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WO2014071250A1 - Procédés de détection et de cartographie de modifications de polymères d'acide nucléique à l'aide de systèmes à nanopore - Google Patents

Procédés de détection et de cartographie de modifications de polymères d'acide nucléique à l'aide de systèmes à nanopore Download PDF

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WO2014071250A1
WO2014071250A1 PCT/US2013/068162 US2013068162W WO2014071250A1 WO 2014071250 A1 WO2014071250 A1 WO 2014071250A1 US 2013068162 W US2013068162 W US 2013068162W WO 2014071250 A1 WO2014071250 A1 WO 2014071250A1
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nucleic acid
nanopore
nucleotide
acid polymer
dna
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Jens H. Gundlach
Ian DERRINGTON
Andrew Laszlo
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University of Washington Center for Commercialization
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University of Washington Center for Commercialization
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification.
  • the name of the text file containing the sequence listing is 43321_SeqList-FINAL.txt.
  • the text file is 19KB; was created on 01 November 2013; and is being submitted via EFS- Web with the filing of the specification.
  • DNA The nucleic acid, DNA, is often referred to as the "blueprint for life.” However, there is more to the code than DNA sequence alone. Modifications can occur on the canonical nucleotide subunits that can affect the functional information embedded in the DNA. For example, epigenetic factors govern the DNA blueprint's transcription and translation into protein. Thus, there is a rapidly growing interest in understanding these modifications, such as epigenetic factors.
  • Epigenetic modifications such as methylation and/or hydroxymethylation of DNA can be natural processes by which normal cells function and carry out or inhibit many cellular functions.
  • epigenetic modifications are known to be involved with normal silencing and/or prevention of gene expression, thereby enabling a cell to essentially turn off one or more genes.
  • the most common epigenetic DNA modification is the methylation of cytosine leading to 5-methylcytosine ( m C).
  • cytosine methylations occur in C-G dinucleotides (CpG; the "CpG” shorthand invokes the relationship that the cytosine and guanine are linked in the same strand by a single phosphate and distinguished the relationship from a C:G pairing of complementary strands, such as in double-stranded DNA).
  • Methylation is associated with gene regulation (i.e., highly methylated DNA tends to be less transcriptionally active) and therefore has implications for cell development, aging, and diseases such as cancer. Further oxidation of the methyl residue results in 5-hydroxymethylcytosine ( h C). Because of its relatively recent discovery in mammalian tissue, the function of h C is less well explored.
  • methylation patterns are tissue specific and change over the life of an organism as it develops or is exposed to certain chemicals and environmental conditions. In some cases, these changes are heritable through multiple generations.
  • nucleotide modifications such as methylation
  • precise mapping of modifications may yield more pertinent information to research and ultimately to clinical diagnosis of gene-regulation-related disease, than sequencing the standard four bases alone.
  • Clinical uses will require fast, inexpensive, and reliable detection methods to map modifications such as methylation. Because such modification patterns vary between cells, it is preferable to use small, native, unamplified DNA samples, making this task suitable for single-molecule techniques.
  • SMRT Single-molecule real-time sequencing
  • T he durations of the pauses are stochastically distributed, and the change in kinetics caused by m C is subtle. Thus, detection requires averaging over dozens of reads, complicating methylation detection.
  • nanochannels have been used as nano-Coulter counters to measure the correlation between DNA methylation and certain histone modifications for single chromatin molecules. However, this method lacks single-nucleotide resolution.
  • the present disclosure provides a method of detecting a nucleotide modification in a nucleic acid polymer.
  • the method comprises applying an electrical field to a nanopore system comprising a first conductive liquid medium in liquid communication with a second conductive liquid medium through a nanopore; translocating the nucleic acid polymer through a nanopore from the first conductive liquid medium to the second conductive liquid medium; detecting an ion current to provide a current pattern associated with a portion of the nucleic acid polymer; and comparing the current pattern to a reference current pattern associated with the same nucleotide sequence as the portion of the nucleic acid polymer without any modifications, wherein a difference between the current pattern and the reference current pattern indicates the presence of a modified nucleotide in nucleic acid polymer.
  • the nucleic acid polymer can be DNA, RNA, mRNA, PNA, or a combination thereof.
  • the DNA is single stranded DNA (ssDNA).
  • the method further comprises identifying the type of nucleotide modification present in the polymer based on a character of the difference between the current pattern and the reference current pattern.
  • the character of the difference comprises the degree of current increase or decrease and/or the duration of the difference.
  • the nucleotide modification is an epigenetic modification or a modification resulting from DNA damage.
  • the nucleotide modification is a 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxycytosine, b-glucosyl-5-hydroxy-methylcytosine, 8-oxoguanine, 2-amino-adenosine, 2-amino-deoxyadenosine, 2-thiothymidine, pyrrolo-pyrimidine, 2-thiocytidine, a thymine dimer, or an abasic lesion.
  • the portion of the nucleic acid polymer comprises one or a plurality of contiguous nucleotides of the nucleic acid polymer. In some embodiments, the portion of the nucleic acid polymer comprises the nucleotide or nucleotide position with the modification. In some embodiments, the portion of the nucleic acid polymer further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional nucleotides adjacent to the nucleotide or nucleotide position with the modification on one or both sides. In some embodiments, at least one additional nucleotide is adjacent at the 5' side of the nucleotide with the modification.
  • At least one additional nucleotide is adjacent at the 3' side of the nucleotide with the modification.
  • the portion of the nucleic acid polymer further comprises at least two additional nucleotides adjacent at the 5' side of the nucleotide with the modification and at least one nucleotide adjacent at the 3' side of the nucleotide with the modification.
  • the nanopore is a solid-state nanopore, protein nanopore, a hybrid solid state-protein nanopore, a biologically adapted solid-state nanopore, or a DNA origami nanopore.
  • the protein nanopore is a ⁇ -barrel pore, such as alpha-hemolysin or Mycobacterium smegmatis porin A (MspA), or a homolog thereof.
  • the protein nanopore sequence is modified from the wild- type sequence to contain at least one amino acid substitution, deletion, or addition. In some embodiments, the at least one amino acid substitution, deletion, or addition results in a net charge change in the nanopore.
  • the electric field is sufficient to cause the electrophoretic translocation of the nucleic acid polymer through the nanopore. In some embodiments, the electric field is between about 40 mV to 1 V.
  • the nanopore is associated with a molecular motor, wherein the molecular motor is capable of moving a nucleic acid polymer into or through the nanopore with an average translocation velocity that is less than the average translocation velocity at which the analyte translocates into or through the nanopore in the absence of the molecular motor.
  • the molecular motor is a polymerase, an exonuclease, a helicase, a topoisomerase, or a translocase. In some embodiments, the molecular motor is phi29.
  • the disclosure provides method of detecting a nucleotide modification in a nucleic acid polymer, as generally described above, but including the step generating a reference nucleic acid polymer that contains the same nucleotide sequence but that does not contain any modifications to the canonical nucleotide structures.
  • the method comprises amplifying a target nucleic acid polymer that potentially contains at least one nucleotide modification to produce a reference nucleic acid polymer that does not contain a nucleotide modification; applying the target and reference nucleic acid polymers to a nanopore system comprising a first conductive liquid medium in liquid communication with a second conductive liquid medium through a nanopore; causing the translocation of the target nucleic acid polymer through the nanopore from the first conductive liquid medium to the second conductive liquid medium; detecting an ion current to provide a target current pattern associated with a portion of the target nucleic acid polymer; causing the translocation of the reference nucleic acid polymer through the nanopore from the first conductive liquid medium to the second conductive liquid medium; detecting an ion current to provide a reference current pattern associated with a portion of the reference nucleic acid polymer, wherein the portion of the target nucleic acid polymer comprises the same nucleotide sequence as the portion of the target nucleic acid polymer;
  • the reference nucleic acid polymer is produced from the target nucleic acid polymer using at least one round of the polymerase chain reaction (PCR).
  • the method further comprises determining the position of the modified nucleotide in the target polymer.
  • the method further comprises identifying the modified nucleotide in the target polymer.
  • the method further comprises determining the sequence of at least a portion of the target nucleic acid polymer comprising the modified nucleotide.
  • the modified nucleotide is identified without knowledge of the nucleotide identity in the unmodified reference sequence.
  • FIGURE 1A-FIGURE 1C schematically illustrate a representative system useful for nanopore-based analysis of nucleic acid modifications according to the present disclosure.
