Nuclease assisted nanopore sequencing

Abstract

The disclosed technology relates to methods, systems, and structures for nuclease assisted nanopore sequencing. In some embodiments, the methods, systems, and structures generally involve nanopore sequencing an oligonucleotide or polynucleotide comprising a plurality of nucleotides. In some embodiments the plurality of nucleotides may have cyclic loops and arresting constructs to facilitate the readout of the nucleotide sequence.

Description

NUCLEASE ASSISTED NANOPORE SEQUENCINGINCORPORATION BY REFERENCE TO RELATED APPLICATION

[0001] This PCT application claims priority to U.S. Provisional Application 63 / 651687, filed May 24, 2024, the entirety of which is incorporated herein for any and all purposes.BACKGROUND

[0002] Some polynucleotide sequencing techniques involve performing a large number of controlled reactions on support surfaces or within predefined reaction chambers. The controlled reactions may then be observed or detected, and subsequent analysis may help identify properties of the polynucleotide involved in the reaction. Examples of such sequencing techniques include next-generation sequencing or massive parallel sequencing involving sequencing-by-ligation, sequencing-by-synthesis, reversible terminator chemistry, or pyrosequencing approaches.

[0003] Some polynucleotide sequencing techniques utilize a nanopore, which can provide a path for an ionic electrical current. For example, as the polynucleotide traverses through the nanopore, it influences the electrical current through the nanopore. Each passing nucleotide, or series of nucleotides, that passes through the nanopore yields a characteristic blockage current. These characteristic electrical currents of the traversing polynucleotide can be recorded to determine the sequence of the polynucleotide.SUMMARY

[0004] The readhead of nanopores (e.g., the constriction region of nanopores) usually “senses” several bases concurrently along the sample DNA strand, increasing the challenge of accurate nanopore sequencing due to many permutations of signals arising from different sequences. For example, the MspA pore reads about 4 bases as a time, giving rise to at least 4A4= 256 different signals that need to be deconvoluted and resolved. Methods that allow for separation of signals, increased ease of deconvolution, or otherwise provide for distinct signals, are therefore desirable in order to increase read speed and accuracy during sequencing.

[0005] In one aspect, the disclosed technology provides a method that allows for spatial separation of individual nucleotide or nucleotide analogues within the readhead of a nanopore, generally by using exonucleases to specifically generate single nucleotides. In some embodiments, the single nucleotides comprise one or more modifications, including arresting constructs and barcoding / reporting moieties. In some embodiments, the arresting constructs interact with inner pore residues to arrest (e.g., halt or slow) translocation of the attached nucleotide through the pore. In some embodiments, an exonuclease or other related enzyme can be conjugated to the entrance of the pore or free floating in solution. Thus, the disclosed technology allows decoding of individual bases. The exonuclease cleaved nucleotides and any attached modifications may produce a distinguishable signal or a distinguishable signal break as each nucleotide in a sequence of nucleotides passes through the nanopore, thereby isolating and / or enhancing the recorded signals from the nucleotides. Thus, the disclosed technology allows improved resolution of the recorded signal. In some embodiments, individual nucleotides and attached modifications may be slowed within the readhead and generate a unique readout when translocating through the pore. In some embodiments, individual nucleotides and attached modifications may be arrested or slowed within the readhead, and translocation occurs only with the application of an increased voltage for a short period of time. In another aspect, linker constructs may join or connect adjacent separated nucleotides, thus preserving nucleotide sequence and order, and creating a synthetic strand to be sequenced at a nanopore readhead. In some embodiments, the linker construct comprises a cyclic loop that may be severed to create an elongated nucleotide. In some embodiments, an adapter can reside within the inner surface of a nanopore, downstream of any exonucleases. In some embodiments, the adapter interacts with nucleotides or modifications on the nucleotide to modulate residence time, capture efficiency, and overall translocation rate of nucleotides through the readhead.

[0006] In another aspect, the disclosed technology provides systems, devices, kits, and methods which allow cleavable linkages along the DNA backbone, synthesis of cleavable cyclic loop nucleotides, barcodes for individual base identification, and polymerase mutation for incorporation of modified nucleotides. In some embodiments, the disclosed technology also provides systems, devices, kits, and methods to allow for exonuclease cleavage of nucleic acid strands, including DNA and RNA, to constituent nucleotides and modified nucleotides thereof,for individual base identification when read on a nanopore system. Systems may be prepared to allow parallel reads in multiple nanopores, such as thousands or millions of nanopores. Accordingly, components of any system may be functionally duplicated to multiply sequencing throughput. Any system may also be adapted with microfluidics or automation.

[0007] The systems, devices, kits, and methods disclosed herein each have several aspects, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the claims, some prominent features will now be discussed briefly. Numerous other examples are also contemplated, including examples that have fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and particularly after reading the section entitled “Detailed Description,” one will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0008] The methods disclosed herein may further include linkers or cyclic loops, including compounds generally having one of the following structures, with or without additional modifications:, or -NH-; Li is a first linking group; L2 is a second linking group; and SP is a spacer.

[0009] The methods disclosed herein can also include an oligonucleotide comprising one of the following structures:S-, or -NH-; Li is a first linking group; L2 is a second linking group; and SP is a spacer.

[0010] In some embodiments, SP comprises one or more of the following moieties: (1) alkyl chains having 5 to 50 carbons, (2) oligonucleotides or modified oligonucleotides having 10 to 100 repeating units, (3) polypeptides having 10 to 100 repeating units, (4) hydrophilic polymers having 10 to 100 repeating units selected from the group consisting of polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine, and (5) hydrophobic polymers having 10 to 100repeating units selected from the group consisting of polylactic acid, polymethylmethacrylate, and polystyrene.

[0011] In some embodiments, each of Li and L2 independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentafluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene.

[0012] In some embodiments, Li and L2 independently further comprises a first linker between the conjugating moiety and X or X’, and a second linker between the conjugating moiety and SP.

[0013] In some embodiments, the first linker and the second linker are independently selected from the group consisting of a polynucleotide having 10 to 100 repeating units, polypeptide having 10 to 100 repeating units, alkyl chains having 10 to 200 carbons, hydrophilic polymers having 10 to 100 repeating units comprising polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, or polyethyleneimine, hydrophobic polymers having 10 to 100 repeating units comprising polylactic acid, polymethylmethacrylate, or polystyrene, and combinations thereof.

[0014] In some embodiments, SP further comprises a side group. In some embodiments, the Base further comprises a side group. In some embodiments, the side group is a linear, a branched or a cyclic polymer. In some embodiments, the side group comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof. In some embodiments, the side group comprises an arresting construct to slow or halt translocation through the nanopore. In some embodiments, SP does not comprise a side group.

[0015] In some embodiments, disclosed herein is also an adapter for interacting with nucleotides or modified versions thereof. Examples of adapters include cyclodextrin (CD), cucurbituril (CB), calixarenes, crown ether, cryptophanes, resorcinarenes, pyrogallolarenes or pillararenes. In some embodiments, adapters can be modified to enhanceassociation with nucleotides or modified nucleotides, including amino groups to interact with phosphate groups, or other groups to modify local microenvironment pKa.

[0016] In some embodiments, any of the aforementioned structures may be cleaved by an exonuclease, endonuclease, or similar enzyme, the cleavage occurring at specific locations along a polyphosphate backbone.

