone.
  • nanopores may perform additional functions, including (i) passing single stranded polynucleotides but not double stranded polynucleotides, and/or (ii) suppressing and/or constraining fluorescent emissions of fluorescent labels during transit of the nanopore.
  • Fig. IB illustrates labeled polynucleotide (100) translocating through nanopore (106) from cis chamber (110) to trans chamber (112) and being illuminated by excitation beam (114), which could be a direct illumination via epi-illumination or evanescent wave illumination.
  • the invention addresses this challenge by providing non-fluorescent quenching agents with affinity to labeled single stranded polynucleotides to reduce or eliminate fluorescent signals from regions outside of desired detection region or zone (124).
  • non-fluorescent quenching agents with affinity to labeled double stranded polynucleotides may be provided to reduce or eliminate fluorescent signals from regions outside of a desired detection zone or region.
  • FIGs. 2A-2C illustrate different embodiments of the invention corresponding to where quenching agents are applied in a nanopore device: trans chamber only (Fig. 2A), cis chamber only (Fig. 2B), or both cis and trans chambers (Fig. 2C).
  • labeled polynucleotide (200) is illustrated translocating nanopore (206) of solid phase membrane (208) from cis chamber (202) to trans chamber (204).
  • Immersed in trans chamber (204) are non- fluorescent quenching agents (205) designated by "Q”.
  • Quenching agents of the invention are soluble under translocation conditions for labeled single stranded polynucleotide (200), and under the same conditions, quenching agents bind to single stranded polynucleotides, such as (200), without substantial sequence specificity.
  • quenching agents of the invention are soluble under translocation conditions for labeled double stranded polynucleotides, and under the same conditions, quenching agents bind to such double stranded polynucleotides, without substantial sequence specificity
  • quenching agents include derivatives of many well-known organic dyes, such as asymmetric cyanine dyes, as well as conjugates of such compounds and oligonucleotides and/or analogs thereof.
  • selection of the type and concentration of quenching agent and the translocation speed define detection zone (210).
  • "detection zone” means a region or volume (which may be contiguous or non-contiguous) from which fluorescent signals are collected to form the raw data from which information, such as sequence information, about a labeled polynucleotide is determined.
  • a "detection zone” is a contiguous region or volume.
  • Fluorescent labels in trans chamber (204) outside of detection zone (210) are substantially quenched by quenching agents (205) bound to the portion of labeled polynucleotide (200) in trans chamber (204).
  • quenching agents comprise an oligonucleotide or analog conjugated to one or more quenching moieties based on organic dyes as described more fully below.
  • Embodiments of Fig. 2A may be employed when, for example, solid phase membrane (208) is or comprises an opaque layer so that fluorescent labels in cis chamber (202) are substantially non-excited.
  • the volume of a detection zone e.g. adjacent to a nanopore exit to the trans chamber, may depend on factors including, but not limited to, translocation speed of the labeled polynucleotide, quenching agent concentration in the trans chamber, affinity of the quenching agent for the polynucleotide, the quenching efficiency of the quenching agent, and the like.
  • Fig. 2B shows substantially the same elements as those in Fig. 2A with the exception that quenching agents (205) are disposed in cis chamber (202). This configuration may be desirable under circumstances where undesired evanescent waves, or like non-radiative light energy, extend to cis chamber (202) and excite fluorescent labels which generate fluorescent signals that are collected. Quenching agents (205) that bind to labeled
  • quenching agents (205) and cross-section of nanopore (206) are selected so that quenching agents (205) are excluded from translocating through nanopore (206). In some embodiments, this may be achieved by using protein nanopore a-hemolysin and quenching agents comprising conjugates of oligonucleotides or analogs thereof and one or more quenching compounds, as described more fully below.
  • Fig. 2C illustrates an embodiment where quenching agents (205) are present in both cis chamber (202) and trans chamber (204), which provides the advantages described for the embodiments of both Figs. 2A and 2B.
  • Fig. 3 illustrates an embodiment which includes the following elements: protein nanopore (300) disposed in lipid bilayer (302); epi-illumination of fluorescent labels with opaque layer (308) in solid phase membrane (306) to prevent or reduce background fluorescence; and quenching agents (310) disposed in trans chamber (326).
  • polynucleotide (320) with fluorescently labeled nucleotides labels being indicated by "f ', as with (322)
  • Oligonucleotide quenchers (310) are disposed in trans chamber (326) under conditions (e.g.
  • Nanopore (300) may be selected so that signals from fluorescent labels are suppressed during transit of the nanopore as described in Huber et al, U.S. patent publication US
  • a fluorescent signal from a single fluorescent label is detected from detection zone (328) during a detection period as the labeled polynucleotide moves through the detection zone.
  • a plurality of fluorescent signals is collected from a plurality of fluorescent labels in detection zone (328) during a predetermined time period.
  • detection period is less than 1 msec, or less than 0.1 msec, or less than 0.01 msec.
  • detection perior is at least 0.01 msec, or at least 0.1 msec, or at least 0.5 msec.
  • Quenching agents of the invention comprise any compound (or set of compounds) that under nanopore sequencing conditions is (i) substantially non-fluorescent, (ii) binds to single stranded nucleic acids, particularly single stranded DNA, and (iii) absorbs excitation energy from other molecules non-radiatively and releases it non-radiatively.
  • quenching agents further bind non-covalently to single stranded DNA.
  • a large variety of quenching compounds are available for use with the invention including, but not limited to, non-fluorescent derivatives of common synthetic dyes such as cyanine and xanthene dyes, as described more fully below. Guidance in selecting quenching compounds may be found in U.S. patents 6,323,337; 6,750,024 and like references, which are incorporated herein by reference.
  • a quenching agent may be a single stranded DNA binding dye that has been covalently modified with a heavy atom that is known to quench fluorescence (such as bromine or iodine), or covalently modified with other groups known to quench fluorescence, such as a nitro group or a azo group.
  • a heavy atom that is known to quench fluorescence (such as bromine or iodine)
  • other groups known to quench fluorescence such as a nitro group or a azo group.
  • An example of dye that is known to bind single stranded DNA is Sybr Green (Zipper et al, (2004), Nucleic Acids Research. 32 (12)).
  • quenching agents comprise a binding moiety and one or more quenching moieties.
  • Binding moieties may include any compound that binds to single stranded nucleic acids without substantial sequence specificity. Binding moieties may comprise peptides or oligonucleotides or analogs of either having modified linkages and/or monomers. Oligonucleotides and their analogs may provide binding to polynucleotides via duplex formation or via non-base paired aptameric binding. In some embodiments, binding moieties comprise an oligonucleotide or analog thereof having a length in the range of from 6 to 60 nucleotides.
  • Such oligonucleotides or analogs may be conjugated to one quenching moiety or to a plurality of quenching moieties.
  • the plurality of quenching moieties conjugated to each oligonucleotide or analog is 2 or 3.
  • Quenching moieties conjugated to a binding moiety may be the same or different.
  • a binding moiety is an oligonucleotide or analog
  • two quenching moieties are conjugated thereto, one at a 5' end and one at a 3' end of the oligonucleotide.
  • Oligonucleotides or analogs having from 2 to 3 quenching moieties may be synthesized using conventional linkage and synthetic chemistries, for example, as disclosed in the references cited herein.
  • Quenching agents comprising single stranded oligonucleotides that bind in a cis chamber may be advantageous in that in some nanopore configurations, such as that illustrated with protein nanopore (300) in Fig. 3, the nanopore may be selected to be sufficiently narrow that it translocates only single stranded nucleic acids; thus, during translocation the nanopore, in effect, strips off bound quenching agents as translocation progresses.
  • a nanopore may be selected that suppresses signal generation (or detection) of fluorescent labels during nanopore transit, followed by free signal generation (or detectability) upon exit into a detection zone (for example (328) in Fig. 3).