  • FIGURE 1A is a diagram of an exemplary template configuration, wherein a hairpin primer oligonucleotide forms a loop and hybridizes to a portion of itself. The remainder of the hairpin primer hybridizes to a 3'-end domain of the template strand. The gap between the 3'-end of the template strand and the 5'-end of the hairpin primer is indicated. Adjacent to the hairpin primer, and hybridizing to an internal domain of the template strand, is a blocking oligomer.
  • FIGURE IB schematically illustrates the use of a molecular motor in connection with a nanopore system to assist the controlled translocation of the nucleic acid template strand through the nanopore for analysis.
  • the nanopore e.g., MspA
  • the nanopore is embedded in a phospholipid bilayer and provides liquid communication between the upper chamber (i.e., cis side) and the lower chamber (i.e., trans side).
  • a voltage (e.g., 180 mV) is applied across the membrane, which causes an ion current to flow through the pore.
  • the molecular motor (shown in dark) is pulled into contact with the vestibule of the nanopore in the cis side, but cannot pass through, thus causing the unzipping of the blocking oligomer from template strand.
  • the blocking oligomer is "unzipped” from the template strand.
  • the short and narrow constriction of MspA concentrates the ion current to resolve the relatively small differences between C, m C, and h C.
  • FIGURE 1C schematically illustrates the progression of template through the nanopore system over time.
  • the single stranded 5'-end of the template strand interacts with, or enters the vestibule of, the nanopore.
  • the molecular motor which is attached to the double stranded portion of the complex, contacts the vestibule of the molecular motor, but does not pass through, thus slowing translocation of the template strand through the nanopore.
  • the force of translocation eventually causes the complete unzipping of the blocking oligomer (the process of which is illustrated in FIGURE IB), thus exposing the 3'-end of the hairpin primer to the molecular motor (e.g., a DNA polymerase such as phi29).
  • the molecular motor pulls the template strand back through the nanopore by enzymatic action where the hairpin primer is elongated based on the template strand.
  • FIGURE 2 graphically illustrates the mean consensus levels and standard deviation of the mean for the normalized current levels (ion current divided by the open- pore current) determined for several events of TGCC quadromers and modified TGCC quadromers (i.e., wherein the C is methylated or hydroxymethylated).
  • the current levels for the TGCC quadromers and modified TGCC quadromers are scaled and offset to each other on the same graph based on their calibration regions (i.e., a recognizable adapter sequence separated from the template strand by a single abasic nucleotide).
  • the unmodified levels are displayed in black and the modified current levels are displayed in gray and with a * symbol.
  • the quadromers and sequences illustrated in this figure are listed from 3 '-5' reflecting the order the data was recorded during phi29 synthesis. This order is the opposite of how DNA is typically listed, thus the CpGs appear as GpCs in this figure.
  • the sequence of the unmodified (top) strand is set forth as SEQ ID NO:5.
  • the sequence of the modified strand is set forth herein as SEQ ID NO:6.
  • the location of hydroxymethylation and methylation in the modified strand TGCC quadromers are indicated by h and m, respectively. Below the levels, the quadromers corresponding to each level are indicated. Note that hydroxymethylation decreases the average current in the affected levels while methylation increases the average current of affected levels (see FIGURE 3).
  • FIGURE 3 graphically illustrates the normalized difference in mean current values for each current level.
  • a control cytosine remains unmodified and shows no significant difference in reads on both strands.
  • the sequence of the illustrated modified strand is set forth herein as SEQ ID NO:6.
  • FIGURE 4 graphically illustrates the normalized difference in mean current values for each level with seven Gaussians fit the data.
  • the Gaussian peaks indicate the centroid of the effect, which corresponds well with the location of the epigenetic modifications.
  • the Gaussian fit for the unmodified GpC region has a very low amplitude relative to the modified GpCs.
  • This mapping difference yields a clear and easily interpretable way of mapping modifications in the nucleic acid polymer.
  • the sequence of the illustrated modified strand is set forth herein as SEQ ID NO:6.
  • FIGURE 5A-FIGURE 5D graphically illustrate the detection of methylation in representative single stranded DNA sequence containing a CpG site.
  • FIGURE 5A and FIGURE 5B illustrate segments of raw current traces for the DNA sequence with unmethylated and methylated cytosine, respectively. Ion current changes as DNA passes through the pore in single-nucleotide steps. The average current values for each current level are horizontal black or horizontal lines, respectively.
  • the traces shown in FIGURE 5A and FIGURE B are for DNA with identical nucleotide sequence.
  • the current trace shown in FIGURE 5A contains a single unmethylated CpG site, whereas the trace in FIGURE 5B contains a single methylated CpG site.
  • FIGURE 5C illustrates the extracted average current values from each level from FIGURE 5A in a solid black line and from FIGURE 5B in a solid gray line, with the difference illustrated with shading.
  • the stochastic duration of current levels has been removed so that the DNA base sequence can be aligned to the observed current levels.
  • the DNA sequence, set forth herein as SEQ ID NO:7 is shown below (from 5'- to 3'-, left to right) with the modified C indicated as ' ,m C".
  • FIGURE 5D illustrates current difference plot. The current levels obtained with methylated DNA were subtracted from the current levels obtained with unmethylated DNA. The effect of a single " pG causes an ion current increase that persists over approximately four steps of the DNA through the pore.
  • FIGURE 6 graphically illustrates the differences in the ion current level sequences resulting from DNA containing methylation or hydroxymethylation and from DNA without methylation or hydroxymethylation.
  • X is an abasic site.
  • FIGURE 6A The sequence illustrated in FIGURE 6A is set forth in SEQ ID NO:8, and the sequence illustrated in FIGURE 6B is set forth in SEQ ID NO:9.
  • the methylated positions are marked by a significant current increase that persists over approximately four steps of the DNA through the pore.
  • the amplitude and shape of the current difference depend on the nucleotides adjacent to the m C. In regions containing no methylation, current differences are insignificant.
  • h C results in a small reduction in current, although the magnitude of the current difference is less than observed for m C.
  • h C results in a current increase.
  • Error bars are the observed SD for single-molecule reads of methylated DNA and indicate the variation in single-molecule reads.
  • the gray boxes along the x axis are the SDs for reads of unmethylated DNA. See TABLE 2, for exact numbers of events.
  • FIGURE 7A and FIGURE 7B graphically illustrates that the DNA sequence context changes the resulting current difference pattern when a modified cytosine replaces a cytosine at a CpG site.
  • FIGURE 7A shows the current difference patterns caused by the sequence XY m CpG, where X and Y are any of the four nucleotides A, C, G, and T.
  • FIGURE 7B shows the current difference patterns caused by the sequence XY h CpG, where X and Y are any of the four nucleotides A, C, G, and T.
  • the right-most column and bottom row of each figure display the current differences averaged over the nucleotides X or Y, respectively.
  • FIGURE 7A illustrates that the maximum difference reaches 7 pA for AA m CpG and is only 1-2 pA when XY contains a thymine.
  • the average maximum difference is approximately 2 pA.
  • the number of levels showing a significant current difference varies from 3 to 5. The difference is maximal when the m C is immediately above the constriction of the nanopore (see FIGURE 8) and the distribution is skewed.
  • FIGURE 7B illustrates that the current deviations due to h C are more complex.
  • FIGURE 8A and FIGURE 8B schematically illustrate spatial methylation sensitivity of MspA.
  • the variable shading indicates the region of higher electric field within MspA.
  • FIGURE 8 A illustrates that when m C is cis of the constriction, it is in a high field region and it modulates the ion current. Other nucleotides that are also within the high field region determine the magnitude of the m C-specific signal.
  • FIGURE 8A illustrates that when m C is trans of the constriction, it is outside the high field region and no longer affects the current.
  • the sequence illustrated in FIGURE 8A is set forth as SEQ ID NO: 12 and the sequence illustrated in FIGURE 8B is set forth as SEQ ID NO: 13.
  • FIGURE 9A-FIGURE 9D graphically illustrate the differences in ion current resulting from multiple adjacent m Cs and h Cs.
  • Current differences [ mo(j ified ⁇ ⁇ unmodified] f° r f° ur DNA strands contain different methylation (and hydroxymethylation) patterns.
  • CpGs rarely occur in such high density, the illustrated data demonstrate that it is possible to discern multiple adjacent m CpGs and h CpGs.
  • Each sequence set forth in FIGURE 9A-FIGURE 9D is set forth herein as SEQ ID NO: 14- 17, respectively.