[0017] Disclosed herein also includes a method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the method comprising: providing a polynucleotide comprising nucleotides, selectively cleaving a P-0 bond on the target polynucleotide using an enzyme to repeatedly generate a separated nucleotide from one end of the target polynucleotide, wherein each separated nucleotide is translocated through the nanopore in order of being generated; and applying a voltage across a readhead to identify a first reporter element in a constriction of a nanopore based on a first electrical response in the system. In some embodiments, the enzyme is an exonuclease. In some embodiments, the enzyme is conjugated to the entrance of the nanopore. In some embodiments, the enzyme is free in solution. In some embodiments, the reading voltage comprises a fixed waveform. In some embodiments, the reading voltage comprises a square waveform. In some embodiments, each of the nucleotides of the target polynucleotide further comprises a modification configured to arrest the separated nucleotide relative to the nanopore as the separated nucleotide is translocating through the nanopore. In some embodiments, the modification is covalently attached to the nucleotide. In some embodiments, the modification is unique to the type of nucleotide to which the modification is attached. In some embodiments, each nucleotide in the target polynucleotide is a cyclic loop nucleotide, wherein the cyclic loop nucleotide comprises a spacer and one or more reporter elements linking a nucleobase and a phosphate group of the nucleotide. In some embodiments, the separated nucleotides are linked together by the spacer. In some embodiments, only one nucleotide resides in the readhead during applying a reading voltage.

[0018] In some embodiments, the nanopore further comprises an adapter inside of the nanopore, wherein the adapter is configured to interact with the separated nucleotide to arrest the separated nucleotide relative to the nanopore during translocation.

[0019] Also disclosed herein is a method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the method comprising: providing atarget polynucleotide comprising cyclic loop nucleotides, wherein each of the cyclic loop nucleotides comprises a spacer linking a nucleobase and a phosphate group of the nucleotides; selectively cleaving a P-0 bond on the target polynucleotide using an enzyme thereby forming an elongated polynucleotide with spaced nucleotides; and applying a reading voltage across a readhead to identify a first reporter element in a constriction of a nanopore based on a first electrical response in the system. In some embodiments, each cyclic loop nucleotide further comprises a modification configured to arrest the spaced nucleotide relative to the nanopore as the elongated nucleotide is translocating through the nanopore. In some embodiments, the nanopore further comprises an adapter inside the nanopore, and the adapter is configured to interact with the spaced nucleotide to arrest the spaced nucleotide relative to the nanopore during translocation.

[0020] In some embodiments, the nanopore is a Mycobacterium smegmatis porin A (MspA), or Alpha hemolysin (aHL).

[0021] Disclosed herein includes a kit for performing a method for determining a sequence of a polynucleotide in a nanopore-based sequencing system, the kit comprising the compound disclosed herein.

[0022] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

[0024] Figure 1 schematically illustrates exonuclease cleavage sites along a polynucleotide.

[0025] Figure 2 schematically illustrates an example of sequencing a polynucleotide strand through a nanopore (prior art).

[0026] Figure 3 schematically illustrates an example of exonuclease processing a polynucleotide strand and subsequent sequencing of individual nucleotides through a nanopore with an adapter.

[0027] Figure 4 schematically illustrates an example of exonuclease processing a modified polynucleotide strand and subsequent sequencing of individual modified nucleotides through a nanopore.

[0028] Figure 5 schematically illustrates an example of exonuclease processing a modified polynucleotide strand and subsequent sequencing of individual modified nucleotides through a nanopore with an adapter.

[0029] Figure 6 schematically illustrates an example of a waveform used in the process of sequencing individual modified nucleotides through a nanopore.

[0030] Figure 7 schematically illustrates an example of a waveform used in the process of sequencing individual modified nucleotides through a nanopore.

[0031] Figure 8 shows cleaved bonds of a polynucleotide chain modified with linker constructs.

[0032] Figures 9 and 10 illustrate example non-covalent adapter interactions with alpha cyclodextrin (Figure 9) and beta cyclodextrin (Figure 10).DETAILED DESCRIPTION

[0033] All patents, applications, published applications and other publications referred to herein are incorporated herein by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference.Definitions

[0034] All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0035] As used herein, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sequence” may include a plurality of such sequences, and so forth.

[0036] The terms comprising, including, containing and various forms of these terms are synonymous with each other and are meant to be equally broad. Moreover, unless explicitly stated to the contrary, examples comprising, including, or having an element or a plurality of elements having a particular property may include additional elements, whether or not the additional elements have that property.

[0037] As used herein, the term “adapter” or “adapters” refers to any chemical molecule (e.g. Cyclodextrin, cucurbituril, pillararenes, etc.) which are known to interact with nucleotides or modified nucleotides.

[0038] As used herein, the term “modified oligonucleotide” or “modified nucleotide” refers to a polymeric chain of nucleobases or nucleotides assembled with moieties comprising a modified nucleobase, modified sugar rings (e.g. LN A, constraint ethyl, ethylene bridged, TNA, 2’-0Me, 2’F, 2’-M0E) or nucleobases attached to a scaffold (e.g. unlock, 4’- thio, CeNA, HNA, TNA, GNA, FNA). The term may also refer to any modification introduced to nucleotides which either: interact with residues comprising the inner pore of a nanopore or interact with adapters located in the microenvironment around the nanopore readhead.

[0039] As used herein, the term “phosphoramidite analogs” refers to any polymer synthesized using phosphoramidite or related chemistries resulting in the formation of phosphodiester, methylphosphonate, or phosphorothioate bonds between each moiety.

[0040] As used herein, the term “modified polyamide” refers to a polymer assembled with individual moieties each having at least one amino group and one carboxylic acid group, resulting in the formation of amide bonds.

[0041] As used herein, the term “nanopore” is intended to mean a hollow structure discrete from, or defined in, and extending across the membrane. The nanopore permits ions, electric current, and / or fluids to cross from one side of the membrane to the other side of the membrane. For example, a membrane that inhibits the passage of ions or water-soluble molecules can include a nanopore structure that extends across the membrane to permit the passage (through a nanoscale opening extending through the nanopore structure) of the ions or water-soluble molecules from one side of the membrane to the other side of the membrane. The diameter of the nanoscale opening extending through the nanopore structure can vary along its length (i.e., from one side of the membrane to the other side of the membrane), but at any point is on the nanoscale (i.e., from about 1 nm to about 100 nm, or to less than 1000 nm).Examples of the nanopore include, for example, biological nanopores, solid-state nanopores, and biological and solid-state hybrid nanopores. In some embodiments, a nanopore refers to a pore having an opening with a diameter at its most narrow point of about 0.3 nm to about 2 nm. For example, a nanopore may be a solid-state nanopore, a graphene nanopore, an elastomer nanopore, or may be a naturally-occurring or recombinant protein that forms a tunnel upon insertion into a bilayer, thin film, membrane, or solid-state aperture, also referred to as a protein pore or protein nanopore herein (e.g., a transmembrane pore). If the protein inserts into the membrane, then the protein is a tunnel-forming protein.

[0042] As used herein, the term “diameter” is intended to mean a longest straight line inscribable in a cross-section of a nanoscale opening through a centroid of the crosssection of the nanoscale opening. It is to be understood that the nanoscale opening may or may not have a circular or substantially circular cross-section (the cross-section of the nanoscale opening being substantially parallel with the cis / trans electrodes). Further, the cross-section may be regularly or irregularly shaped.

[0043] As used herein, “cis” refers to the side of a nanopore opening through which an analyte or modified analyte enters the opening or across the face of which the analyte or modified analyte moves.