  • Oligonucleotides or analogs may be provided as a single species or they may be provided as mixtures of a plurality of oligonucleotides or analogs with different sequences, and therefore, different binding specificities.
  • oligonucleotides or analogs are random sequence polymers; that is, they are provided as mixtures of every possible sequence of a given length.
  • such oligonucleotides or analogs may be represented by the formulas, "NNNNNN” for 6-mers, or "NNNNNNNN” for 8-mers, wherein N may be A, C, G or T, or an analog thereof.
  • oligonucleotides in reference to oligonucleotides means an oligonucleotide that contains one or more nucleotide analogs.
  • a "nucleotide analog” is a nucleotide that may have a modified linkage moiety, sugar moiety or base moiety.
  • oligonucleotide analogs that may be used with the invention include, but are not limited to, peptide nucleic acids (PNAs), locked nucleic acids (LNAs)(2'-0-methyl RNA), phosphorothioate oligonucleotides, bridged nucleic acids (BNAs), or the like.
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • BNAs bridged nucleic acids
  • oligonucleotide binding moieties comprise universal bases; that is, they contain one or more nucleotide analogs that can replace any of the four natural nucleotides without destabilizing base-pair interactions. Nucleotide analogs having universal base properties are described in Loakes, Nucleic Acids Research, 29(12): 2437-2447 (2001), which is incorporated herein by reference. In some embodiments, oligonucleotide binding moieties comprise 2'-deoxyinosine, 7-deaza-2'-deoxyinosine, 2-aza-2'-deoxyinosine, 3- nitropyrrole nucleotides, 5-nitroindole nucleotides, or the like.
  • quenching agents may comprise a combination of two or more compounds that act together to quench undesired fluorescent signals of a single stranded labeled polynucleotide.
  • a quenching agent may comprise an oligonucleotide (e.g., polydeoxyinosine) that may form a duplex with the labeled polynucleotide and separately a double stranded intercalator that is a quencher.
  • any synthetic dye that can detectably quench fluorescent signals of the fluorescent labels of a labeled polynucleotide is an acceptable quenching moiety for the purposes of the invention.
  • the quenching moieties possess an absorption band that exhibits at least some spectral overlap with an emission band of the fluorescent labels on a labeled polynucleotide.
  • This overlap may occur with emission of the fluorescent label (donor) occurring at a lower or even higher wavelength emission maximum than the maximal absorbance wavelength of the quenching moiety (acceptor), provided that sufficient spectral overlap exists. Energy transfer may also occur through transfer of emission of the donor to higher electronic states of the acceptor.
  • One of ordinary skill in the art determines the utility of a given quenching moiety by examination of that dye's excitation bands with respect to the emission spectrum of the fluorescent labels being used.
  • fluorescence quenching in the invention occurs through Fluorescence Resonance Energy Transfer (FRET or through the formation of charge transfer complexes) between a fluorescent label and a quenching moiety of the invention.
  • FRET Fluorescence Resonance Energy Transfer
  • the spectral and electronic properties of the donor and acceptor compounds have a strong effect on the degree of energy transfer observed, as does the separation distance between the fluorescent labels on the labeled polynucleotide and the quenching moiety. As the separation distance increases, the degree of fluorescence quenching decreases.
  • a quenching moiety may be optionally fluorescent, provided that the maximal emission wavelength of the dye is well separated from the maximal emission wavelength of the fluorescent labels when bound to labeled polynucleotides.
  • the quenching moiety is only dimly fluorescent, or is substantially non-fluorescent, when covalently conjugated to a oligonucleotide or analog.
  • substantially non-fluorescent indicates that the fluorescence efficiency of the quenching moiety in an assay solution as described for any of the methods herein is less than or equal to 5 percent, preferably less than or equal to 1 percent.
  • the covalently bound quenching moiety exhibits a quantum yield of less than about 0.1, more preferably less than about 0.01.
  • oligonucleotide of the invention is quenched more than 50% relative to the same
  • the fluorescent labels are quenched more than 90% relative to the unlabeled oligonucleotide.
  • the nucleic acid stains are quenched more than 95% relative to the unlabeled oligonucleotide.
  • a quenching moiety may be a pyrene, an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7-nitrobenz-2-oxa-l,3-diazole (NBD), a cyanine, a carbocyanine, a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a coumarin (including hydroxycoumarins and aminocoumarins and fluorinated and sulfonated derivatives thereof (as described in U.S.
  • quenching moieties that are substantially non-fluorescent dyes include in particular azo dyes (such as DABCYL or DABSYL dyes and their structural analogs), triarylmethane dyes such as malachite green or phenol red, 4',5z-diether substituted fluoresceins (U.S. Pat. No. 4,318,846 (1982)), or asymmetric cyanine dye quenchers (PCT Int. App. WO 99 37,717 (1999)).
  • azo dyes such as DABCYL or DABSYL dyes and their structural analogs
  • triarylmethane dyes such as malachite green or phenol red
  • 4',5z-diether substituted fluoresceins U.S. Pat. No. 4,318,846 (1982)
  • PCT Int. App. WO 99 37,717 (1999) PCT Int. App. WO 99 37,717 (1999)
  • the synthetic dye is optionally a fluorescein, a rhodol (U.S. Pat. No. 5,227,487 to Haugland, et al. (1993), incorporated by reference), or a rhodamine.
  • fluorescein includes benzo- or dibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.
  • rhodol includes seminaphthorhodafluors (U.S. Pat. No. 4,945,171 to Haugland, et al.
  • Xanthenes include fluorinated derivatives of xanthene dyes (Int. Publ. No. WO 97/39064, Molecular Probes, Inc. (1997), incorporated by reference), and sulfonated derivatives of xanthene dyes (Int. Publ. No. WO 99/15517, Molecular Probes, Inc. (1999), incorporated by reference).
  • oxazines include resorufms
  • the quenching moiety is an substantially nonfluorescent derivative of 3- and/or 6-amino xanthene that is substituted at one or more amino nitrogen atoms by an aromatic or heteroaromatic ring system, e.g. as described in U.S. patent
  • quenching dyes typically have absorption maxima above 530 nm, have little or no observable fluorescence and efficiently quench a broad spectrum of luminescent emission, such as is emitted by chemilumiphores, phosphors, or fluorophores.
  • the quenching dye is a substituted rhodamine.
  • the quenching compound is a substituted rhodol.
  • a quenching moiety may comprise one or more non- fluorescent quenchers known as Black Hole Quenchers TM compounds (BHQs) described in the following patents, which are incorporated herein by reference: 7,019,129; 7,109,312; 7,582,432; 8,410,025; 8,440,399; 8,633,307; 8,946,404; 9,018,369; or 9,139,610.
  • BHQs Black Hole Quenchers TM compounds
  • an epi-illumination system in which excitation beam delivery and optical signal collection occurs through a single objective, may be used for direct illumination of labels on a polymer analyte or donors on nanopores.
  • the basic components of a confocal epi-illumination system for use with the invention is illustrated in Fig. 4.
  • Excitation beam (402) is directed to dichroic (404) and onto (412) objective lens (406) which focuses (410) excitation beam (402) onto layered membrane (400), in which labels are excited directly to emit an optical signal, such as a fluorescent signal, or are excited indirectly via a FRET interaction to emit an optical signal.
  • optical signal is collected by objective lens (406) and directed to dichroic (404), which is selected so that it passes light of optical signal (411) but reflects light of excitation beam (402).
  • Optical signal (411) passes through lens (414) which focuses it through pinhole (416) and onto detector (418).
  • optical signal (411) comprises fluorescent signals from multiple fluorescent labels further optical components, filters, beam splitters, monochromators, or the like, may be provided for further separating the different fluorescent signals from different fluorescent labels.