  • FIGURE 9A which illustrates data from a strand containing one m C and one h C (as indicated at the bottom), demonstrates that one can simultaneously detect ⁇ Kl and h C in a single strand.
  • FIGURE 9B illustrates the current difference resulting from a strand with identical sequence to that shown in FIGURE 9A, but containing four m Cs as well as two h Cs (indicated at the bottom). As demonstrated, even with this density of modified CpGs, individual m Cs and h Cs can be resolved.
  • FIGURE 9C illustrates the current differences resulting from a strand with adjacent m CpG sites. The modification density results in wide and large current difference profiles. The current difference profiles for individual m Cs seemingly superimpose.
  • FIGURE 9D illustrates the current differences for a strand with identical sequence to that in FIGURE 9C but with two " s replaced by two h Cs (indicated at the bottom).
  • the effects of m C and h C counteract one another.
  • the result is approximately a superposition of the signals shown in FIGURE 7.
  • FIGURE 1 OA-FIGURE 10D graphically illustrate the classification power for individual-level positions surrounding CpG sites.
  • FIGURE 10A illustrates the t-test value for each level and each measured sequence context (XYCpG), testing the unmethylated hypothesis XYCpG, against the methylated hypothesis XY ⁇ pG. Darker values indicate that a level has more power to call the methylation status of the CpG.
  • FIGURE 10B similarly illustrates the predictive power of single levels to call hydroxymethylation.
  • FIGURE IOC similarly illustrates the predictive power of single levels to call methylation and hydroxymethylation.
  • the lower plots in FIGURE 1 OA-FIGURE IOC show the t-test value for each position, averaged over all sequence contexts.
  • FIGURE 10D illustrates the classification frequency using just one specific level at the indicated positions.
  • FIGURE 10D, part (i) illustrates the rates that strands containing a m C were called correctly as m CpG (indicated as " m C"), or incorrectly as h CpG (indicated as " h C”) and unmethylated CpG (indicated with "C").
  • the present disclosure generally relates to methods for measuring, diagnosing, visualizing, and/or detecting modifications in nucleic acids through the use of nanopore-based analysis.
  • the methods and compositions are useful for accurately detecting, distinguishing, and mapping epigenetic modifications in nucleic acids such as 5-methylcytosine ( m C) and 5-hydroxymethylcytosine ( h C).
  • Nanopore analysis is an emerging single-molecule technique that has shown promise for DNA sequencing and analysis. As is described in more detail below, in nanopore sequencing, a thin membrane containing a single nanometer- sized pore divides a salt solution into two wells, cis and trans. A voltage across the membrane causes an ion current through the pore.
  • This current can also facilitate the interaction of analytes, such as DNA, with the nanopore, in some cases driving the analyte through the pore from one side to the other.
  • analytes such as DNA
  • the nucleotides at the narrowest section of the pore modulate the ion current.
  • Solid-state nanopores have been used to detect the bulk presence of ⁇ Kl and h C in double-stranded DNA (dsDNA) (Wanunu, M., et al., "Discrimination of Methylcytosine From Hydroxymethylcytosine in DNA Molecules," Journal of the American Chemical Society 133(3):486-492, 2011). Recently, solid-state nanopores were also used to detect dsDNA complexed with methyl-binding proteins and thereby indirectly measured the approximate location of individual methylation sites (Shim, J., et al., Detection and Quantification of Methylation in DNA Using Solid-State Nanopores,” Scientific Reports 3: 1389, 2013).
  • the present inventors have developed an approach using nanopore-based analysis that can detect, map, and distinguish (i.e., accurately identify) multiple, distinct DNA modifications within a single DNA template polymer.
  • the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) was used to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA with single-nucleotide resolution.
  • MspA Mycobacterium smegmatis porin A
  • DNAP phi29 DNA polymerase
  • a comparison of the current levels generated with DNA containing methylated or hydroxymethylated CpG sites to current levels obtained with unmethylated copies of the same DNA sequence resulted in a surprisingly precise indication of methylated or hydroxymethylated CpG sites.
  • the detection efficiency in a quasi-random DNA strand was 97.5 + 0.7% for methylation and 97 + 0.9% for hydroxymethylation.
  • the disclosed approach can be applied to detection and mapping of modifications, such as epigenetic modifications or the results of DNA damage, that occur in genomic DNA or RNA. Such information can be valuable for clinical uses, such as assessing associations of such changes with risk or presence of disease.
  • the present disclosure provides a method of detecting a nucleotide modification in a nucleic acid polymer.
  • the method comprises applying an electrical field to a nanopore system comprising a first conductive liquid medium in liquid communication with a second conductive liquid medium through a nanopore.
  • the nucleic acid polymer is translocated through a nanopore from the first conductive liquid medium to the second conductive liquid medium.
  • An ion current is detected to provide a current pattern associated with a portion of the nucleic acid polymer.
  • the current pattern is compared to a reference current pattern associated with the same nucleotide sequence as the portion of the nucleic acid polymer without any modifications, wherein a difference between the current pattern and the reference current pattern indicates the presence of a modified nucleotide in the polymer.
  • nucleic acid can refer to a deoxyribonucleotide polymer (DNA), ribonucleotide polymer (RNA, including mRNA), peptide nucleic acids (PNAs) and phosphorothioate DNA, in either single- or double-stranded form.
  • DNA deoxyribonucleotide polymer
  • RNA ribonucleotide polymer
  • PNAs peptide nucleic acids
  • phosphorothioate DNA in either single- or double-stranded form.
  • the nucleic acid subunits for each distinct nucleic acid polymer-type are commonly known.
  • the structure of the canonical polymer subunits of DNA are referred to herein as adenine (A), guanine (G), cytosine (C), and thymine (T).
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • these are generally referred to herein as nucleotides or nucleotide residues.
  • U uracil
  • T thymine
  • the present disclosure is directed to detecting modifications that can occur within the nucleic acid polymers, and in some embodiments, modifications that specifically occur to the individual subunits of the nucleic acid polymers, i.e., the individual nucleotides.
  • modification encompasses any chemical change in the structure of the nucleic acid polymer subunit that results in a noncanonical subunit structure. Such chemical changes can results from, for example, epigenetic modifications (such as to genomic DNA or RNA), or damage resulting from radiation, chemical, or other means.
  • nucleotide modification do not refer to simple additions or deletions of canonical nucleotides to the sequence of the polymer. Nor do the terms refer to substitutions of one canonical nucleotide for another canonical nucleotide for that polymer-type.
  • uracil (U) is considered a noncanonical nucleotide structure for DNA polymers (and, conversely, thymine (T) is considered a noncanonical nucleotide structure for RNA polymers).
  • the present disclosure is directed to the detection of nucleotide modifications, as defined.
  • the disclosure encompasses the detection of a noncanonical nucleotide structure within a nucleic acid polymer, which results from the act of modification.
  • Any of the foregoing noncanonical subunits include analog structures.
  • noncanonical nucleic acid subunits include uracil (for DNA), thymine (for RNA), 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine, b-glucosyl-5-hydroxy-methylcytosine, 8-oxoguanine, 2-amino-adenosine, 2-amino- deoxyadenosine, 2-thiothymidine, pyrrolo-pyrimidine, 2-thiocytidine, or an abasic lesion.
  • An abasic lesion is a location along the deoxyribose backbone but lacking a base.
  • noncanonical structures can incorporate more than one nucleic acid subunits, such as thymine dimers.
  • a single target nucleic acid polymer can comprise a combination of any of the foregoing polymers and/or polymer subunits.
  • the polymer analyte is a combination of any two or more of DNA, RNA, PNA.
  • the present disclosure addresses the detection of modified nucleic acid subunits that are noncanonical (i.e., have been modified from the canonical structure) with reference to the canonical subunit structures for the two or more types of nucleic acids that make up the single polymer.
  • Nanopore specifically refers to a pore having an opening with a diameter at its most narrow point of about 0.3 nm to about 2 nm.
  • Nanopores useful in the present disclosure include any pore capable of permitting the linear translocation of a nucleic acid polymer from one side to the other at a velocity amenable to monitoring techniques, such as techniques to detect current fluctuations.
  • the nanopore comprises a protein.
  • proteins can be ⁇ -barrel pores, outer membrane proteins (often of bacterial origin), ⁇ -toxin porings, and transport proteins.