[0044] As used herein, “trans” refers to the side of a nanopore opening through which an analyte or modified analyte (or fragments thereof) exits the opening or across the face of which the analyte or modified analyte does not move.

[0045] As used herein, the term “biological nanopore” is intended to mean a nanopore whose structural portion is made from materials of biological origin. Biological origin refers to a material derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Biological nanopores include, for example, polypeptide nanopores and polynucleotide nanopores.

[0046] As used herein, a “moiety” is one of two or more parts into which something may be divided, such as, for example, the various parts of a tether, a molecule or a probe.

[0047] As used herein, a “reporter” is composed of one or more reporter elements. Reporters include what are known as “tags” and “labels.” The linker construct or cyclic loop (when including reporter moiety) or nucleobase residue of the elongated polymer can beconsidered a reporter. Reporters serve to parse the genetic information of the target nucleic acid.

[0048] As used herein, a “linker” is a molecule or moiety that joins two molecules or moieties and provides spacing between the two molecules or moieties such that they are able to function in their intended manner. For example, a linker can comprise a diamine hydrocarbon chain that is covalently bound through a reactive group on one end to an oligonucleotide analog molecule and through a reactive group on another end to a solid support, such as, for example, a bead surface. Coupling of linkers to nucleotides and substrate constructs of interest can be accomplished through the use of coupling reagents that are known in the art (see, e.g., Efimov et al., Nucleic Acids Res. 27: 4416-4426, 1999). Methods of derivatizing and coupling organic molecules are well known in the arts of organic and bioorganic chemistry. A linker may also be cleavable or reversible.

[0049] As used herein, the term “polypeptide nanopore” is intended to mean a protein / polypeptide that extends across the membrane, and permits ions, electric current, polymers such as DNA or peptides, or other molecules of appropriate dimension and charge, and / or fluids to flow therethrough from one side of the membrane to the other side of the membrane. A polypeptide nanopore can be a monomer, a homopolymer, or a heteropolymer. Structures of polypeptide nanopores include, for example, an a-helix bundle nanopore and a 0-barrel nanopore. Example polypeptide nanopores include a-hemolysin, Mycobacterium smegmatis porin A (MspA), gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-pilus, etc. The protein a-hemolysin is found naturally in cell membranes, where it acts as a pore for ions or molecules to be transported in and out of cells. Mycobacterium smegmatis porin A (MspA) is a membrane porin produced by Mycobacteria, which allows hydrophilic molecules to enter the bacterium. MspA forms a tightly interconnected octamer and transmembrane beta-barrel that resembles a goblet and contains a central pore.

[0050] As used herein, a “peptide” refers to two or more amino acids joined together by an amide bond (that is, a “peptide bond”). Peptides comprise up to or include 50 amino acids. Peptides may be linear or cyclic. Peptides may be a, 0, y, 5, or higher, or mixed. Peptides may comprise any mixture of amino acids as defined herein, such as comprising any combination of D, L, a, 0, y, 5, or higher amino acids.

[0051] As used herein, a “protein” refers to an amino acid sequence having 51 or more amino acids.

[0052] A polypeptide nanopore can be synthetic. A synthetic polypeptide nanopore includes a protein-like amino acid sequence that does not occur in nature. The protein-like amino acid sequence may include some of the amino acids that are known to exist but do not form the basis of proteins (i.e., non-proteinogenic amino acids). The protein-like amino acid sequence may be artificially synthesized rather than expressed in an organism and then purified / isolated.

[0053] The nanopores disclosed herein may be hybrid nanopores. A “hybrid nanopore” refers to a nanopore including materials of both biological and non-biological origins. An example of a hybrid nanopore includes a polypeptide-solid-state hybrid nanopore and a polynucleotide-solid-state nanopore.

[0054] As used herein, an “exonuclease” refers to enzymes that selectively cleave nucleotides sequentially from the end of a polynucleotide chain. For some exonucleases, a hydrolyzing reaction breaks phosphodi ester bonds at either the 3’ or 5’ end of a polynucleotide chain.

[0055] The application of the electric potential difference across a nanopore may force the translocation of a nucleic acid through the nanopore. In some embodiments, a square waveform may be used to push one or more nucleotides through the nanopore, wherein each “pulse” on a square waveform forces the translocation of one or more nucleotides through the nanopore. One or more signals are thus generated that correspond to the translocation of the nucleotide through the nanopore. Accordingly, as a target polynucleotide, or as a mononucleotide or a probe derived from the target polynucleotide or mononucleotide, transits through the nanopore, the current across the membrane changes due to base-dependent (or probe dependent) blockage of the constriction, for example. The signal from that change in current can be measured using any of a variety of methods. Each signal is unique to the species of nucleotide(s) (or linker constructs with a reporter moiety region) in the nanopore, such that the resultant signal can be used to determine a characteristic of the polynucleotide. For example, the identity of one or more species of nucleotide(s) (or probe) that produces a characteristic signal can be determined.

[0056] As used herein, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is a ribose, and in deoxyribonucleotides (DNA), the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present at the 2' position in ribose. The nitrogen containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-l atom of deoxyribose is bonded to N-l of a pyrimidine or N-9 of a purine. The phosphate groups may be in the mono- , di-, or tri-phosphate form. These nucleotides are natural nucleotides, but it is to be further understood that non-natural nucleotides, modified nucleotides or analogs of the aforementioned nucleotides can also be used.

[0057] As used herein, “nucleobase” is a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. A nucleobase can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted at the 8 position with methyl or bromine, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7- deaza-adenine, N4-ethanocytosine, 2,6- diaminopurine, N6-ethano-2,6-diaminopurine, 5- methylcytosine, 5-(C3-C6)- alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510 and PCT applications WO 92 / 002258, WO 93 / 10820, WO 94 / 22892, and WO 94 / 24144, and Fasman (“Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, 1989, CRC Press, Boca Raton, LO), all herein incorporated by reference in their entireties.

[0058] The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids inmanner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphorothiolate DNA. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, diTP, 2-amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate, pyrrolo-pyrimidine triphosphate, and 2-thiocytidine, as well as the alphathiotriphosphates for all of the above, and 2'-O-methyl-ribonucleotide triphosphates for all the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

[0059] As used herein, the term “signal” is intended to mean an indicator that represents information. Signals include, for example, an electrical signal and an optical signal. The term “electrical signal” refers to an indicator of an electrical quality that represents information. The indicator can be, for example, current, voltage, tunneling, resistance, potential, voltage, conductance, or a transverse electrical effect. An “electronic current” or “electric current” refers to a flow of electric charge. In an example, an electrical signal may be an electric current passing through a nanopore, and the electric current may flow when an electric potential difference is applied across the nanopore.

[0060] As used herein, the term “driving force” is intended to mean an electrical current that allows a polynucleotide to translocate through the nanopore. In some embodiments, the electrical current electric current may flow when an electric potential difference is applied across the nanopore.

[0061] As used herein, the term “holding force” is intended to mean a resistance that slows and / or stops a polynucleotide to translocate through the nanopore. In some embodiments, the holding force is overcome by the application of a driving force. Thus, the driving force overcomes / overrides the resistance that slows and / or stops a polynucleotide, thereby allowing the polynucleotide to translocate through the nanopore.