  • labels on nucleotides may be excited by an evanescence field using an apparatus similar to that shown in Fig. 5, described in Soni et al, Review of Scientific Instruments, 81: 014301 (2010); and in U.S.
  • Array of apertures (500) (which may include protein nanopores inserted in a lipid bilayer), may be formed in silicon nitride layer (502), which may have a thickness in the range of from 20-100 nm. Silicon nitride layer (502) may be formed on a silicon support layer (503).
  • Second chamber (506) may be formed by silicon nitride layer (502), silicon dioxide layer (504) which determines the height of second chamber (506), and surface (508) of glass slide (510). Silicon dioxide layer (504) may have a thickness in the range of from 50-100 nm.
  • a desired evanescent field (507) extending from surface (508) across silicon nitride layer (502) may be established by directing light beam (512) at an appropriate angle relative to glass slide (510) so that TIR occurs.
  • cis(-) conditions may be established in first chamber (516) and trans(+) conditions may be established in second chamber (506) with electrodes operationally connected to first and second chambers (506 and 521).
  • a series of optical signals may be measured from a resolution limited area wherein each optical measurement comprises a plurality of component signals from different adjacent nucleotides (whose order in the polynucleotide cannot be determined from a single measurement because, for example, the component signals are generated from within a diffraction limited area).
  • optically-based nanopore analysis of polynucleotides (i) generates a time series of optical measurements that comprise overlapping contributions from sequences of more than one labeled nucleotide, thereby making it difficult, if not impossible, to determine an ordering of the nucleotides from a single measurement, and (ii) by selecting optical labels for nucleotides which generate distinguishable signals, the optical measurements can be separated into contributions from different labels on different kinds of nucleotides, which allows overlapping measurements to be converted into sequence information.
  • a method of the invention may be implemented by the following steps: (a) translocating a polynucleotide through a nanopore, wherein different kinds of nucleotides of the polynucleotide are labeled with different optical labels that generate distinguishable optical signals and wherein the nanopore constrains the nucleotides to move single file through a detection zone that encompasses a plurality of nucleotides, wherein the detection zone is characterized by an absence of quenching agents bound to the
  • a time-ordered set of optical measurements are recorded.
  • Optical measurements at adjacent time points are overlapping in the sense that each optical measurement contains contributions from labels of adjacent nucleotides.
  • three nucleotides generate signals at each time point (for example, B, C and D of polynucleotide ...-A-(B-C-D)-...
  • sequence information may be determined from the time-ordered set of optical signal measurements when it is separated into a plurality of time- ordered sets of nucleotide-specific signals. Algorithms similar to those used in sequencing-by- hybridization (SBH) to reconstruct target polynucleotide sequences from hybridization data may be used to reconstruct target polynucleotides here, e.g. U.S.
  • Fig. 6 illustrates one embodiment of a step for determining nucleotide sequence information from a time-ordered set of overlapping optical signals based on a simple model of nanopore translocation.
  • the simple model assumes that optical measurements at each time step (except at the entry and exit of a polynucleotide from a nanopore) each contain signal contributions from the same number of nucleotides (referred to in Fig. 6 as an "n-tuple" to indicate that a measurement would contain contributions from n nucleotides). It is understood that more complex models may allow for differing numbers of contributing nucleotides in each measurement, for local variations in translocation speed, deviations in linear movement of nucleotides, and other like phenomena.
  • optical measurements at different times may have contributions from different numbers of nucleotides.
  • the differing number of nucleotides are ordered along a segment of the target polynucleotide.
  • the step of determining illustrated by Fig. 6 assumes that a labeled polynucleotide has passed through a nanopore and that a time ordered set of optical measurements has been made, including separation of optical signals into nucleotide-specific signals (600). The entry and exit of a polynucleotide are treated differently since there are necessarily different numbers of nucleotides in the detection zone(s) upon entry and exit.
  • preparation of labeled polynucleotides for analysis may include insertion of a plurality of predetermined labeled nucleotides at one or both ends of such labeled polynucleotides for the purpose of generating a known sequence of optical signals to aid in a sequence determination step.
  • predetermined labeled nucleotides would be similar to key sequences in Ion Torrent or 454 sequencing, e.g. U.S. patent 7,575,865, which is incorporated by reference.
  • time index, i is set to zero; the index, j, for candidate sequences at the current time, i, is set to 1 (602); and the initial n-tuple of the set of nucleotide-specific time-ordered optical signals is examined (604).
  • Such examination comprises first determining from the measurement at time i all possible n-tuples of nucleotides that are consistent with the measurement, then determining from those n-tuples which ones that properly overlap candidate sequence Si. New candidate sequences Si+1 are formed (and a sequence Si is extended) by each properly overlapping n-tuple for the set consistent with the measurement (606).
  • New extended candidate sequences, Si+1, are stored and the index giving the number of candidate sequences at time i+1, Ji+1, is updated (608). This step is repeated until every candidate sequence, Si, has been examined (610), and a similar examination is carried out at each time, i, until each optical measurement in the time- ordered set has been examined.
  • Nanopores used with the invention may be solid-state nanopores, protein nanopores, or hybrid nanopores comprising protein nanopores or organic nano tubes such as carbon or graphene nanotubes, configured in a solid-state membrane, or like framework.
  • Important features of nanopores include constraining polynucleotide analytes, such as labeled polynucleotides so that their monomers pass through a signal generation region (or equivalently, an excitation zone, or detection zone, or the like) in sequence.
  • a nanopore contrains the movement of a polynucleotide analyte, such as a polynucleotide, so that nucleotides pass through a detection zone (or excitation region) in single file.
  • additional functions of nanopores include (i) passing single stranded nucleic acids while not passing double stranded nucleic acids, or equivalently bulky molecules and/or (ii) constraining fluorescent labels on nucleotides so that fluorescent signal generation is suppressed or directed so that it is not collected.
  • nanopores used in connection with the methods and devices of the invention are provided in the form of arrays, such as an array of clusters of nanopores, which may be disposed regularly on a planar surface.
  • clusters are each in a separate resolution limited area so that optical signals from nanopores of different clusters are distinguishable by the optical detection system employed, but optical signals from nanopores within the same cluster cannot necessarily be assigned to a specific nanopore within such cluster by the optical detection system employed.
  • Solid state nanopores may be fabricated in a variety of materials including but not limited to, silicon nitride (S1 3 N 4 ), silicon dioxide (S1O 2 ), and the like.
  • the fabrication and operation of nanopores for analytical applications, such as DNA sequencing, are disclosed in the following exemplary references that are incorporated by reference: Ling, U.S. patent 7,678,562; Hu et al, U.S. patent 7,397,232; Golovchenko et al, U.S. patent 6,464,842; Chu et al, U.S. patent 5,798,042; Sauer et al, U.S. patent 7,001,792; Su et al, U.S.
  • the invention comprises nanopore arrays with one or more light-blocking layers, that is, one or more opaque layers.
  • nanopore arrays are fabricated in thin sheets of material, such as, silicon, silicon nitride, silicon oxide, aluminum oxide, or the like, which readily transmit light, particularly at the thicknesses used, e.g. less than 50-100 nm. For electrical detection of analytes this is not a problem.
  • light transmitted through an array invariably excites materials outside of intended reaction sites, thus generates optical noise, for example, from nonspecific background fluorescence, fluorescence from labels of molecules that have not yet entered a nanopore, or the like.
  • an opaque layer may be a metal layer.
  • metal layer may comprise Sn, Al, V, Ti, Ni, Mo, Ta, W, Au, Ag or Cu.
  • metal layer may comprise Al, Au, Ag or Cu.
  • such metal layer may comprise aluminum or gold, or may comprise solely aluminum.