  • Exemplary pores include alpha-hemolysin, Mycobacterium smegmatis porin A (MspA) and related porins such as from Norcadia facinica, membrane outer protein (OmpATb), membrane outer protein F (OmpF), membrane outer protein G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), lysenin, anthrax toxin and leukocidins, and homologs thereof, or other porins, as described in U.S. Pub. No.
  • a "homolog,” as used herein, is a gene or gene product from another bacterial species that has a similar structure and evolutionary origin.
  • homologs of wild-type MspA such as MppA, PorMl, PorM2, and Mmcs4296, can serve as the nanopore in the present invention.
  • Protein nanopores have the advantage that, as biomolecules, they self-assemble and are essentially identical to one another.
  • protein nanopores can be wild-type or can be modified to contain at least one amino acid substitution, deletion, or addition.
  • the at least one amino acid substitution, deletion, or addition results in a different net charge of the nanopore.
  • the different in net charge increases the difference of net charge as compared to the first charged moiety of the polymer analyte.
  • the at least one amino acid substitution, deletion, or addition results in a nanopore that is less negatively charged.
  • the resulting net charge is negative (but less so), is neutral (where it was previously negative), is positive (where it was previously negative or neutral), or is more positive (where it was previously positive but less so).
  • MspA nanopores can be modified with amino acid substitutions to result in a MspA mutant with a mutation at position 93, a mutation at position 90, position 91, or both positions 90 and 91, and optionally one or more mutations at any of the following amino acid positions: 88, 105, 108, 118, 134, or 139, with reference to the wild type amino acid sequence.
  • the MspA contains the mutations D90N/D91N/D93N, with reference to the wild type sequence positions (referred to therein as "MIMspA” or “Ml-NNN”). In another embodiment, the MspA contains the mutations D90N/D91N/D93N/D118R/D134R/E139K, with reference to the wild type sequence positions (referred to therein as "M2MspA”). See U.S. Pub. No. 2012/0055792.
  • Such mutations can result in a MspA nanopore that comprises a vestibule having a length from about 2 to about 6 nm and a diameter from about 2 to about 6 nm, and a constriction zone having a length from about 0.3 to about 3 nm and a diameter from about 0.3 to about 3 nm, wherein the vestibule and constriction zone together define a tunnel.
  • the amino acid substitutions described in these examples provide a greater net positive charge in the vestibule of the nanopore, further enhancing the energetic favorability of interacting with a negatively charged analyte polymer end.
  • the nanopores can include or comprise DNA-based structures, such as generated by DNA origami techniques.
  • DNA origami-based nanopores for analyte detection, see PCT Pub. No. WO2013083983, incorporated herein by reference.
  • the nanopore can be a solid state nanopore.
  • Solid state nanopores can be produced as described in U.S. Patent Nos. 7,258,838 and 7,504,058, incorporated herein by reference in their entireties. Solid state nanopores have the advantage that they are more robust and stable. Furthermore, solid state nanopores can in some cases be multiplexed and batch fabricated in an efficient and cost-effective manner. Finally, they might be combined with micro-electronic fabrication technology.
  • the nanopore comprises a hybrid protein/solid state nanopore in which a nanopore protein is incorporated into a solid state nanopore.
  • the nanopore is a biologically adapted solid-state pore.
  • the nanopore comprises a vestibule and a constriction zone that together form a tunnel.
  • a "vestibule” refers to the cone-shaped portion of the interior of the nanopore whose diameter generally decreases from one end to the other along a central axis, where the narrowest portion of the vestibule is connected to the constriction zone.
  • a vestibule may generally be visualized as "goblet-shaped.” Because the vestibule is goblet- shaped, the diameter changes along the path of a central axis, where the diameter is larger at one end than the opposite end. The diameter may range from about 2 nm to about 6 nm.
  • the diameter is about, at least about, or at most about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or any range derivable therein.
  • the length of the central axis may range from about 2 nm to about 6 nm.
  • the length is about, at least about, or at most about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or any range derivable therein.
  • diameter When referring to "diameter” herein, one can determine a diameter by measuring center-to-center distances or atomic surface-to-surface distances.
  • a “constriction zone” refers to the narrowest portion of the tunnel of the nanopore, in terms of diameter, that is connected to the vestibule.
  • the length of the constriction zone can range, for example, from about 0.3 nm to about 20 nm. Optionally, the length is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, or any range derivable therein.
  • the diameter of the constriction zone can range from about 0.3 nm to about 2 nm.
  • the diameter is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, or any range derivable therein.
  • the range of dimension can extend up to about 20 nm.
  • the constriction zone of a solid state nanopore is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1,2 13, 14, 15, 16, 17, 18, 19, or 20 nm, or any range derivable therein.
  • the nanopore is disposed within a membrane, thin film, or lipid bilayer, which can separate the first and second conductive liquid media, which provides a nonconductive barrier between the first conductive liquid medium and the second conductive liquid medium.
  • the nanopore thus, provides liquid communication between the first and second conductive liquid media.
  • the pore provides the only liquid communication between the first and second conductive liquid media.
  • the liquid media typically comprises electrolytes or ions that can flow from the first conductive liquid medium to the second conductive liquid medium through the interior of the nanopore. Liquids employable in methods described herein are well-known in the art. Descriptions and examples of such media, including conductive liquid media, are provided in U.S. Patent No.
  • the first and second liquid media may be the same or different, and either one or both may comprise one or more of a salt, a detergent, or a buffer. Indeed, any liquid media described herein may comprise one or more of a salt, a detergent, or a buffer. Additionally, any liquid medium described herein may comprise a viscosity-altering substance or a velocity-altering substance.
  • the nanopore is capable of interacting with the nucleic acid analyte polymer serving as the target or focus of a modification analysis herein.
  • the polymer and nanopore are capable of interacting such that the polymer can translocate through the nanopore from a first conductive liquid medium to a second conductive liquid medium.
  • the translocation is preferably in a linear fashion, through the pore to the other side.
  • the terms "interact" or "interacting" indicate that the analyte moves into at least an interior portion of the nanopore and, optionally, moves into the constriction zone so as to maximally effect the measurable current through the nanopore.
  • the terms "through the nanopore” or “translocate” are used to convey that at least some portion of the polymer analyte enters one side of the nanopore and moves to and out of the other side of the nanopore.
  • the first and second conductive liquid media located on either side of the nanopore are referred to as being on the cis and trans regions, where the analyte polymer to be measured generally translocates first from the cis region to the trans region through the nanopore.
  • the analyte polymer to be measured can translocate from the trans region to the cis region through the nanopore.
  • the nanopore system used incorporated a molecular motor, a blocking oligo, and a hairpin primer.
  • the blocking oligo is unzipped from the template strand as the template strand passes linearly from the cis to the trans side.
  • the molecular motor pulls the strand backwards through the nanopore, from the trans to the cis side, by virtue of the polymerase action that is "primed" by the hairpin primer.
  • the entire length of the polymer does not pass through the pore, but sub-portions or segments of the polymer complete the pass through the nanopore for analysis.
  • the analyte nucleic acid polymer can be translocated through the nanopore using a variety of mechanisms.
  • the analyte polymer and/or reference sequence can be electrophoretically translocated through the nanopore by virtue of the electrical field that is applied to the system.
  • some nanopore systems also incorporate structural elements to apply an electrical field across the nanopore-bearing membrane or film.
  • the system can include a pair of drive electrodes that drive current through the nanopores.
  • the system can include one or more measurement electrodes that measure the current through the nanopore. These can be, for example, a patch-clamp amplifier or a data acquisition device.
  • nanopore systems can include an Axopatch-IB patch-clamp amplifier (Axon Instruments, Union City, CA) to apply voltage across the bilayer and measure the ionic current flowing through the nanopore.
  • the electrical field is sufficient to translocate a polymer analyte through the nanopore.
  • the voltage range that can be used can depend on the type of nanopore system being used.
  • the applied electrical field is between about 20 mV and about 260 mV, for protein-based nanopores embedded in lipid membranes.
  • the applied electrical field is between about 40 mV and about 200 mV.
  • the applied electrical field is between about 100 mV and about 200 mV.
  • the applied electrical field is about 180 mV.
  • the applied electrical field can be in a similar range as described, up to as high as 1 V.
  • nanopore systems can include a component that translocates a polymer through the nanopore enzymatically.
  • a molecular motor can be included to influence the translocation of polymers through the nanopore.
  • a molecular motor can be useful for facilitating entry of a polymer into the nanopore and/or facilitating or modulating translocation of the polymer through the nanopore.