[0062] As used herein, the term “modification” is intended to mean a moiety attached to a nucleotide. A modification provides a resistance (in the form of a “holding force”) that slows and / or stops a polynucleotide to translocate through the nanopore unless the resistance due to the modification is overcome by a “driving force.” The resistance provided by the modification is due to a property of the modification (e.g., size, geometry, and / or non-covalent interaction with the nanopore). Modifications can operate as a ratchet or a brake for the polypeptide translocation through a nanopore. A modification can be attached to any part of the nucleotide and can also be attached to the nucleotide at two locations forming a loop.

[0063] The aspects and examples set forth herein and recited in the claims can be understood in view of the above definitions.Overview

[0064] A common drawback with nanopore sequencers is that the nanopore is only sensitive to multiple bases of a DNA strand in the nanopore, as opposed to reading a single base one at a time. For example, the MspA nanopore has a constriction region which serves as a readhead of at least 4 nucleotides (termed a “k-mer”), resulting in at least 256 (4A4) different permutations of 4-mer sequences that need to be deconvoluted. For a k-mer of 5 bases, the number of possible signals is 4A5= 1,024. A longer readhead will result in an exponential increase in the number of signals to be differentiated, which complicates the sequencing readout and increases the complexity of base calling, thus reducing accuracy. Another issue with nanopore sequencers is that the speed of translocation of natural single stranded DNA is on the order of >10 million nucleotides per second, way above the rate that is compatible with electronics and detectors. Among the advantages of the methods described herein are techniques to arrest (halt or slow) translocation to achieve controlled reading of individual or fewer groups of bases, and allow for single base reading.

[0065] Exonuclease assisted sequencing has been demonstrated previously. Individual nucleic acid molecules could be sequenced by a modified protein nanopore as they are released by exonucleases. While the use of an adapter to improve the frequency of nucleotides being captured has been demonstrated, approximately 1 out of every 1000 single nucleotides that collided with the pore mouth reached the adapter. Furthermore, many blockade events are too short-lived to be detected. Thus, many single nucleotides would translocate through the pore without corresponding detection, resulting in deletions of a nucleotide in generated sequences. Presented herein are methods of effectively arresting (slowing or halting) translocation. In some embodiments, the method involves the application of an “advancement” pulse or signal to translocate nucleotides through a nanopore.

[0066] In some embodiments, disclosed are methods involving exonucleases to arrest translocation while also reducing read complexity of signals generated at the nanoporereadhead. In some embodiments, exonucleases are used to digest (or cleave) polynucleotide strands. In some embodiments, the polynucleotide strands comprise nucleotides, or modified nucleotides. In some embodiments, the nanopore microenvironment comprises an adapter. In some embodiments, a square waveform may be generated to translocate a nucleotide or modified nucleotide across a nanopore. In some embodiments, the exonuclease digested polynucleotides comprise one or more cyclic loops. The effect of the disclosed technology allows having 1 nucleotide or linked nucleotide residing in the readhead at any point in time, successfully reducing the diversity of reads, enabling more accurate sequencing at a lower cost. Alternatively, the rate of translocation of any nucleotide across a nanopore may be attenuated such that the rate is slowed, or even halted such that a nucleotide is translocated only upon generation of a specific electrical signal. In some embodiments, the disclosed technology provides high throughput, cheaper and more accurate DNA sequencing.System and Method

[0067] Figure 1 schematically illustrates sites along a polynucleotide chain’s polyphosphate backbone for exonucleases or related enzymes to attack a P-0 bond. In some embodiments, an exonuclease located in solution, or located at a specific site relative to or inside a nanopore, can process and selectively cut specific P-0 bonds on a polynucleotide, including polynucleotides bearing modifications (such as reporters) and / or cyclic loops.

[0068] Figure 2 schematically illustrates an example of sequencing a polymer, or strand, wherein a strand of polynucleotides is fed through a nanopore. A strand 202 comprising nucleotides is fed through a nanopore 204, which nanopore resides in a lipid bilayer 206. The strand 202 translocates through the nanopore 204, passing through the readhead 208. The k- mer length as each successive nucleotide translocates through the nanopore can generate multiple signals, increasing complexity of base calling and signal deconvolution. Diversity of reads is dependent on the K-mer resident within or occupying the nanopore at any given time.

[0069] Figure 3 schematically illustrates an example of sequencing a polymer, or strand, wherein the strand is sequentially processed through an exonuclease. A strand 202 is processed by exonuclease 302, attached to nanopore 204. The resulting excised nucleotide(s) 304 are then individually translocated through the nanopore readhead 208. Moreover, as depicted in Figure 3, an adapter 306, which interacts with nucleotides, is present within the microenvironment of nanopore 204. It can be appreciated that the adapter 306 slows down therate of translocation of individual nucleotides 304, by interacting with moieties present within the nucleotide molecular structure, thus increasing resident time in the nanopore for each nucleotide.

[0070] Figure 4 schematically illustrates an example of sequencing a polymer, or strand, wherein the strand is sequentially processed through an exonuclease, and the strand is modified with a modification, which is depicted as a tail off of A, T, C, and G. A modified strand 402 is processed by exonuclease 302, attached to nanopore 204. The resulting excised modified nucleotide(s) 404 are then individually translocated through the nanopore readhead 208. The modifications present on the modified nucleotide(s) are configured to arrest the translocation of the modified nucleotide(s) 304 by interacting, or otherwise sterically blocking against residues within the microenvironment of the nanopore 204 or the readhead 208. Arrest of nucleotide translocation may require the use of electric current, or other signals, to sequentially process and translocate each modified nucleotide.

[0071] Figure 5 schematically illustrates an example of sequencing a polymer, or strand, wherein the strand is sequentially processed through an exonuclease, the strand is modified with a modification, and an adapter is present within the microenvironment of the nanopore. A modified strand 402 is processed by exonuclease 302, attached to nanopore 204. The resulting excised modified nucleotide(s) 404 are then individually translocated through the nanopore readhead 208. The modifications present on the modified nucleotide(s) are configured to arrest the translocation of the modified nucleotide(s) 304 by interacting, or otherwise sterically blocking residues within the microenvironment of the nanopore 204 or the readhead 208. Moreover, an adapter 306, which interacts with modified nucleotide(s) and / or modifications, is present within the microenvironment of nanopore 204. It can be appreciated that the adapter 306 slows down the rate of translocation of individual modified nucleotide(s) 404 by interacting with moieties present within the nucleotide molecular structure, thus increasing resident time in the nanopore for each nucleotide. Arrest of nucleotide translocation may require the use of electric current, or other signals, to sequentially process and translocate each modified nucleotide.

[0072] Figures 6 and 7 illustrate sample waveforms which may be configured for the overall rate of the arrest of translocation. Figure 6 illustrates a fixed waveform, wherein the modification on the nucleotide interacts transiently with inner pore residues, and translocationis subsequently slowed. In contrast, Figure 7 illustrates a square waveform, wherein the modification on the nucleotide blocks translocation at a first voltage, and translocation occurs with the application of an increased voltage (second voltage) for a short period of time, which effectively pulls the nucleotide through a constriction at or near the readhead.

[0073] Figure 8 illustrates the addition of a cyclic loop to a sample polynucleotide. Polynucleotides with cyclic loop structures can be processed by an exonuclease to cleave specific bonds, including bonds along the polyphosphate backbone. Due to cyclic loop modifications which connect adjacent nucleotides, the result of cleavage is an elongated polymer comprising modified nucleotides, the elongated polymer preserving the sequence and order of constituent nucleotides compared to the parent polynucleotide.