  • the thickness of an opaque layer may vary widely and depends on the physical and chemical properties of material composing the layer. In some embodiments, the thickness of an opaque layer may be at least 5 nm, or at least 10 nm, or at least 40 nm. In other embodiments, the thickness of an opaque layer may be in the range of from 5-100 nm; in other embodiments, the thickness of an opaque layer may be in the range of from 10-80 nm.
  • An opaque layer need not block (i.e. reflect or absorb) 100 percent of the light from an excitation beam. In some embodiments, an opaque layer may block at least 10 percent of incident light from an excitation beam; in other embodiments, an opaque layer may block at least 50 percent of incident light from an excitation beam.
  • Opaque layers or coatings may be fabricated on solid state membranes by a variety of techniques known in the art.
  • Material deposition techniques may be used including chemical vapor deposition, electrodeposition, epitaxy, thermal oxidation, physical vapor deposition, including evaporation and sputtering, casting, and the like.
  • atomic layer deposition may be used, e.g. U.S. patent 6,464,842; Wei et al, Small, 6(13): 1406- 1414 (2010), which are incorporated by reference.
  • a 1-100 nm channel or aperture may be formed through a solid substrate, usually a planar substrate, such as a membrane, through which an analyte, such as single stranded DNA, is induced to translocate.
  • a 2-50 nm channel or aperture is formed through a substrate; and in still other embodiments, a 2-30 nm, or a 2-20 nm, or a 3-30 nm, or a 3-20 nm, or a 3-10 nm channel or aperture if formed through a substrate.
  • Biological nanopores provide reproducible narrow bores, or lumens, especially in the 1- 10 nanometer range, as well as techniques for tailoring the physical and/or chemical properties of the nanopore and for directly or indirectly attaching groups or elements, such as fluorescent labels, which may be FRET donors or acceptors, by conventional protein engineering methods.
  • Solid- state nanopores may be combined with a biological nanopore to form a so-called “hybrid" nanopore that overcomes some of these shortcomings, thereby providing the precision of a biological pore protein with the stability of a solid state nanopore.
  • hybrid nanopore provides a precise location of the nanopore which simplifies the data acquisition greatly.
  • clusters may also be formed by disposing protein nanopores in lipid bilayers supported by solid phase membrane containing an array of apertures.
  • an array may comprise apertures fabricated (e.g. drilled, etched, or the like) in solid phase support.
  • the geometry of such apertures may vary depending on the fabrication techniques employed.
  • each such aperture is associated with, or encompassed by, a separate resolution limited area; however, in other embodiments, multiple apertures may be within the same resolution limited area.
  • the cross-sectional area of the apertures may vary widely and may or may not be the same as between different clusters, although such areas are usually substantially the same as a result of conventional fabrication approaches.
  • apertures have a minimal linear dimension (e.g. diameter in the case of circular apertures) in the range of from 10 to 200 nm, or have areas in the range of from about 100 to 3x104 nm2. Across the apertures may be disposed a lipid bilayer.
  • the distribution of protein nanopores per aperture may be varied, for example, by controlling the concentration of protein nanopores during inserting step.
  • clusters of nanopores may comprise a random number of nanopores.
  • clusters containing one or more apertures on average have a number of protein nanopores that is greater than zero; in other embodiments, such clusters have a number of protein nanopores that is greater than 0.25; in other
  • such clusters have a number of protein nanopores that is greater than 0.5; in other embodiments, such clusters have a number of protein nanopores that is greater than 0.75; in other embodiments, such clusters have a number of protein nanopores that is greater than 1.0.
  • methods and devices of the invention comprise a solid phase membrane, such as a SiN membrane, having an array of apertures therethrough providing communication between a first chamber and a second chamber (also sometimes referred to as a "cis chamber” and a “trans chamber”) and supporting a lipid bilayer on a surface facing the second, or trans, chamber.
  • a solid phase membrane such as a SiN membrane
  • diameters of the aperture in such a solid phase membrane may be in the range of 10 to 200 nm, or in the range of 20 to 100 nm.
  • such solid phase membranes further include protein nanopores inserted into the lipid bilayer in regions where such bilayer spans the apertures on the surface facing the trans chamber.
  • such protein nanopores are inserted from the cis side of the solid phase membrane using techniques described herein.
  • such protein nanopores have a structure identical to, or similar to, a-hemolysin in that it comprises a barrel, or bore, along an axis and at one end has a "cap” structure and at the other end has a “stem” structure (using the terminology from Song et al, Science, 274: 1859-1866 (1996)).
  • insertion into the lipid bilayer results in the protein nanopore being oriented so that its cap structure is exposed to the cis chamber and its stem structure is exposed to the trans chamber.
  • the present invention may employ hybrid nanopores in clusters, particularly for optical-based nanopore sequencing of polynucleotides.
  • Such nanopores comprise a solid-state orifice, or aperture, into which a protein biosensor, such as a protein nanopore, is stably inserted.
  • a charged polynucleotide may be attached to a protein nanopore (e.g. alpha hemolysin) by conventional protein engineering techniques after which an applied electric field may be used to guide a protein nanopore into an aperture in a solid- state membrane.
  • the aperture in the solid-state substrate is selected to be slightly smaller than the protein, thereby preventing it from translocating through the aperture. Instead, the protein will be embedded into the solid-state orifice.
  • Solid state, or synthetic, nanopores may be preprared in a variety of ways, as exemplified in the references cited above.
  • a helium ion microscope may be used to drill the synthetic nanopores in a variety of materials, e.g. as disclosed by Yang et al, Nanotechnolgy, 22: 285310 (2011), which is incorporated herein by reference.
  • a chip that supports one or more regions of a thin-film material, e.g. silicon nitride, that has been processed to be a free-standing membrane is introduced to the helium ion microscope (HIM) chamber.
  • HIM helium ion microscope
  • HIM motor controls are used to bring a free-standing membrane into the path of the ion beam while the microscope is set for low magnification. Beam parameters including focus and stigmation are adjusted at a region adjacent to the free-standing membrane, but on the solid substrate. Once the parameters have been properly fixed, the chip position is moved such that the free-standing membrane region is centered on the ion beam scan region and the beam is blanked.
  • the HIM field of view is set to a dimension (in ⁇ ) that is sufficient to contain the entire anticipated nanopore pattern and sufficient to be useful in future optical readout (i.e. dependent on optical magnification, camera resolution, etc.).
  • the ion beam is then rastered once through the entire field of view at a pixel dwell time that results in a total ion dose sufficient to remove all or most of the membrane autofluorescence.
  • the field of view is then set to the proper value (smaller than that used above) to perform lithographically-defined milling of either a single nanopore or an array of nanopores.
  • the pixel dwell time of the pattern is set to result in nanopores of one or more predetermined diameters, determined through the use of a calibration sample prior to sample processing. This entire process is repeated for each desired region on a single chip and/or for each chip introduced into the HIM chamber.
  • a device for implementing the above methods for analyzing polynucleotides typically includes a set of electrodes for establishing an electric field across the layered membrane and nanopores.
  • Single stranded nucleic acids are exposed to nanopores by placing them in an electrolyte in a first chamber, which is configured as the "cis" side of the layered membrane by placement of a negative electrode in the chamber.
  • the negatively single stranded nucleic acids are captured by nanopores and translocated to a second chamber on the other side of the layered membrane, which is configured as the "trans" side of membrane by placement of a positive electrode in the chamber.
  • the speed of translocation depends in part on the ionic strength of the electrolytes in the first and second chambers and the applied voltage across the nanopores.
  • a translocation speed may be selected by preliminary calibration measurements, for example, using predetermined standards of labeled single stranded nucleic acids that generate signals at different expected rates per nanopore for different voltages.