  • the translocation velocity, or an average translocation velocity is less than the translocation velocity that would occur without the molecular motor.
  • the molecular motor can be an enzyme.
  • Illustrative, nonlimiting examples useful for nanopore systems include polymerases, exonucleases, a Klenow fragment, helicases (such as hel308/Mbu, T7hp4A, RecD, XpD), translocases, and topoisomerases.
  • a DNA polymerase such as phi29 can be used to facilitate movement in both directions. See Cherf, G.M., et al., "Automated Forward and Reverse Ratcheting of DNA in a Nanopore at 5- A Precision," Nature Biotechnology 30:344-348, 2012; and Manrao et al., 2012, both of which are incorporated herein by reference in their entireties.
  • An embodiment of the present system that utilizes a molecular motor is also schematically illustrated in FIGURES IB and 1C.
  • the present aspect of the disclosure includes the step of detecting an ion current to provide a current patter associated with a portion of the nucleic acid polymer.
  • characteristics of the nucleic acid polymer analyte, or subunit(s) thereof can be determined based on the effect of the polymer, or subunit(s) thereof, on a measurable signal when interacting with the nanopore, such as interactions with the outer rim, vestibule, or constriction zone of the nanopore.
  • the output signal produced by the nanopore system is any measurable signal that provides a multitude of distinct and reproducible signals depending on the physical characteristics of the polymer or polymer subunit(s).
  • the polymer subunit(s) that determine(s) or influence(s) a measurable signal is/are the subunit(s) residing in the "constriction zone," i.e., the three- dimensional region in the interior of the pore with the narrowest diameter.
  • the number of polymer subunits that influence the co- passage of electrolytes and, thus, a current output signal can vary.
  • the ionic current level through the pore is an output signal that can vary depending on the particular polymer subunit(s) residing in the constriction zone of the nanopore at any given time.
  • the current levels can vary to create a trace, or "current pattern,” of multiple output signals corresponding to the contiguous sequence of the polymer subunits that have affected the current at each iterative step.
  • This detection of current levels, or “blockade” events have been used to characterize a host of information about the structure polymers, such as DNA, passing through, or held in, a nanopore in various contexts.
  • a "blockade” is evidenced by a change in ion current that is clearly distinguishable from noise fluctuations and is usually associated with the presence of an analyte molecule, e.g., one or more polymer subunits, within the nanopore such as in the constriction zone.
  • the strength of the blockade, or change in current will depend on a characteristic of the polymer subunit(s) present. Accordingly, in some embodiments, a “blockade” is defined against a “blockade reference” current level.
  • the blockade reference current level corresponds to the current level when the nanopore is unblocked (i.e., has no analyte structures present in, or interacting with, the nanopore).
  • the blockade reference current level corresponds to the current level when the nanopore has a known analyte (e.g., a known analyte polymer subunit) residing in the nanopore.
  • the current level returns spontaneously to the blockade reference level (if the nanopore reverts to an empty state, or becomes occupied again by the known analyte).
  • the current level proceeds to a level that reflects the next iterative translocation event of the polymer analyte domain through the nanopore, and the particular subunit(s) residing in the nanopore change(s).
  • the blockade is established when the current is lower than the blockade reference current level by an amount of about 1-100% of the blockade reference current level. It will be understood that the blockade reference current level can immediately precede the blockade event or, alternatively, be separated from the blockade event by a period of time with intervening current measurements.
  • the ionic current may be lower than the blockade reference current level by a threshold amount of about, at least about, or at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range derivable therein, of the blockade reference current level when a polymer analyte domain subunit enters the nanopore.
  • a threshold amount of about, at least about, or at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range derivable therein, of the blockade reference current level when a polymer analyte domain subunit enters the nanopore.
  • the blockade With respect to the blockade reference current level defined by the presence of a known analyte (e.g., known polymer subunit(s)), the blockade is established when the current is lower or higher than the reference level by an amount of about 1-100% of the reference current level. It will be understood that the blockade reference current level can immediately precede the blockade event or, alternatively, be separated from the blockade event by a period of time with intervening current measurements.
  • a known analyte e.g., known polymer subunit(s)
  • the ionic current may be lower or higher than the blockade reference current level by threshold of about, at least about, or at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range derivable therein, of the blockade reference current level when a polymer analyte domain subunit enters the nanopore.
  • "Deep blockades" can be identified as intervals where the ionic current is lower (or higher) by at least 50% of the blockade reference level. Intervals where the current drops by less than 50% of the blockade reference level can identified as "partial blockades.”
  • the current level in a blockade remains at the reduced (or elevated) level for at least about 1.0 ⁇ 8.
  • the present inventors have determined that the measureable current pattern, specifically one or more blockades in a trace, are associated with structure(s) of one or more contiguous nucleotides of a nucleic acid polymer that reside in the constriction zone of the nanopore during translocation. Furthermore, the inventors have determined that slight modifications, such as methylation and hydroxymethylation, on one or more specific nucleotide residues differentially effect the current flow as compared to a polymer with the same nucleotide sequence, but with unmodified nucleotides. The influence on the current flow is detectable and, as demonstrated below, can be used to accurately detect the modification and map it to the specific nucleotide.
  • the current pattern associated with a portion of the nucleic acid polymer is compared to a "reference current pattern" (as distinct from the "blockade reference level") associated with the same nucleotide sequence as the portion of the nucleic acid polymer, but wherein sequence associate with the reference current pattern does not have any modifications to any of the nucleotides.
  • the presence of a modified nucleotide in the analyte polymer (or a portion thereof) is indicated by a difference between the current pattern and the reference current pattern is detected.
  • the construction zone i.e., the shape of the narrowest portion of the pore tunnel
  • the output signal for each iterative step during translocation i.e., the signal reflecting the passage of a single nucleotide
  • the output signal for each iterative step during translocation is often affected by multiple contiguous nucleotides in the polymer sequence, specifically those that reside in the constriction zone at each iterative passage step.
  • each blockade event in the trace is influenced mostly by a quadromer (or 4-mer) of contiguous nucleotides that reside in the constriction zone at that time.
  • each individual nucleotide in the sequence can ultimately contribute to four blockade events if it passes completely through the constriction zone.
  • the inventors have shown that the specific difference in the signal that results from a modification of a single nucleotide is similarly observed over four blockade events and the profile of the difference in current signal over at least four blockade events is indicative of the specific type of modification and the location of the modification.
  • the specific profile of the difference in current signal over at least four blockade events is also influenced by the sequence context of the quadromer, i.e., the surrounding nucleotide sequence of the quadromer.
  • the determination of the presence of a modified nucleotide can depend on the whether the difference between the analyte polymer current pattern and the reference current pattern is associated with a particular portion of the analyte polymer.
  • the current difference is associated with a portion of the nucleic acid analyte polymer that comprises one or a plurality of contiguous nucleotides of the nucleic acid polymer.
  • the portion of the nucleic acid analyte polymer comprises two or more contiguous nucleotides of the nucleic acid polymer, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides of the nucleic acid polymer. In some embodiments, the portion of the nucleic acid analyte polymer comprises 3, 4, 5, or 6 contiguous nucleotides of the nucleic acid polymer. In some embodiments, the portion of the nucleic acid polymer comprises the nucleotide or nucleotide position with the modification.
  • the portion of the nucleic acid polymer also comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides immediately adjacent to the nucleotide or nucleotide position with the modification.
  • the additional contiguous nucleotides can be immediately adjacent at the 5'-, the 3'-, or both sides of the nucleotide or nucleotide position with the modification.
  • the portion of the nucleic acid polymer includes at least one nucleotide immediately 5'- to the modified nucleotide. In some embodiments, the portion of the nucleic acid polymer includes at least one nucleotide immediately 3'- to the modified nucleotide.
  • the portion of the nucleic acid polymer includes at least one nucleotide immediately 5'- and at least one nucleotide immediately 3' to the modified nucleotide.
  • the two nucleotides positioned immediately 5' to the modified nucleotide had the most influence on the current signal difference, followed by the single nucleotide position immediately 3' to the modified nucleotide. Persons of skill in the art would be able to determine the positions of maximal influence on the signal difference for any nanopore of interest.
  • the inventors discovered that the identity of the specific nucleotides that occupy each position in the nucleic acid polymer portion influence the identifiable character of the current difference.
  • every sequence variation of XY ⁇ G (and XY h CG) were tested and the various current profiles (and differences with the reference sequences) were catalogued (see, e.g., FIGURES 7A and 7B).