[0074] By “translocation,” it is meant that an analyte (e.g., DNA) enters one side of an opening of a nanopore and move to and out of the other side of the opening. It is contemplated that any embodiment herein comprising translocation may refer to electrophoretic translocation or non-electrophoretic translocation, unless specifically noted. An electric field may move an analyte (e.g., a polynucleotide) or modified analyte. By “interacts,” it is meant that the analyte (e.g., DNA) or modified analyte moves into and, optionally, through the opening, where “through the opening” (or “translocates”) means to enter one side of the opening and move to and out of the other side of the opening. Optionally, methods that do not employ electrophoretic translocation are contemplated. In some embodiments, physical pressure causes a modified analyte to interact with, enter, or translocate (after alteration) through the opening. In some embodiments, a magnetic bead is attached to an analyte or modified analyte on the trans side, and magnetic force causes the modified analyte to interact with, enter, or translocate (after alteration) through the opening. Other methods for translocation include, but are not limited to, gravity, osmotic forces, temperature, and other physical forces such as centripetal force.

[0075] In some embodiments, the nanopore may comprise a solid-state material, such as silicon nitride, modified silicon nitride, silicon, silicon oxide, or graphene, or a combination thereof. In some embodiments, the nanopore is a protein that forms a tunnel upon insertion into a bilayer, membrane, thin film, or solid-state aperture. In some embodiments, the nanopore is located in the thickness of a lipid bilayer. In some embodiments, the nanopore is located in an artificial membrane comprising a mycolic acid. The nanopore may be aMycobacterium smegmatis porin (Msp) having a vestibule and a constriction zone that define the tunnel. The Msp porin may be a mutant MspA porin. In some embodiments, amino acids at positions 90, 91, and 93 of the mutant MspA porin are each substituted with asparagine. Some embodiments may comprise altering the translocation velocity or sequencing sensitivity by removing, adding, or replacing at least one amino acid of an Msp porin. A “mutant MspA porin” is a multimer complex that has at least or at most 70, 75, 80, 85, 90, 95, 98, or 99 percent or more identity, or any range derivable therein, but less than 100%, to its corresponding wildtype MspA porin and retains tunnel-forming capability. A mutant MspA porin may be recombinant protein. Optionally, a mutant MspA porin is one having a mutation in the constriction zone or the vestibule of a wild-type MspA porin. Optionally, a mutation may occur in the rim or the outside of the periplasmic loops of a wild-type MspA porin. A mutant MspA porin may be employed in any embodiment described herein.

[0076] A “vestibule” refers to the cone-shaped portion of the interior of an Msp porin 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 also be referred to as a “goblet.” The vestibule and the constriction zone together define the tunnel of an Msp porin. A “constriction zone” or the “readhead” refers to the narrowest portion of the tunnel of an Msp porin, in terms of diameter, that is connected to the vestibule. The length of the constriction zone may range from about 0.3 nm to about 2 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 may range from about 0.3 nm to about 2 nm. Optionally, 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. A “tunnel” refers to the central, empty portion of an Msp porin that is defined by the vestibule and the constriction zone, through which a gas, liquid, ion, or analyte may pass. A tunnel is an example of an opening of a nanopore.

[0077] Various conditions such as light and the liquid medium that contacts a nanopore, including its pH, buffer composition, detergent composition, and temperature, may affect the behavior of the nanopore, particularly with respect to its conductance through thetunnel as well as the movement of an analyte with respect to the tunnel, either temporarily or permanently.

[0078] In some embodiments, the disclosed system for nanopore sequencing comprises an Msp porin having a vestibule and a constriction zone that define a tunnel, wherein the tunnel is positioned between a first liquid medium and a second liquid medium, wherein at least one liquid medium comprises an analyte polynucleotide, and wherein the system is operative to detect a property of the analyte. The system may be operative to detect a property of any analyte comprising subjecting an Msp porin to an electric field such that the analyte interacts with the Msp porin. The system may be operative to detect a property of the analyte comprising subjecting the Msp porin to an electric field such that the analyte electrophoretically translocates through the tunnel of the Msp porin. In some embodiments, the system comprises an Msp porin having a vestibule and a constriction zone that define a tunnel, wherein the tunnel is positioned in a lipid bilayer between a first liquid medium and a second liquid medium, and wherein the only point of liquid communication between the first and second liquid media occurs in the tunnel. Moreover, any Msp porin described herein may be comprised in any system described herein. In some embodiments, the system may further comprise an amplifier or a data acquisition device. The system may further comprise one or more temperature regulating devices in communication with the first liquid medium, the second liquid medium, or both. The system described herein may be operative to translocate an analyte through an Msp porin tunnel either electrophoretically or otherwise.

[0079] In some embodiments, polymerase may be used to integrate various modifications into nucleotides, or polynucleotide strands. Polymerase is used as an enzyme generally for joining 3'-OH 5 '-triphosphate nucleotides, oligomers, and their analogs. Polymerases include, but are not limited to, DNA-dependent DNA polymerases, DNA- dependent RNA polymerases, RNA- dependent DNA polymerases, RNA-dependent RNA polymerases, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase I, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, VentR® DNA polymerase (New England Biolabs), Deep VentR® DNA polymerase (New England Biolabs), Bst DNA Polymerase Large Fragment, Stoeffel Fragment, 90N DNA Polymerase, 90N DNA polymerase, Pfu DNA Polymerase, Tfl DNA Polymerase, Tth DNA Polymerase, RepliPHIPhi29 Polymerase, Tli DNA polymerase, eukaryotic DNA polymerase beta, telomerase, Therminator™ polymerase (New England Biolabs), KOD HiFi™ DNA polymerase (Novagen), K0D1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered by bioprospecting, and polymerases cited in US 2007 / 0048748, US 6,329,178, US 6,602,695, and US 6,395,524 (incorporated by reference). These polymerases include wild-type, mutant isoforms, and genetically engineered variants. “Encode” or “parse” are verbs referring to transferring from one format to another, and refer to transferring the genetic information of the target template base sequence into an arrangement of reporters.

[0080] After the polymerization process is completed, cleavage at predetermined locations (such as shown in Figure 8) opens the loops and increases the distances between adjacent nucleotides. In some embodiments, cleavage can occur along P-0 bonds and be mediated by an exonuclease. Cleavage of the polymer strand can be designed to occur at any part of the backbone as long as it occurs within the loop structure between the two positions where the linker construct is attached to the nucleotide structure. Cleavage of the polymer strand along the backbone opens the loops and elongates the polymer strand, leaving the linker construct conjoining the backbone phosphate and the sugar. In embodiments where the linker construct contains a modification configured to interact with the nanopore, the modifications may slow or halt the translocation of the elongated polymer and allow the nucleotides to be read by the nanopore one at a time.

[0081] In embodiments where a reporter moiety (such as a reporter barcode) is a part of a linker construct, the cleaved product, i.e., the elongated polymer, exposes a series of reporter moiety, each of which reports the identity of the base to which it corresponds. In embodiments where the linker construct also contains a modification configured to interact with the nanopore, the elongated polymer can be sequenced on the nanopore one barcode at a time.Cleavable Cyclic Loop Nucleotides

[0082] Cleavable cyclic loop nucleotides are nucleotides / nucleotide analogs that are modified to include a linker construct attached to two positions of the nucleotide / nucleotide analog structure. Although “cleavable cyclic loop nucleotide” is used, it includes both modifiednatural nucleotide and modified nucleotide analogs. The nucleotides and nucleotide analogs useful as described herein include the following compounds:wherein X is NHR, OR, or CH2R; R is H, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl; and Base is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil. A linker construct may be conjugated to the nucleotide / nucleotide analog to form a cleavable cyclic loop nucleotide.