  • a translocation speed may be selected based on the signal rates from such calibration measurements. Consequently, from such measurements a voltage may be selected that permits, or maximizes, reliable nucleotide identifications, for example, over an array of nanopores.
  • such calibrations may be made using nucleic acids from the sample of templates being analyzed (instead of, or in addition to, predetermined standard sequences). In some embodiments, such calibrations may be carried out in real time during a sequencing run and the applied voltage may be modified in real time based on such measurements, for example, to maximize the acquisition of nucleotide- specific signals.
  • Controlling translocation speeds of polynucleotides through nanopores is necessary to permit collection of data from which sequence information can be obtained. Translocation speeds depend in part on the voltage difference (or electrical field strength) across a nanopore and conditions in the reaction mixture of the first chamber where nucleic acid polynucleotides are exposed to the nanopores (e.g. disposed in a solid phase membrane making up one wall of the first chamber). Nucleic acid polynucleotide capture rates by nanopores depend on concentration of such polynucleotides.
  • conventional reaction mixture conditions for nanopore sequencing may be employed with the invention, for example, 1M KCl (or equivalent salt, such as NaCl, LiCl, or the like) and a pH buffering system (which, for example, ensures that proteins being used, e.g. protein nanopores, nucleases, or the like, are not denatured).
  • a pH buffering system may be used to keep the pH substantially constant at a value in the range of 6.8 to 8.8.
  • a voltage difference across the nanopores may be in the range of from 70 to 200 mV. In other embodiments, a voltage difference across the nanopores may be in the range of from 80 to 150 mV.
  • An appropriate voltage for operation may be selected using conventional measurement techniques. Current (or voltage) across a nanopore may readily be measured using commercially available instruments. A voltage difference may be selected so that
  • translocation speed is within a desired range.
  • a range of translocation speeds comprises those speeds less than 1000 nucleotides per second.
  • a range of translocation speeds is from 10 to 800 nucleotides per second; in other embodiments, a range of translocation speeds is from 10 to 600 nucleotides per second; in other
  • a range of translocation speeds is from 200 to 800 nucleotides per second; in other embodiments, a range of translocation speeds is from 200 to 500 nucleotides per second.
  • self- and mutually quenching fluorescent labels may be used in addition to quenching agents in order to reduce fluorescent emissions outside of those from labels on nucleotides exiting nanopores.
  • Use of such fluorescent labels is disclosed in U.S. patent publication 2016/0122812, which is incorporated by reference.
  • monomers are labeled with fluorescent labels that are capable of at least three states while attached to a target polynucleotide: (i) A substantially quenched state wherein fluorescence of an attached fluorescent label is quenched by a fluorescent label on an immediately adjacent monomer; for example, a fluorescent label attached to a polynucleotide in accordance with the invention is substantially quenched when the labeled polynucleotide is free in conventional aqueous solution for studying and manipulating the polynucleotide, (ii) A sterically constrained state wherein a labeled polynucleotide is translocating through a nanopore such that the free- solution movements or alignments of an attached fluorescent label is disrupted or limited so that there is little or no detectable fluorescent signal generated from the fluorescent label, (iii) A transition state wherein a fluorescent label attached to a polynucleotide transitions from the sterically constrained state to the quenched state as the fluorescent label exits the nano
  • this example is an application of the discovery that during the transition interval a fluorescent label (on an otherwise substantially fully labeled and self-quenched polynucleotide) is capable of generating a detectable fluorescent signal.
  • a fluorescent label on an otherwise substantially fully labeled and self-quenched polynucleotide
  • the fluorescent signal generated during the transition interval is due to the presence of a freely rotatable dipole in the fluorescent label emerging from the nanopore, which renders the fluorescent label temporarily capable of generating a fluorescent signal, for example, after direct excitation or via FRET.
  • the dipoles are limited in their rotational freedom thereby reducing or limiting the number of emitted photons.
  • the polynucleotide is a polynucleotide, usually a single stranded polynucleotide, such as, DNA or RNA, but especially single stranded DNA.
  • the invention includes a method for determining a nucleotide sequence of a polynucleotide by recording signals generated by attached fluorescent labels as they exit a nanopore one at a time as a polynucleotide translocates through the nanopore. Upon exit, each attached fluorescent label transitions during a transition interval from a constrained state in the nanopore to a quenched state on the polynucleotide in free solution.
  • a step of the method of the invention comprises exciting each fluorescent label as it is transitioning from a constrained state in the nanopore to a quenched state on the polynucleotide in free solution.
  • the fluorescent label is capable of emitting a detectable fluorescent signal indicative of the nucleotide it is attached to.
  • the invention includes an application of the discovery that fluorescent labels and nanopores may be selected so that during translocation of a
  • nanopores are selected that have a bore, or lumen, with a diameter in the range of from 1 to 4 nm; in other embodiments, nanopores are selected that have a bore or lumen with a diameter in the range of from 2 to 3 nm. In some embodiments, such bore diameters are provided by a protein nanopore.
  • such nanopores are used to force fluorescent labels into a constrained state in accordance with the invention, so that whenever a fluorescent label exits a nanopore, it transitions from being substantially incapable of generating a fluorescent signal to being detectable and identifiable by a fluorescent signal it can be induced to emit.
  • fluorescent labels attached to each of a sequence of monomers of a polynucleotide may be detected in sequence as they suddenly generate a fluorescent signal in a region immediately adjacent to a nanopore exit (a "transition zone” or “transition volume” or “detection zone”).
  • organic fluorescent dyes are used as fluorescent labels with nanopores of the above diameters.
  • At least one such organic fluorescent dye is selected from the set consisting of xanthene dyes, rhodamine dyes and cyanine dyes.
  • Some embodiments for determining a monomer sequence of a polynucleotide may be carried out with the following steps: (a) translocating a polynucleotide through a nanopore, wherein monomers of the polynucleotide are labeled with fluorescent labels wherein the nanopore constrains fluorescent labels within its bore into a constrained state such that substantially no detectable fluorescent signal is generated therein; (b) exciting the fluorescent label of each monomer upon exiting the nanopore; (c) measuring a fluorescent signal in a detection zone generated by the exiting fluorescent label to identify the monomer to which the fluorescent label is attached; (d) quenching fluorescent signals from excited fluorescent labels outside of the detection zone, and (d) determining a monomer sequence of the polynucleotide from a sequence of fluorescent signals.
  • fluorescent labels are acceptors of a
  • substantially quenched as used above means a fluorescent label generates a fluorescent signal at least thirty percent reduced from a signal generated under the same conditions, but without adjacent mutually quenching labels. In some embodiments, “substantially quenched” as used above means a fluorescent label generates a fluorescent signal at least fifty percent reduced from a signal generated under the same conditions, but without adjacent mutually quenching labels.
  • a nucleotide sequence of a target polynucleotide is determined by carrying out four separate reactions in which copies of the target polynucleotide have each of its four different kinds of nucleotide (A, C, G and T) labeled with a single fluorescent label.
  • a nucleotide sequence of a target polynucleotide is determined by carrying out four separate reactions in which copies of the target polynucleotide have each of its four different kinds of nucleotide (A, C, G and T) labeled with one fluorescent label while at the same time the other nucleotides on the same target polynucleotide are labeled with a second fluorescent label. For example, if a first fluorescent label is attached to A's of the target polynucleotide in a first reaction, then a second fluorescent label is attached to C's, G's and T's (i.e.
  • the first label is attached to C's of the target polynucleotide and the second fluorescent label is attached to A's, G's and T's (i.e. to the "not-C" nucleotides) of the target polynucleotide. And so on, for nucleotides G and T.