  • Such data can be used as a reference, or "look-up" up table, to assist the mapping and identification of the specific modifications that are detected.
  • the present method further comprises identifying the type of modification present in the nucleic acid polymer based on a character of the difference between the current pattern and the reference current pattern.
  • the character is the duration of the difference, e.g., how much time or many blockade events the difference is observed for.
  • the character is the degree of change, e.g., how much the current pattern differs from the reference pattern in terms of increase or decrease.
  • the character can also combine a particular range of current increase or decrease over a particular time (or number of blockade events).
  • knowledge of the specific sequence of the nucleic acid portion can contribute to the determination of whether the difference is indicative of a modified nucleotide.
  • the comparable current patterns for the portion of the analyte nucleic acid polymer and reference current pattern must be associated with the same sequence, whether or not known.
  • a reference nucleic acid polymer can be replicated or amplified from the analyte nucleic acid polymer using conventional techniques that replicate the (potentially unknown) sequence, but that do not replicate the modification. Such techniques include using the polymerase chain reaction.
  • the reference current pattern can be obtained in a number of ways. In some embodiments, the reference current pattern can be generated de novo by similarly applying a reference nucleic acid polymer to nanopore analysis to generate a current pattern.
  • the reference current pattern was previously determined and is available in a reference or look-up table.
  • the reference current pattern can be derived from the modeling signals that would be expected from the structure of the reference portion, i.e., the sequence without modification.
  • the present disclosure provides a method that involves generating a reference current pattern for comparison.
  • this aspect provides a method detecting a nucleotide modification in a nucleic acid polymer, comprising the step of amplifying a target nucleic acid polymer that potentially contains at least one nucleotide modification to produce a reference nucleic acid polymer that does not contain a nucleotide modification.
  • the method further comprises applying the target and reference nucleic acid polymers to a nanopore system comprising a first conductive liquid medium in liquid communication with a second conductive liquid medium through a nanopore.
  • the target nucleic acid polymer is caused to translocate through the nanopore from the first conductive liquid medium to the second conductive liquid medium, and an ion current is detected to provide a target current pattern associated with a portion of the target nucleic acid polymer.
  • the reference nucleic acid polymer is caused to translocate of through the nanopore from the first conductive liquid medium to the second conductive liquid medium, and an ion current is detected to provide a reference current pattern associated with a portion of the reference nucleic acid polymer, wherein the portion of the target nucleic acid polymer comprises the same nucleotide sequence as the portion of the target nucleic acid polymer.
  • the target current pattern is compared to the reference current pattern, wherein a difference between the current pattern and the reference current pattern indicates the presence of a modified nucleotide in the target nucleic acid polymer.
  • the technique of replication is limited to approaches that provide reference nucleic acid polymers that contain the same nucleotide sequences as the portion of the target nucleic acid polymer, but that does not retain the modifications to be detected.
  • the reference nucleic acid polymer is produced from the target nucleic acid polymer using at least one round of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the following describes the application of a nanopore system to detect and map methylated and hydroxymethylated cytosine nucleotides in DNA polymer analyte.
  • the M2-NNN-MspA protein was purified from Mycobacterium smegmatis as previously described in Butler et al (2008).
  • DNA oligonucleotides were synthesized at Stanford University Protein and Nucleic Acid Facility and purified at their facility using column purification methods. See Table 1 for sequence of ssDNA strands used. The location of the modifications is indicated in all figures and in Table 1 with "m” or "h”.
  • DNA templates, primers and blocking oligomers were mixed at relative molar concentrations of 1: 1: 1.2 and annealed by incubating at 95 °C for 3 min followed by slow-cooling to below 30 °C. DNA and phi29 DNAP were stored at -20 °C until immediately before use.
  • An Axopatch 200B integrating patch clamp amplifier (Axon Instruments) applied a 180 mV voltage across the bilayer (trans side positive) and measured the ionic current through the pore.
  • M2-NNN MspA was added to the grounded cis compartment, yielding a concentration of -2.5 ng/ml. Once a single pore inserted, the compartment was flushed with experimental buffer to avoid further insertions.
  • Annealed DNA as shown in FIGURE 1A, was then added to the experimental volume to achieve a final concentration near -1 ⁇ .
  • DNAP DNA Polymerase
  • EDTA and DTT were added to the front well to final concentrations of ImM each. The EDTA and DTT bind up contaminant divalent ions and create the reducing conditions which phi29 requires for functionality.
  • phi29 DNAP is added to a final concentration of 1.5 ⁇
  • a dNTP mixture is added to ⁇ of each of the four standard dNTPs
  • MgCl 2 is added to a final concentration of lOmM.
  • FIGURES IB and 1C The interaction of phi29 DNAP with the MspA nanopore and hybridized template construct is illustrated schematically in FIGURES IB and 1C.
  • the reproducible current levels were generated indicating the nucleotide- by- nucleotide movement of the ssDNA template strand length- wise through the constriction zone of the nanopore.
  • the template strand threads through the nanopore from the cis to the trans side of the pore, while the blocking oligo unzips from the template strand.
  • the 3'-end of the hairpin primer is exposed to the phi29 DNAP active site, which enables the polymerase to extend the primer.
  • this extension action of the polymerase overcomes the original translocation direction of the template strand, resulting in the template strand being "pulled” back through the nanopore from the trans side to the cis side.
  • the electrical field is monitored for the current flowing through the nanopore. Fluctuations of current are indicative of the specific one or more nucleotides of DNA residing in the most constricted portion of the nanopore opening.
  • each blockade event is determined by a quadromer of contiguous nucleotides within the constriction zone of the nanopore.
  • sequence of the quadromer changes leading to a fluctuation in current level.
  • the current fluctuations are recorded to generate a "trace" of current fluctuations over time, with each nucleotide movement associated with a distinct blockade event.
  • Established relative blockades for the different combinations of nucleotides allow for the reconstruction of the DNA sequence with its associated levels. See International PCT Pub. No. WO2013/159042, incorporated herein by reference in its entirety.
  • FIGURE 2 It is noted that the sequence illustrated in FIGURES 2-4 (and set forth herein as SEQ ID NOS:5 and 6) are internal portions of the sequences set forth in SEQ ID NOS: l and 2, respectively, but are illustrated from 3' to 5' to correspond to the temporal acquisition of the data as the phi29 DNAP pulled the template strand into the cis side of the pore.
  • FIGURE 3 which shows the normalized current differences (modified TGCC signal minus the TGCC signal)
  • the introduction of h C tended to result in a lower current level over the associated quadromers
  • the introduction of m C resulted in a consistently higher current level in the associated quadromers.
  • An unmodified CpG present in the modified TGCC template strand notably resulted in a similar current signal as the reference TGCC strand.
  • a statistical analysis wherein Gaussian peaks were fit to the data illustrated in FIGURE 3 resulted in the Gaussian peaks corresponding closely to the locations of the epigenetic modifications in the modified TGCC template.
  • the signal changes resulting from these epigenetic modifications can be precisely correlated to the location of and type of modification within the DNA polymer using a nanopore-based analysis.
  • the following is a description of an expanded study a nanopore-based analysis to detect and distinguish multiple epigenetic modifications that appear in DNA polymers within a variety of different sequence contexts.
  • DNAP phi29 DNA polymerase
  • FIGURES 5A and 5B show raw current traces for unmethylated and methylated DNA, respectively.
  • the extracted average current levels are shown in FIGURE 5C for unmethylated DNA in black and methylated DNA in light gray.
  • FIGURE 5C shows the difference between the methylated and unmethylated current level sequences.
  • FIGURES 6A-6D show the average current level differences in 20 or more single-molecule comparisons for four different DNA constructs. Across all such comparisons, m C consistently increases current relative to C, whereas h C generally decreased current relative to C.
  • the current difference caused by a m C or a h C was found to be strongly affected by the sequence context in which it is embedded.
  • the nucleotides immediately adjacent to a m C or h C have the greatest influence on the size and shape of the current difference.
  • the nucleotides on the 5' side were varied and the nucleotide on the 3' side of the C was fixed as a G because of the biological relevance of CpG sites.
  • it was observed that the nucleotide two positions to the 5' side of the modified cytosine have a bigger influence than the nucleotide two positions toward the 3' side, which have a lesser effect (see FIGURE 10).
  • FIGURES 7A and 7B Results for all 16 XY m CpGs and XY h CpGs are summarized in FIGURES 7A and 7B.