[0083] The linker construct can be conjugated at one end to the nucleotide / nucleotide analog at by means of either a P-N, P-O, or P-C bond or other P-X heteroatom bond. The other end can be conjugated to (i) any position on the nucleobase in all the structure above, (ii) the 5’-carbon of the ribose sugar in structures NT-1, NT-3, and NT-4, (iii) the N-atom adjacent to the 5’-carbon in structure NT-4, (iv) a 5’-allyl modification in structure NT-2, or (v) any position on the ribose ring. Examples of covalent conjugation chemistries include amine-NHS ester, amine-imidoester, amine-pentafluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehydealkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene. Once the linker construct is conjugated to the nucleotide / nucleotide analog, it forms the cyclic loop portion of the cleavable cyclic loop nucleotide. Thus the cyclic loop comprises an -L1-SP-L2- moiety.

[0084] Examples of the cleavable cyclic loop nucleotides include:wherein, or -NH-; Li is a first linking group; L2 is a second linking group; SP is a spacer; and Base is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil.

[0085] Further examples of the cleavable cyclic loop nucleotide compound that may be included in the nanopore sequencing system or the kit for nanopore sequencing are:wherein, or -NH-; Li is a first linking group; L2 is a second linking group; SP is a spacer; and Base is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil.

[0086] In some embodiments, each of the first linking group Li and the second linking group L2 independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentafluorophenyl ester, aminehydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiolpyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehydealkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene. Li and L2 may or may not be the same.

[0087] In some embodiments, each of the first linking group Li and the second linking group L2 may independently further comprise a linker. A first linker may be present between the conjugating moiety and X or X’ (alpha phosphate), and a second linker may be present between the conjugating moiety and SP. In some embodiments, the linker may be selected from the group consisting of hydrophilic polymers (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine), hydrophobic polymers (polylactic acid, polymethylmethacrylate, polystyrene), oligonucleotides, peptides, polypeptides, aliphatic chains (C5 to C50) and combinations thereof. In some embodiments, the first and the second linkers may independently comprisepeptides, polypeptides, alkyl chains, polyethylene glycol, or combinations thereof. In some embodiments, one or more linkers may be absent.

[0088] In some embodiments, SP comprises a polymer. In some embodiments, SP comprises oligonucleotides, modified oligonucleotides, hydrophilic polymers (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine), hydrophobic polymers (polylactic acid, polymethylmethacrylate, polystyrene), polypeptides, modified polyamide, simple aliphatic chains (C5 to C50), substituted aliphatic chains (the substituent may include halo such as chloro, bromo or fluoro, alkyl such as methyl, ethyl or propyl, or aromatic groups such as phenyl or pyridyl), or combinations thereof.

[0089] In some embodiments, phosphoramidite analogs may include:

[0090] Phosphoramidite analogs can be assembled into a polymer using an oligonucleotide synthesis process.

[0091] In some embodiments, polypeptides can comprise both natural and unnatural amino acid residues, including residues selected from the following:L / D-Unnatural amino acids

[0092] In some embodiments, modified oligonucleotides may comprise modified nucleotides and / or modified nucleobases. In some embodiments, examples of modified nucleotides and modified nucleobases include, but are not limited to:Modified nucleotides

[0094] In some embodiments, the spacer may comprise a reporter moiety that correspond to the specific nucleobase. In other embodiments, the spacer may not include a reporter moiety.

[0095] In some embodiments, SP comprises one or more of the following moieties: (1) alkyl chains having 5 to 50 carbons, (2) oligonucleotides, modified oligonucleotides or phosphoramidite analogs having 10 to 100 repeating units, (3) polypeptides having 10 to 100 repeating units, (4) hydrophilic polymers having 10 to 100 repeating units selected from the group consisting of polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine, and (5) hydrophobic polymers having 10 to 100 repeating units selected from the group consisting of polylactic acid, polymethylmethacrylate, and polystyrene. In some embodiments, the alkyl chains may be substituted or unsubstituted.

[0096] In some embodiments, the cleavable cyclic loop nucleotide may further comprise a side group configured to interact with the nanopore. The side group may comprise a linear, a branched or a cyclic polymer, wherein the polymer is selected from a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, and combinations thereof. In some embodiments, the side group may be a side branch attached to the cleavable cyclic loop nucleotide. In some embodiments, the side group may be a side branch attached to the nucleobase of the nucleotide / nucleotide analog. Insome embodiments, the side group may be a side branch attached to the cyclic loop - on either the spacer SP or the linking group Li or L2.

[0097] When the side group is attached to the spacer SP portion of the cyclic loop, the modification may be attached to any part of the spacer, for example, in the middle of the spacer chain, on either ends of the spacer chain, or anywhere in between. In embodiments where the side group is attached to the middle of the cyclic loop structure, the cyclic loop may be a symmetrical loop. In embodiments where the side group is attached to other part of the cyclic loop structure, the cyclic loop may be an asymmetrical loop.

[0098] The side group may be attached to the cleavable cyclic loop nucleotide through a third linking group L3. In some embodiments, L3 may be a moiety selected from the group consisting of hydrophilic polymers (polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine), hydrophobic polymers (polylactic acid, polymethylmethacrylate, polystyrene), oligonucleotides, peptides, polypeptides, aliphatic chains (C5 to C50), aromatic groups (phenyl or pyridyl) and combinations thereof.

[0099] Cleavable cyclic loop nucleotides comprise certain cleavable sites in bonds which can be broken under controlled conditions such as, for example, conditions for selective cleavage of a phosphorothiolate bond, a photocleavable bond, a phosphoramidate bond, a phosphoramide bond, a 3'-O-B- D-ribofuranosyl-2' bond, a thioether bond, a selenoether bond, a sulfoxide bond, a disulfide bond, deoxyribosyl-5'-3' phosphodiester bond, or a ribosyl-5'-3' phosphodiester bond, as well as other cleavable bonds known in the art. A selectively cleavable bond can be an intra-tether bond or between or within a probe or a nucleobase residue or can be the bond formed by hybridization between a probe and a template strand. Selectively cleavable bonds are not limited to covalent bonds, and can be non-covalent bonds or associations, such as those based on hydrogen bonds, hydrophobic bonds, ionic bonds, pi-bond ring stacking interactions, Van der Waals interactions, and the like.

[0100] For example, in some embodiments, the cleavable sites include the P-Y bond / linkage in cleavable cyclic loop nucleotide structures (I) and (II), the P-N bond / linkage in structure (III), and the O-C (5’-C of the ribose sugar) bond / linkage in structures (IV) and (V). These bonds are shown as bolded bonds below and can be cleaved under conditions known in the art.wherein X, X’, Y, Li, L2, SP and Base are as defined above.Daughter Strand

[0101] Compound (la), (Ila), (Illa), (IVa), and (Va) may be used as cyclic loop nucleotides for synthesizing a daughter strand. The daughter strand comprises at least one of the compounds (I), (II), (III), (V), and (V). In some embodiments, the daughter strand is formed by linking a plurality of compounds (I), a plurality of compound (II), a plurality of compound (III), a plurality of compound (IV), or a plurality of compound (V) or a combination of these together into a polynucleotide or an oligonucleotide. The daughter strand may comprise one of the following structures:wherein X, X’, Y, Li, L2, SP, and Base are as defined above, and the adjacent Bases on the daughter strand may be different or the same.Cleavage of Cyclic Loop Nucleotide

[0102] The daughter strand can further be subject to a condition disclosed herein suitable for cleaving the cyclic loop nucleotides, elongating the backbone to form an elongated polymer:wherein X, X’, Y, Li, L2, SP, and Base are as defined above, and the adjacent Bases on the elongated polymer strand may be different or the same.