  • the same labeling scheme may be expressed in terms of conventional terminology for subsets of nucleotide types; thus, in the above example, in a first fluorescent label is attached to A's and a second fluorescent label is attached to B's; in a second reaction, a first fluorescent label is attached to C's and a second fluorescent label is attached to D's; in a third reaction, a first fluorescent label is attached to G's and a second fluorescent label is attached to H's; and in a fourth reaction, a first fluorescent label is attached to T's and a second fluorescent label is attached to Vs.
  • a polymer such as a polynucleotide or peptide
  • a polymer may be labeled with a single fluorescent label attached to a single kind of monomer, for example, every T (or substantially every T) of a polynucleotide is labeled with a fluorescent label, e.g. a cyanine dye.
  • a collection, or sequence, of fluorescent signals from the polynucleotide may form a signature or fingerprint for the particular polynucleotide. In some such embodiments, such fingerprints may or may not provide enough information for a sequence of monomers to be determined.
  • a feature of the invention is the labeling of substantially all monomers of a polynucleotide analyte with fluorescent dyes or labels that are members of a mutually quenching set.
  • the use of the term “substantially all” in reference to labeling polynucleotide analytes is to acknowledge that chemical and enzymatic labeling techniques are typically less than 100 percent efficient.
  • “substantially all” means at least 80 percent of all monomer have fluorescent labels attached.
  • “substantially all” means at least 90 percent of all monomer have fluorescent labels attached.
  • substantially all means at least 95 percent of all monomer have fluorescent labels attached.
  • Mutually quenching sets of fluorescent dyes have the following properties: (i) each member quenches fluorescence of every member (for example, by FRET or by static or contact mechanisms), and (ii) each member generates a distinct fluorescent signal when excited and when in a non-quenched state. That is, if a mutually quenching set consists of two dyes, Dl and D2, then (i) Dl is self-quenched (e.g. by contact quenching with another Dl molecule) and it is quenched by D2 (e.g. by contact quenching) and (ii) D2 is self- quenched (e.g. by contact quenching with another D2 molecule) and it is quenched by Dl (e.g.
  • members of a mutually quenching set comprise organic fluorescent dyes that components or moieties capable of stacking interactions, such as aromatic ring structures.
  • Exemplary mutually quenching sets of fluorescent dyes, or labels may be selected from rhodamine dyes, fluorescein dyes and cyanine dyes.
  • a mutually quenching set may comprise the rhodamine dye, TAMRA, and the fluorescein dye, FAM.
  • mutually quenching sets of fluorescent dyes may be formed by selecting two or more dyes from the group consisting of Oregon Green 488, Fluorescein-EX, fluorescein isothiocyanate, Rhodamine Red-X, Lissamine rhodamine B, Calcein, Fluorescein, Rhodamine, one or more BODIPY dyes, Texas Red, Oregon Green 514, and one or more Alexa Fluors.
  • Respresentative BODIPY dyes include BODIPY FL, BODIPY R6G, BODIPY TMR,
  • Alexa Fluors include Alexa Fluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750 and 790.
  • a monomer sequence of a target polynucleotide is determined by carrying out separate reactions (one for each kind of monomer) in which copies of the target polynucleotide have each different kind of monomer labeled with a mutually- or self-quenching fluorescent label.
  • a monomer sequence of a target polynucleotide is determined by carrying out separate reactions (one for each kind of monomer) in which copies of the target polynucleotide have each different kind of monomer labeled with a different mutually quenching fluorescent label selected from the same mutually quenching set.
  • a selected monomer (say, monomer X) is labeled with a first mutually quenching dye and every other kind of monomer (i.e., not-monomer X) is labeled with a second mutually quenching dye from the same set.
  • steps of the embodiment generate a sequence of two different fluorescent signals, one indicating monomer X and another indicating not-monomer X.
  • a single fluorescent label (for example, attached to a single kind of monomer in a polynucleotide comprising multiple kinds of monomers) may be used that is self-quenching when attached to adjacent monomers (of the same kind) on a
  • polynucleotide such as adjacent nucleotides of a polynucleotide.
  • exemplary self-quenching fluorescent labels include, but are not limited to, Oregon Green 488, fluorescein-EX, FITC, Rhodamine Red-X, Lissamine rhodamine B, calcein, fluorescein, rhodamine, BODIPYS, and Texas Red, e.g. which are disclosed in Molecular Probes Handbook, 11th Edition (2010).
  • nucleotide as few as two different kinds of nucleotide are labeled with different optical labels that generate distinguishable optical signals for the selected kinds of nucleotide in both sense strands and antisense strands of target polynucleotides.
  • C's and T's of the complementary strands of each target polynucleotide may be replaced by labeled analogs, wherein the labels of the C and T analogs are capable of generating distinct optical signals.
  • Optical signatures are then generated by translocating the labeled strands through nanopores where nucleotides of the strands are constrained to pass sequentially through an optical detection region where their labels are caused to generate optical signals.
  • information from optical signatures from both sense and antisense strands are combined to determine a nucleotide sequence of target polynucleotides.
  • the selected kinds of nucleotides of target polynucleotides are replaced by labeled nucleotide analogs in an extension reaction using a nucleic acid polymerase.
  • Labeled strands of target polynucleotides are translocated through nanopores that constrain the nucleotides of strands to move single file through an optical detection region where they are excited so that they produce an optical signal.
  • a collection of optical signals for an individual strand is referred to herein as an optical signature of the strand.
  • a strand and its complement i.e.
  • a single optical signature may include optical signals from optical labels on nucleotides from both the sense strand and the antisense strand.
  • different strands of a target polynucleotide may separately generate two different optical signatures which may be combined, or used together, for analysis, as mentioned above.
  • Such separately analyzed strands may be associated after generation of optical signatures, for example, by using molecular tags (which may be, for example, oligonucleotide segments attached to target polynucleotides in a known position, length and sequence pattern and diversity to permit ready association).
  • optical signature of the invention may comprise mixed optical signals in that the signal detected in each detection interval may comprise contributions from multiple optical labels emitting within a resolution limited area or volume; that is, they may (for example) be mixed FRET signals, as described by Huber et al, U.S. patent publication US20160076091, which is incorporated herein by reference.
  • methods of the invention may be implemented with the following steps: (a) copying a strand of a double stranded
  • two kinds of nucleotide are labeled, which may be C's and T's, C's and G's, C's and A's, T's and G's, T's and A's, or G's and A's.
  • pyrimidine nucleotides are labeled.
  • purine nucleotides are labeled.
  • selected kinds of nucleotides of a strand are labeled by incorporating labeled analog dNTPs of the selected kind of nucleotides in a primer extension reaction using a nucleic acid polymerase.
  • selected kinds of nucleotides of a strand are labeled by incorporating analog dNTPs of the selected kinds of nucleotides in an extension reaction, wherein the analog dNTPs are derivatized with orthogonally reactive functionalities that allow attachment of different labels to different kinds of nucleotides in a subsequent reaction.
  • analog dNTPs are derivatized with orthogonally reactive functionalities that allow attachment of different labels to different kinds of nucleotides in a subsequent reaction.
  • three kinds of nucleotide are labeled, which may include labeling C's with a first optical label, T's with a second optical label, and G's and A's with a third optical label.
  • the following groups of nucleotides may be labeled as indicated: C's and G's with a first optical label and second optical label, respectively, and T's and A's with a third optical label; C's and A's with a first optical label and second optical label, respectively, and T's and G's with a third optical label; T's and G's with a first optical label and second optical label, respectively, and C's and A's with a third optical label; A's and G's with a first optical label and second optical label, respectively, and T's and C's with a third optical label.
  • optical labels are fluorescent acceptor molecules that generate a fluorescent resonance energy transfer (FRET) signal after energy transfer from a donor associated with a nanopore.
  • FRET fluorescent resonance energy transfer
  • donors may be optically active nanoparticles, such as, quantum dots, nanodiamonds, or the like.