  • the maximum difference is up to 7 pA depending on sequence context. On average, the maximum difference caused by m C is approximately 2.5 pA (FIGURE 7A, Bottom Right Panel).
  • FIGURE 7A Bottom Right Panel
  • four nucleotides within MspA's constriction affect each current level (see, e.g., schematic illustrations in FIGURES IB and FIGURE 8), with the two nucleotides centered in the pore's constriction affecting the current the most (Manrao et al., 2011; Manrao et al., 2012).
  • the present results are consistent in that the replacement of C for m C or h C affects approximately four consecutive current levels.
  • the current difference is maximal when the m C is positioned immediately to cis of the constriction and the shape of the difference peak exhibits skewness.
  • the schematic in FIGURE 8 shows how sequence context dependence arises.
  • MspA's cross-section is shown in solid black, and variable gray shading indicates the region of high electric field.
  • Nucleotides within the region of high electric field affect the ion current.
  • m C or h C pass through the pore, their location relative to the pore constriction determines how much they affect the current.
  • All nucleotides within the high-field region of the constriction will influence the current, and therefore alter the influence of a ⁇ Kl and h C modification.
  • the nucleotide to the 3' of the CpG is also relevant, albeit to a smaller extent.
  • FIGURE 4 the 3' side of the current difference peak caused by m C is reduced.
  • the data in FIGURE 7 demonstrate that the four nucleotides X, Y, C, and G dominate the magnitude of the current difference caused by m CpG and h CpG (see also FIGURE 10).
  • FIGURES 9A-9D shows a construct with several modified Cs spaced only five nucleotides apart.
  • the current difference peaks associated with the four m Cs and two h Cs are still easily distinguishable.
  • the difference peak is wider and higher than the signal for just one m CpG within the same context.
  • Placing a h CpG immediately adjacent to a " pG (FIGURE 9D) reduces the signal of the nearby m CpGs. The signal is approximately a superposition of the individual " and h C signals.
  • Methylated sites with smaller current differences such as CT m CpG and TC m CpG, were detected with lower accuracy: ⁇ 86% and ⁇ 88%, respectively (see TABLE 2 for individual context-dependent detection rates).
  • h C true-positive rates were lower than for m C.
  • m C was distinct from h C; "KIpGs were miscalled as h CpGs in 3 out of 478 occurrences, whereas h CpGs were never miscalled as roCpGs in 609 reads.
  • true negatives included non-CpG regions in addition to CpGs tested above, resulting in a higher true- negative detection rate than in the method described in the preceding paragraph. Rates from these two methods are not directly comparable.
  • a Bayesian classification measure was used to find m Cs, yielding similar detection efficiencies (see the Materials and Methods in the below Examples section).
  • m C detection without reference to DNA sequence is useful for hypermethylation or hypomethylation detection and is comparable to other nanopore methylation detection techniques (Wanunu, M., et al., "Discrimination of Methylcytosine From Hydroxymethylcytosine in DNA Molecules," Journal of the American Chemical Society 133(3):486-492, 2011; Shim et al., 2013).
  • the nanopore strand sequencing method used in this work produces a second read of the same DNA molecule because of the bi-directional movement of the template strand through the nanopore (Manrao et al., 2012). Using this second read can improve calling accuracies. In contrast to other m C and h C detection techniques that rely on m C- specific chemical reactions and/or enzymatic kinetics, the present system detects the methylation directly.
  • the present methylation detection method does not require de novo sequencing with the nanopore to detect methylation. Given a previously measured reference current sequence for unmethylated DNA and known context-dependent methylation patterns as in FIGURES 7 A and 7B, one can then take a single read of a methylated DNA molecule and detect methylation with confidence for most sequence contexts. Because PCR does not copy certain epigenetic modifications such as methylation, nanopore reads of amplified copies would serve as the unmethylated reference. Genomic DNA would then be extracted, given adapters to enable polymerase control, and then be presented to the pore. Individual reads of methylated DNA could then be aligned to the current level reference using a Smith- Waterman alignment algorithm (Manrao et al., 2012).
  • phi29 DNAP was used as a molecular motor to control the motion of DNA through a single MspA pore established in an unsupported phospholipid bilayer.
  • the buffer was 300 mM KC1, 10 mM Hepes buffered at pH 8.00 + 0.05. Currents were recorded on an Axopatch 200B amplifier with custom Labview software (National Instruments) at a voltage bias of 180 mV.
  • DNA template, primer, and blocking oligomer were mixed together in a 1: 1: 1.2 ratio to a final concentration of 50 ⁇ .
  • DNA was then annealed by heating to 95 °C for 5 min, cooling to 60 °C for 2 min, and then cooling to 4 °C.
  • Experimental concentrations were ⁇ 500 nM for DNA, ⁇ 500 nM for phi29 DNAP, ⁇ 500 ⁇ for dNTPs, -10 mM for MgC12, and ⁇ 1 mM for DTT.
  • This sequence creates a reproducible current motif that signals the end of the read. This region was used to calibrate currents and, thus, to control for small changes in buffer conductivity due to evaporation or temperature variation.
  • the sequence of interest followed this calibration sequence.
  • the DNA was designed to contain a variety of nucleotides adjacent to the CpGs. Each strand had at least three CpGs embedded in a random sequence, sufficiently spaced so that their current signatures did not overlap. In each strand, three of these CpGs were uniformly either unmethylated, methylated, or hydroxymethylated. Additionally, eight different DNA sequences were examined (PAN Laboratories, Stanford University, Stanford, CA) containing various methylation patterns (TABLE 2 for sequences used). Some experiments were performed with a mixture of methylated, hydroxymethylated, and unmethylated DNA. Without calibration, these strands could still be sorted by methylation- specific currents.
  • blockade events were determined using a thresholding method on current data.
  • a feed-forward neural network removed events that did not correspond with phi29 polymerase activity. Once appropriate events were determined, raw current levels were discerned using a custom-written graphical user interface. Current level transition boundaries were selected, and the median current levels were extracted in the time order that they occurred for each event. The phi29 DNAP occasionally exhibited backstepping, causing repeated levels that were removed. Consensus current level sequences were found for each sequence type, and event levels associated with that sequence were automatically aligned using a Needleman-Wunsch algorithm. For experiments with DNA mixtures, a quality score from the Needleman-Wunsch algorithm was used to distinguish DNA with different types of methylation.
  • Events are found using a threshold detection algorithm, described in Butler et al. (2008). Events with durations less than 1 second or with average currents greater than 75 pA or less than 15 pA were rejected.
  • a feed-forward neural network consisting of 5 layers, each layer containing 20 neurons, was employed. The features used in the neural network were the event duration, the event average current and variance, and the outputs of a K-means clustering algorithm. The neural network was trained with -200 events. The neural network removed 100% of the events that were not associated with polymerase activity.
  • DNA type classification After extracting current level sequences, we constructed consensus current level sequences specific to unmethylated, methylated, and hydroxymethylated DNA constructs. These consensus current level sequences were constructed using events from experiments containing a single sequence and methylation pattern of DNA, without a mixture of other methylation patterns. Alignment to form consensus level sequences was performed using a Needleman-Wunsch algorithm with an affine gap, as used in Manrao et al., 2012. An event classification algorithm was developed to sort events from experiments that had mixtures of unmethylated, methylated, and hydroxymethylated DNA. The algorithm aligned each event to the consensus levels extracted above and produced a similarity score used to classify each event.
  • the classification method was tested on 297 events from experiments containing a single sequence and methylation pattern of DNA, yielding a classification accuracy of 99.7%.
  • This score-based whole-event classification algorithm was used to separate methylated, hydroxymethylated, and unmethylated events in experiments that were run with mixtures of DNA with various methylation patterns.
  • the consensus levels for unmethylated, methylated, or hydroxymethylated were updated as these events were classified.
  • FIGURES 5A-5D and FIGURES 6A-6D With events classified and current levels aligned, we constructed the level differences, shown in FIGURES 5A-5D and FIGURES 6A-6D. From the known CpG location the level differences surrounding the CpG were extracted, as shown in FIGURE 7. These level differences are used in the Bayesian methylation classifier described below. Level differences near the CpG were used to distinguish m CpG, hCpG, and unmethylated CpG's. We estimated the ability of a single level near the CpG to accurately call the type of CpG's methylation with a t-test that compared the three hypotheses: methylated, hydroxymethylated, or unmethylated (FIGURES 1 OA- IOC).
  • FIGURES 10A, 10B, and IOC also show the average of the t-test for different contexts, yielding the classification power of a given position for all contexts XYCpG.