[0103] The cyclic loops can be designed symmetrically or asymmetrically, depending on the conjugation chemistry on the nucleotide. In some embodiments, each of the first linking group Li and the second linking group L2 independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, aminepentafluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol- maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azidephosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azidenorbornene. Li and L2 may or may not be the same.

[0104] In some embodiments, each of the first linking group Li and the second linking group L2 may independently further comprise a linker. A first linker may be present between the conjugating moiety and a base, and a second linker may be present between the conjugating moiety and another portion of a nucleotide. In some embodiments, the linker may be selected from the group consisting of hydrophilic polymers (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine), hydrophobic polymers (polylactic acid, polymethylmethacrylate, polystyrene), oligonucleotides, peptides, polypeptides, aliphatic chains (C5 to C50) and combinations thereof. In some embodiments, the first and the second linkers may independently comprisepeptides, polypeptides, alkyl chains, polyethylene glycol, or combinations thereof. In some embodiments, one or more linkers may be absent.

[0105] In some embodiments, SP comprises a polymer. In some embodiments, the polymer comprises oligonucleotides, modified oligonucleotides, hydrophilic polymers (polyethylene glycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine), hydrophobic polymers (polylactic acid, polymethylmethacrylate, polystyrene), polypeptides, aliphatic chains (C5 to C50), substituted aliphatic chains (small molecules such as chloro, bromo or fluoro, alkyl such as methyl, ethyl or propyl, or aromatic groups such as phenyl or pyridyl), or combinations thereof. In some embodiments, modified oligonucleotides may include oligonucleotides that do not have a baseattached to the sugar, such as ' ° cya. In some embodiments, modified oligonucleotidesan oligonucleotide synthesis process. In some embodiments, the spacer may comprise a reporter moiety that correspond to the specific nucleobase. In other embodiments, the spacer may not include a reporter moiety.

[0106] In some embodiments, the cyclic loop may comprise one or more barcodes. Examples include, but not limited to, nucleosidic bases, non-nucleosidic bases, peptides or other synthetic polymers such as polyethyleneglycol, polyvinylalcohol, polyacrylamide, polyvinylpyrrolidone, polyethyleneimine, etc.

[0107] In some embodiments, disclosed herein is a method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the method comprising providing a target polynucleotide comprising nucleotides, selectively cleaving a P- O bond on the target polynucleotide using an enzyme to repeatedly generate a separatednucleotide from one end of the target polynucleotide, wherein each separated nucleotide is translocated through the nanopore in order of being generated; and applying a reading voltage across a readhead to identify a first reporter element in a constriction of a nanopore based on a first electrical response in the system.

[0108] In some embodiments, the enzyme is an exonuclease. In some embodiments, the enzyme is either conjugated to the entrance of the nanopore or free in solution. In some embodiments, the reading voltage comprises a fixed waveform. In some embodiments, the reading voltage comprises a square waveform. In some embodiments, each of the nucleotides of the target polynucleotide further comprises a modification configured to arrest the separated nucleotide relative to the nanopore as the separated nucleotide is translocating through the nanopore. In some embodiments, the modification is covalently attached to the nucleotide. In some embodiments, the modification is unique to the type of the nucleotide to which the modification is attached. In some embodiments, each nucleotide in the target polynucleotide is a cyclic loop nucleotide, wherein the cyclic loop nucleotide comprises a spacer linking a nucleobase and a phosphate group of the nucleotide. In some embodiments, the separated nucleotides are linked together by the spacer. In some embodiments, only one nucleotide resides in the readhead during reading. In some embodiments, only 2, 3, 4, 5, 10, nucleotides, or any value in between, are present in the readhead prior to translocation or reading. In some embodiments, the nanopore further comprises an adapter inside of the nanopore, and the adapter is configured to interact with the separated nucleotide to arrest the separated nucleotide relative to the nanopore during translocation.

[0109] In some embodiments, provided herein is a method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the method comprising: providing a target polynucleotide comprising cyclic loop nucleotides, wherein each of the cyclic loop nucleotides comprises a spacer linking a nucleobase and a phosphate group of the nucleotides; selectively cleave a P-0 bond on the target polynucleotide using an enzyme thereby forming an elongated polynucleotide with spaced nucleotides; and applying a reading voltage across a readhead to identify a first reporter element in a constriction of a nanopore based on a first electrical response in the system. In some embodiments, each cyclic loop nucleotide further comprises a modification configured to arrest the spaced nucleotide relative to the nanopore as the elongated nucleotide is translocating through the nanopore. Insome embodiments, the nanopore further comprises an adapter inside of the nanopore, and the adapter is configured to interact with the spaced nucleotide to arrest the spaced nucleotide relative to the nanopore during translocation. In some embodiments, the enzyme is an exonuclease. In some embodiments, the enzyme is either conjugated to the entrance of the nanopore or free in solution. In some embodiments, the reading voltage comprises a fixed waveform. In some embodiments, the reading voltage comprises a square waveform. In some embodiments, each of the nucleotides of the target polynucleotide further comprises a modification configured to arrest the separated nucleotide relative to the nanopore as the separated nucleotide is translocating through the nanopore. In some embodiments, the modification is covalently attached to the nucleotide. In some embodiments, the modification is unique to the type of the nucleotide to which the modification is attached. In some embodiments, each nucleotide in the target polynucleotide is a cyclic loop nucleotide, wherein the cyclic loop nucleotide comprises a spacer linking a nucleobase and a phosphate group of the nucleotide. In some embodiments, the separated nucleotides are linked together by the spacer. In some embodiments, only one nucleotide resides in the readhead during reading. In some embodiments, 2, 3, 4, 5, 10, nucleotides, or any value in between, are present in the readhead prior to translocation or reading. In some embodiments, the nanopore further comprises an adapter inside of the nanopore, and the adapter (e.g. a macrocyclic structure such as shown in Figures 9 and 10 below) may be configured to interact with the separated nucleotide to arrest the separated nucleotide relative to the nanopore during translocation. In some embodiments, the nanopore is a Mycobacterium smegmatis porin A (MspA). In some embodiments, the nanopore is an Alpha hemolysin (aHL).Non-Covalent Adapter Interactions

[0110] Figures 9 and 10 illustrate example non-covalent adapter interactions with alpha cyclodextrin (Figure 9) and beta cyclodextrin (Figure 10). The type of interaction varies depending upon the base that is interacting with the cyclodextrin. For example, shown in the figures are the interactions of adenosine, guanosine, uridine, and cytidine. Some bases may interact the strongest in a non-covalent manner (such as through hydrogen bonding) on the rim of the cyclodextrin and some bases may interact more strongly with the interior of the cyclodextrin. For example, the interaction between alpha cyclodextrin and uridine or cytidineis shown as an inclusion complex interaction in the interior or base of the alpha cyclodextrin. On the other hand, the interaction of alpha cyclodextrin and adenosine or guanosine is shown as a no-inclusion interaction where the non-covalent interaction is on the rim of the cyclodextrin via hydrogen bonding. This is due to the larger size of the purine nitrogeneous bases which are unable to fit into the interior cavity of the alpha cyclodextrin. For Figure 10, all of the interactions shown with beta cyclodextrin are inclusion complex interactions. In some cases, one or more water molecules may facilitate non-covalent interactions with the cyclodextrins.Additional Notes

[0111] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0112] Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

[0113] It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if such value or sub-range were explicitly recited. For example, a range from about 2 nm to about 20 nm should be interpreted to include not only the explicitly recited limits of from about 2 nm to about 20 nm, but also to include individual values, such as about 3.5 nm, about 8 nm, about 18.2 nm, etc., and sub-ranges, such as from about 5 nm to about 10 nm, etc. Furthermore, when “about” and / or “substantially”are / is utilized to describe a value, this is meant to encompass minor variations (up to + / - 10%) from the stated value.