  • a single quantum dot is attached to a nanopore and is excited to fluoresce using an excitation beam whose wavelength is sufficiently separated, usually lower (i.e. bluer), so that it does not contribute to FRET signals generated by acceptors.
  • a quantum dot is selected whose emission wavelength overlaps the absorption bands of both acceptor molecules to facilitate FRET interactions.
  • two donors may be used for each excitation zone of a nanopore, wherein the emission wavelength of each is selected to optimally overlap the absorption band of a different one of the acceptor molecules.
  • double stranded target polynucleotide (700) (SEQ ID NO: 1) consists of sense strand (701) and complementary antisense strand (702), to which is ligated (703) "Y" adaptors (704) and (706) using conventional methods, e.g. Weissman et al, U.S. patent 6,287,825; Schmitt et al, U.S. patent publication US2015/004468; which are incorporated herein by reference.
  • Arms (708) and (710) of adaptors (704 and 706, respectively) include primer binding sites to which primers (716) and (718) are annealed (705).
  • Double stranded portions (712) and (714) may include tag sequences, e.g. one or both may include randomers of predetermined length and composition, which may be used for later re- association of the strands, for example, to obtain sequence information from the respective optical signatures of the strands.
  • primers (716) and (718) After annealing primers (716) and (718), they may be extended (707) by a nucleic acid polymerase in the presence of (for example, as illustrated) labeled dUTP analogs (labels shown as open circles in the incorporated nucleotides) and labeled dCTP analogs (labels shown as filled circles in the incorporated nucleotides) and natural unlabeled dGTPs and dATPs (with neither unlabeled dTTP nor unlabeled dCTP being present so that the analogs are fully substituted in the extended strands).
  • labeled dUTP analogs labels shown as open circles in the incorporated nucleotides
  • labeled dCTP analogs labels shown as filled circles in the incorporated nucleotides
  • natural unlabeled dGTPs and dATPs with neither unlabeled dTTP nor unlabeled dCTP being present so that the analog
  • extension products (720) and (722) are illustrated for an alternative embodiment employing three labels.
  • Incorporated labeled dUTP analogs are shown as open circles and incorporated labeled dCTP analogs are shown as filled circles, as above.
  • nucleic acid polymerases for use with the invention include, but are not limited to, Vent exo-, Taq, E. coli Pol I, Tgo exo-, Klenow fragment exo-, Deep Vent exo-, and the like.
  • exemplary nucleic acid polymerases include, but are not limited to, Vent exo- and Klenow fragment exo-.
  • Exemplary fluorescent labels for dNTP analogs include, but are not limited to, Alexa 488, AMCA, Atto 655, Cy3, Cy5, Evoblue 30, fluorescein, Gnothis blue 1, Gnothis blue 2, Gnothis blue 3, Dy630, Dy635, MR121, rhodamine, Rhodamine Green, Oregon Green, TAMRA, and the like.
  • Exemplary fluorescent labels for dUTP analogs include, but are not limited to, Alexa 488, AMCA, Atto 655, Cy3, Cy5, Dy630, Dy665, Evoblue 30, Evoblue 90, fluorescein, Gnothis blue 1, Gnothis blue 2, Gnothis blue 3, MR121, Oregon Green, rhodamine,
  • Exemplary fluorescent labels for dCTP analogs include, but are not limited to, Atto 655, Cy5, Evoblue 30, Gnothis blue 3, rhodamine, Rhodamine Green, TAMRA, and the like.
  • Exemplary fluorescent labels for dATP analogs include, but are not limited to, Atto 655, Cy5, Evoblue 30, Gnothis blue 3, Rhodamine Green, and the like.
  • Exemplary fluorescent labels for dGTP analogs include, but are not limited to, Evoblue 30, Gnothis blue 3, Rhodamine Green, and the like.
  • Exemplary pairs of fluorescent labels for dUTP analogs and dCTP analogs include, but are not limited to, (TAMRA,
  • Rhodamine Green (Atto 655, Evoblue 30), (Evoblue 30, Atto 655), (Evoblue 30, Gnothis blue 3), (Evoblue 30, Rhodamine Green), (Gnothis blue 1, Rhodamine Green), (Gnothis blue 2, Atto 655), Gnothis blue 3, Cy5), and the like.
  • Fig. 7C illustrates an embodiment in which two labels are used and sense and antisense strands are linked by means of hairpin adaptor (730), for example, as taught in U.S. patent publications US 2015/0152492 and US 2012/0058468, which are incorporated herein by reference. Tailed adaptor (732) and hairpin adaptor (730) are ligated to target
  • polynucleotide (700) (SEQ ID NO: 1).
  • extension reaction produces extension product (735) which includes segment (736), the labeled complement of strand (701) and segment (738), the labeled reverse complement of strand (701).
  • sequence of target polynucleotide (700) (SEQ ID NO: 1) can be determined.
  • sequence of hairpin (730) may be selected so that a predetermined pattern of labels is incorporated during the extension reaction, which may be used to assist in the analysis of the optical signature, e.g. by indicating where segment (736) ends and where segment (738) begins, or the like.
  • kits for carrying out the methods of the invention.
  • kits include one or more quenching agents appropriate for quenching fluorescent signals generated by fluorescent labels attached to target polynucleotides.
  • kits may be designed to comprise quenching agents whose absorption bands maximally overlap the emission bands of fluorescent labels on target polynucleotides.
  • quenching agents of kits comprise a polynucleotide binding moiety conjugated to a quenching moiety.
  • polynucleotide binding moieties comprise one or more oligonucleotides or analogs thereof.
  • quenching agents of kits are provided at predetermined concentrations. In some embodiments, such predetermined concentrations of quenching agents are selected to determine a volume of a detection zone.
  • Electrode field means a non-propagating electromagnetic field; that is, it is an electromagnetic field in which the average value of the Poynting vector is zero.
  • FRET Fluorescence, resonant energy transfer
  • FRET means a non- radiative dipole-dipole energy transfer mechanism from an excited donor fluorophore to an acceptor fluorophore in a ground state.
  • the rate of energy transfer in a FRET interaction depends on the extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and acceptor molecules, Lakowitz, Principles of Fluorescence Spectroscopy, Third Edition (Springer, 2006).
  • FRET interactions of particular interest are those which result a portion of the energy being transferred to an acceptor, in turn, being emitted by the acceptor as a photon, with a frequency lower than that of the light exciting its donor (i.e. a "FRET signal”).
  • FRET distance means a distance between a FRET donor and a FRET acceptor over which a FRET interaction can take place and a detectable FRET signal produced by the FRET acceptor.
  • Kit refers to any delivery system for delivering materials or reagents for carrying out a method of the invention.
  • delivery systems include systems and/or compounds (such as dilutants, surfactants, carriers, or the like) that allow for the storage, transport, or delivery of reaction reagents (e.g., fluorescent labels, such as mutually quenching fluorescent labels, fluorescent label linking agents, enzymes, quenching agents, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • reaction reagents e.g., fluorescent labels, such as mutually quenching fluorescent labels, fluorescent label linking agents, enzymes, quenching agents, etc. in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay etc.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. Such contents may be delivered to the intended recipient together or separately.
  • Nanopore means any opening positioned in a substrate that allows the passage of analytes through the substrate in a predetermined or discernable order, or in the case of polymer analytes, passage of their monomeric units through the substrate in a pretermined or discernible order. In the latter case, a predetermined or discernible order may be the primary sequence of monomeric units in the polymer.
  • nanopores include proteinaceous or protein based nanopores, synthetic or solid state nanopores, and hybrid nanopores comprising a solid state nanopore having a protein nanopore embedded therein.
  • a nanopore may have an inner diameter of 1-10 nm or 1-5 nm or 1-3 nm.