  • the plots of classification power resemble the current difference plots shown in the right-most bottom plot in FIGURES 7A and 7B, but include level variance information and are independent of sign.
  • the magnitude of the classification power indicates how distinguishable m CpG is from CpG, h CpG is from CpG, and all three are from each other in FIGURES 10A, 10B, and IOC, respectively. It is observed that level positions -1, 0 and 1, corresponding to the two levels on the 5' side of the modified C site and one level to the 3' side of the modified C, have the highest discrimination power.
  • This table encompasses two similar tables, A and B. Within each table, the calling frequencies and detected count of XYCpG's are presented. Each row gives the calling frequency for a m C, h C, or C called as a m C, h C or C, as indicated for each row. The highlighted rows indicate CpG sites called as non-methylated CpG due to positive "KIpG or h CpG detection within 2 levels. Each column indicates the context within which its calling frequencies were obtained. Numbers in parentheses are the count of observed CpG levels within the given context and 5 type of CpG.
  • the fourth row in the first column states that in 81% of 75 observed events, an AA h CG was accurately called as an AA h CG.
  • the final column provides the average and the standard error of the calling frequency obtained by bootstrap resampling 1/5 of the observed events for each construct 5 times.
  • TABLE 3A has the results classification using three levels (-1, 0 , and 1) and TABLE 3B has the results classification using two levels (-1 and 0).
  • Bayesian probability classifier as described below, was used. This started with P(XYZ ⁇ ⁇ ; ⁇ ), which is the probability of the sequence hypothesis XYZ given set of current differences ⁇ A j ⁇ .
  • the letters X and Y are each any standard nucleotide and Z is a C, m C, or h C.
  • P( ⁇ A ⁇ ⁇ XYZ) was defined, which is the probability of observing current difference knowing the sequence XYZ at location i. This was modeled as
  • ⁇ ( ⁇ ; I XYZ) exp(-(A ; - A XYZ i ) ⁇ 2 ⁇ XYZ J ) , (equation 1) where A XYZ i and ⁇ ⁇ ⁇ are the mean and variance of the level difference at position i for context XYZ (as shown in FIGURES 7 A and 7B).
  • Equation 3 The highest probability for equation 3 was used to classify the set of current differences as belonging to the sequence hypothesis XYZ.
  • the prior probability can be taken to be the expected probability of CpG methylation or hydroxymethylation for a given sample, or to be 1/3 as was chosen for the samples. Because XYZ were compared over all hypotheses, the factor ⁇ ( ⁇ ; ⁇ ) does not matter, and only the product of probabilities from Equation 1 were used.
  • Equation 3 Given the known sequence and location of the CpG site with known context XY, Equation 3 was used and considered only C, m C, or h C. Classification of the CpG was given by the highest value of PiXYZ). Classification frequency was calculated as the number of classifications divided by the number of expected classifications for the given XYZ context.
  • Equation 3 Given an unknown sequence, but the observed level differences, Equation 3 was extended to consider all dinucleotide hypotheses and all cytosine variants C, m C, or h C. The known level differences were compared to all sets of level differences within a given event, and observed peaks in probabilities for given XYZ hypotheses. Classification was given by the highest probability along the progression of level differences.
  • a peak detection algorithm was used to identify methylation sites without using sequence specific knowledge (i.e., events were not compared using known current difference patterns for various sequence contexts). Such detection can identify methylation sites independent of the sequence of the examined DNA with reasonable accuracy.
  • the peak detection required level differences that reach a maximal height of 1.1 pA and that have a separation of at least 6 levels from any adjacent peaks. With these parameters, -93% true-positive methylation within 2 levels of the known methylation position were identified, and >99% true-negative (non-methylation) were identified. Increasing the requisite peak height improved true-negative detection, at the cost of true- positive detection. While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

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Abstract

La présente invention concerne en général des procédés de mesure, de diagnostic, de visualisation et/ou de détection de modifications dans des acides nucléiques à l'aide d'une analyse sur la base de nanopore. Les procédés comprennent la translocation du polymère d'acide nucléique à travers un nanopore, la détection d'un motif de courant ionique associé à une partie du polymère d'acide nucléique et la comparaison du motif de courant à un motif de courant de référence associé à la même séquence nucléotidique que la partie du polymère d'acide nucléique mais qui ne contient aucune modification. Dans certains modes de réalisation, les procédés et compositions sont utiles pour la détection, la distinction et la cartographie précises de modifications épigénétiques dans des acides nucléiques tels que la 5-méthylcytosine (mC) et la 5-hydroxyméthylcytosine (hC). Dans certains modes de réalisation, les procédés comprennent l'amplification d'un polymère d'acide nucléique cible ayant des modifications épigénétiques attendues pour générer un polymère d'acide nucléique de référence ayant la même séquence nucléotidique mais sans aucune modification épigénétique.
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US10822652B2 (en) * 2013-08-30 2020-11-03 University Of Washington Through Its Center For Commercialization Selective modification of polymer subunits to improve nanopore-based analysis
EP3137490B1 (fr) 2014-05-02 2021-01-27 Oxford Nanopore Technologies Limited Pores mutants
WO2015175789A1 (fr) * 2014-05-14 2015-11-19 Mcruer Robert N Régulation de translocation pour la détection par un nanopore
US10457979B2 (en) 2014-05-14 2019-10-29 Stratos Genomics, Inc. Translocation control for sensing by a nanopore
US10676782B2 (en) 2014-05-14 2020-06-09 Stratos Genomics, Inc. Translocation control for sensing by a nanopore
WO2015197355A1 (fr) * 2014-06-25 2015-12-30 Joseph Prosser Séquenceur
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WO2016141221A1 (fr) * 2015-03-03 2016-09-09 Stratos Genomics, Inc. Renvoi du séquençage des protéines se liant à des polynucléotides à des applications connexes
US20180044725A1 (en) * 2015-03-03 2018-02-15 Stratos Genomics, Inc. Polynucleotide binding protein sequencing
WO2016164363A1 (fr) * 2015-04-06 2016-10-13 The Regents Of The University Of California Procédé permettant de déterminer l'emplacement de bases dans un polynucléotide
US10760117B2 (en) 2015-04-06 2020-09-01 The Regents Of The University Of California Methods for determining base locations in a polynucleotide
WO2017027518A1 (fr) * 2015-08-10 2017-02-16 Stratos Genomics, Inc. Séquençage de molécule unique d'acide nucléique avec des complexes de chimiorécepteurs
WO2020043082A1 (fr) * 2018-08-28 2020-03-05 Nanjing University Nanopore protéique pour l'identification d'un analyte
WO2020168286A1 (fr) * 2019-02-14 2020-08-20 University Of Washington Systèmes et procédés pour analyse fondée sur des nanopores d'acides nucléiques améliorée
US20220366313A1 (en) * 2019-02-14 2022-11-17 University Of Washington Systems and methods for improved nanopore-based analysis of nucleic acids
US12321837B2 (en) * 2019-02-14 2025-06-03 University Of Washington Systems and methods for improved nanopore-based analysis of nucleic acids
US20200333290A1 (en) * 2019-04-16 2020-10-22 The Board Of Trustees Of The University Of Illinois Classification of epigenetic biomarkers and/or dna conformational superstructures via use of atomically thin nanopores
US11860123B2 (en) * 2019-04-16 2024-01-02 The Board Of Trustees Of The University Of Illinois Classification of epigenetic biomarkers and/or DNA conformational superstructures via use of atomically thin nanopores
CN113470751A (zh) * 2021-06-30 2021-10-01 南方科技大学 一种蛋白纳米孔氨基酸序列的筛选方法、蛋白纳米孔及其应用
WO2023030146A1 (fr) * 2021-08-30 2023-03-09 四川大学 Méthode d'analyse de compositions de polypeptides basées sur des nanopores à mspa modifiés par des ions cuivre
CN115877018A (zh) * 2022-08-05 2023-03-31 四川大学华西医院 一种孔蛋白在制备检测去氢表雄酮硫酸酯的试剂盒中的应用
WO2024156993A1 (fr) * 2023-01-25 2024-08-02 Oxford Nanopore Technologies Plc Étalonnage d'un dispositif de réseau de nanopores
WO2025155921A1 (fr) * 2024-01-17 2025-07-24 Government Of The United States Of America, As Represented By The Secretary Of Commerce Mesure de dommages induits par un rayonnement à un biopolymère

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