  • protein nanopores include but are not limited to, alpha-hemolysin, voltage-dependent mitochondrial porin (VDAC), OmpF, OmpC, MspA and LamB (maltoporin), e.g. disclosed in Rhee, M. et al., Trends in
  • a nanopore protein may be labeled at a specific site on the exterior of the pore, or at a specific site on the exterior of one or more monomer units making up the pore forming protein.
  • Pore proteins are chosen from a group of proteins such as, but not limited to, alpha-hemolysin, MspA, voltage-dependent mitochondrial porin (VDAC), Anthrax porin, OmpF, OmpC and LamB (maltoporin).
  • a synthetic nanopore, or solid- state nanopore may be created in various forms of solid substrates, examples of which include but are not limited to silicones (e.g. Si3N4, Si02), metals, metal oxides (e.g. A1203) plastics, glass, semiconductor material, and combinations thereof.
  • a synthetic nanopore may be more stable than a biological protein pore positioned in a lipid bilayer membrane.
  • a synthetic nanopore may also be created by using a carbon nanotube embedded in a suitable substrate such as but not limited to polymerized epoxy.
  • Carbon nanotubes can have uniform and well- defined chemical and structural properties. Various sized carbon nanotubes can be obtained, ranging from one to hundreds of nanometers.
  • the surface charge of a carbon nanotube is known to be about zero, and as a result, electrophoretic transport of a nucleic acid through the nanopore becomes simple and predictable (Ito, T. et al., Chem. Commun. 12 (2003): 1482-83).
  • the substrate surface of a synthetic nanopore may be chemically modified to allow for covalent attachment of the protein pore or to render the surface properties suitable for optical nanopore sequencing. Such surface modifications can be covalent or non-covalent. Most covalent modification include an organosilane deposition for which the most common protocols are described: 1) Deposition from aqueous alcohol.
  • Silanes can be applied to substrates under dry aprotic conditions by chemical vapor deposition methods. These methods favor monolayer deposition. In closed chamber designs, substrates are heated to sufficient temperature to achieve 5mm vapor pressure. Alternatively, vacuum can be applied until silane evaporation is observed. 3) Spin-on deposition.
  • Nanopores of a nanopore array may be spaced regularly, for example, in a rectilinear pattern, or may be spaced randomly. In a preferred embodiment, nanopores are spaced regularly in a rectilinear pattern in a planar solid phase substrate.
  • Polymer means a plurality of monomers connected into a linear chain.
  • polymers comprise more than one type of monomer, for example, as a polynucleotide comprising A's, C's, G's and T's, or a polypeptide comprising more than one kind of amino acid.
  • Monomers may include without limitation nucleosides and derivatives or analogs thereof and amino acids and derivatives and analogs thereof.
  • polymers are polynucleotides, whereby nucleoside monomers are connected by phosphodiester linkages, or analogs thereof.
  • Polynucleotide or “oligonucleotide” are used interchangeably and each mean a linear polymer of nucleotide monomers or analogs thereof. Monomers making up
  • polynucleotides and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • monomers and their intemucleosidic linkages may be naturally occurring or may be analogs thereof, e.g. naturally occurring or non-naturally occurring analogs.
  • Non-naturally occurring analogs may include PNAs, phosphorothioate
  • intemucleosidic linkages bases containing linking groups permitting the attachment of labels, such as fluorophores, or haptens, and the like.
  • labels such as fluorophores, or haptens, and the like.
  • oligonucleotide or polynucleotide require enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill would understand that oligonucleotides or polynucleotides in those instances would not contain certain analogs of intemucleosidic linkages, sugar moieties, or bases at any or some positions.
  • Polynucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are usually referred to as
  • oligonucleotides to several thousand monomeric units. Whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (upper or lower case), such as
  • polynucleotides comprise the four natural nucleosides (e.g. deoxyadenosine, deoxycytidine, deoxyguanosine,
  • deoxythymidine for DNA or their ribose counterparts for RNA linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g. including modified bases, sugars, or intemucleosidic linkages. It is clear to those skilled in the art that where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g.
  • oligonucleotide and polynucleotide may refer to either a single stranded form or a double stranded form (i.e. duplexes of an oligonucleotide or polynucleotide and its respective complement). It will be clear to one of ordinary skill which form or whether both forms are intended from the context of the terms usage.
  • Primer means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed.
  • Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase.
  • the sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide.
  • primers are extended by a DNA polymerase.
  • Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following references that are incorporated by reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003).
  • a surface of a nanopore array may be partitioned, or subdivided, into non-overlapping regions, or substantially non-overlapping regions, corresponding to resolution limited areas. The size of such subdivisions corresponding to resolution limited areas may depend on a particular optical detection system employed.
  • a resolution limited area is in the range of from 300 nm2 to 3.0 ⁇ 2; in other embodiments, a resolution limited area is in the range of from 1200 nm2 to 0.7 ⁇ 2; in other embodiments, a resolution limited area is in the range of from 3x104 nm2 to 0.7 ⁇ 2, wherein the foregoing ranges of areas are in reference to a surface of a nanopore or nanowell array.
  • the visible spectrum means wavelengths in the range of from about 380 nm to about 700 nm.
  • Sequence determination includes determination of partial as well as full sequence information of the polynucleotide. That is, the terms include sequences of subsets of the full set of four natural nucleotides, A, C, G and T, such as, for example, a sequence of just A's and C's of a target polynucleotide. That is, the terms include the determination of the identities, ordering, and locations of one, two, three or all of the four types of nucleotides within a target polynucleotide.
  • the terms include the determination of the identities, ordering, and locations of two, three or all of the four types of nucleotides within a target polynucleotide.
  • sequence determination may be accomplished by identifying the ordering and locations of a single type of nucleotide, e.g. cytosines, within the target polynucleotide "catcgc . . . " so that its sequence is represented as a binary code, e.g. " 100101 . . . " representing "c-(not c)(not c)c-(not c)-c . . . " and the like.
  • sequence determination may be accomplished by identifying the ordering and locations of a single type of nucleotide, e.g. cytosines, within the target polynucleotide "catcgc . . . " so that its sequence is represented as a binary code, e.g. " 100101 . . . " representing "c-(not c)
  • the terms may also include subsequences of a target polynucleotide that serve as a fingerprint for the target polynucleotide; that is, subsequences that uniquely identify a target polynucleotide, or a class of target polynucleotides, within a set of polynucleotides, e.g. all different RNA sequences expressed by a cell.

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Abstract

La présente invention concerne des procédés et des compositions pour l'analyse de polynucléotides à base optique à l'aide de nanopores, des agents d'extinction étant utilisés pour réduire ou éliminer des signaux optiques parasites générés par des marqueurs polynucléotidiques à l'extérieur de zones de détection. Dans certains modes de réalisation, l'invention peut être mise en oeuvre par les étapes suivantes, consistant à : effectuer une translocation d'un polynucléotide à travers un nanopore, différents types de nucléotides du polynucléotide étant marqués par différentes étiquettes fluorescentes qui génèrent des signaux fluorescents pouvant être distingués et le nanopore contraignant les nucléotides à se déplacer en une file unique à travers une zone de détection ; exciter les marqueurs fluorescents ; éteindre des signaux fluorescents provenant d'étiquettes fluorescentes excitées à l'extérieur de la zone de détection à l'aide d'un ou plusieurs agents d'extinction non fluorescents ; détecter des signaux fluorescents émis par les marqueurs fluorescents lorsque les marqueurs fluorescents passent à travers la zone de détection ; et déterminer une séquence nucléotidique à partir des signaux fluorescents détectés.
EP17841831.5A 2016-08-19 2017-07-25 Séquençage par nanopores à base optique à l'aide d'agents d'extinction Withdrawn EP3500684A4 (fr)

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