[0114] While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

[0115] While certain examples have been described, these examples have been presented by way of example only, and are not intended to limit the scope of the disclosure. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms. Furthermore, various omissions, substitutions and changes in the systems and methods described herein may be made without departing from the spirit of the disclosure. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the disclosure.

[0116] Features, materials, characteristics, or groups described in conjunction with a particular aspect, or example are to be understood to be applicable to any other aspect or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing examples. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0117] Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations, one or more features from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a sub-combination or variation of a sub-combination.

[0118] Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order shown or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted or described can be incorporated in the example methods and processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some examples, the actual steps taken in the processes illustrated and / or disclosed may differ from those shown in the figures. Depending on the example, certain of the steps described above may be removed or others may be added. Furthermore, the features and attributes of the specific examples disclosed above may be combined in different ways to form additional examples, all of which fall within the scope of the present disclosure. Also, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products. For example, any of the components for an energy storage system described herein can be provided separately, or integrated together (e.g., packaged together, or attached together) to form an energy storage system.

[0119] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0120] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain examples include, while other examples do not include, certain features, elements, and / or steps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without user inputor prompting, whether these features, elements, and / or steps are included or are to be performed in any particular example.

[0121] Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain examples require the presence of at least one of X, at least one of Y, and at least one of Z.

[0122] Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0123] The scope of the present disclosure is not intended to be limited by the specific disclosures of preferred examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.

[0124] Although the foregoing invention has been described in terms of certain preferred embodiments, other embodiments will be apparent to those of ordinary skill in the art. Additionally, other combinations, omissions, substitutions and modification will be apparent to the skilled artisan, in view of the disclosure herein. Accordingly, the present invention is not intended to be limited by the recitation of the preferred embodiments, but is instead to be defined by reference to the appended claims.

[0125] The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner and unless otherwise indicated refers to the ordinary meaning as would be understood by one of ordinary skill in the art in view of the specification. Furthermore, embodiments may comprise, consist of, consist essentially of, several novel features, no single one of which is solely responsible for its desirable attributes or is believed to be essential to practicing the embodiments herein described. As used herein, the section headings are for organizational purposes only and are not to be construed as limitingthe described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein.

[0126] Although this disclosure is in the context of certain embodiments and examples, those of ordinary skill in the art will understand that the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the embodiments and obvious modifications and equivalents thereof. In addition, while several variations of the embodiments have been shown and described in detail, other modifications, which are within the scope of this disclosure, will be readily apparent to those of ordinary skill in the art based upon this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for, one another in order to form varying modes or embodiments of the disclosure. Thus, it is intended that the scope of the present disclosure herein disclosed should not be limited by the particular disclosed embodiments described above.

Claims

WHAT IS CLAIMED IS:

1. A method for determining a sequence of a target polynucleotide in a nanoporebased sequencing system, the method comprising: providing a target polynucleotide, selectively cleaving a P-0 bond on the target polynucleotide using an enzyme to repeatedly generate a separated nucleotide from one end of the target polynucleotide, wherein each separated nucleotide is translocated through a nanopore in the order of being generated; and applying a reading voltage across the nanopore to identify the separated nucleotide as it translocates through the nanopore based on a first electrical response in the system.

2. The method of Claim 1, wherein the enzyme is an exonuclease.

3. The method of Claim 1 , wherein the enzyme is either conjugated to the entrance of the nanopore or free in solution.

4. The method of any one of Claims 1 to 3, wherein the reading voltage comprises a fixed waveform.

5. The method of any one of Claims 1 to 3, wherein the reading voltage comprises a square waveform.

6. The method of any one of Claims 1 to 5, wherein each of the nucleotides of the target polynucleotide further comprises a modification configured to arrest the separated nucleotide relative to the nanopore as the separated nucleotide is translocating through the nanopore.

7. The method of Claim 6, wherein the modification is covalently attached to the nucleotide.

8. The method of Claim 6 or 7, wherein the modification is unique to the type of the nucleotide to which the modification is attached.

9. The method of any one of Claims 1 to 8, wherein each nucleotide in the target polynucleotide is a cyclic loop nucleotide, wherein the cyclic loop nucleotide comprises a spacer linking a nucleobase and a phosphate group of the nucleotide.

10. The method of Claim 9, wherein the separated nucleotides are linked together by the spacer.

11. The method of any one of Claims 1 to 10, wherein only one nucleotide resides in the readhead during reading.

12. The method of any one of Claims 1 to 11, wherein the nanopore further comprises an adapter inside of the nanopore, and the adapter is configured to interact with the separated nucleotide to arrest the separated nucleotide relative to the nanopore during translocation.

13. A method for determining a sequence of a target polynucleotide in a nanoporebased sequencing system, the method comprising: providing a target polynucleotide comprising cyclic loop nucleotides, wherein each of the cyclic loop nucleotides comprises a spacer linking a nucleobase and a phosphate group of the nucleotides; selectively cleave a P-0 bond on the target polynucleotide using an enzyme thereby forming an elongated polynucleotide with spaced nucleotides; and applying a reading voltage across a readhead to identify a first reporter element in a constriction of a nanopore based on a first electrical response in the system.

14. The method of Claim 13, each cyclic loop nucleotide further comprises a modification configured to arrest the spaced nucleotide relative to the nanopore as the elongated nucleotide is translocating through the nanopore.

15. The method of Claim 13 or 14, wherein the nanopore further comprises an adapter inside of the nanopore, and the adapter is configured to interact with the spaced nucleotide to arrest the spaced nucleotide relative to the nanopore during translocation.

16. The method of any one of Claims 13 to 15, wherein the enzyme is an exonuclease or endonuclease.

17. The method of any one of Claims 13 to 15, wherein the enzyme is either conjugated to the entrance of the nanopore or free in solution.

18. The method of any one of Claims 13 to 17, wherein the reading voltage comprises a fixed waveform.

19. The method of any one of Claims 13 to 17, wherein the reading voltage comprises a square waveform.

20. The method of any one of Claims 13 to 19, wherein each of the nucleotides of the target polynucleotide further comprises a modification configured to arrest the separated-sonucleotide relative to the nanopore as the separated nucleotide is translocating through the nanopore.

21. The method of Claim 20, wherein the modification is covalently attached to the nucleotide.

22. The method of Claim 20 or 21, wherein the modification is unique to the type of the nucleotide to which the modification is attached.

23. The method of any one of Claims 13 to 22, wherein each nucleotide in the target polynucleotide is a cyclic loop nucleotide, wherein the cyclic loop nucleotide comprises a spacer and one or more reporter elements linking a nucleobase and a phosphate group of the nucleotide.

24. The method of Claim 23, wherein the separated nucleotides are linked together by the spacer.

25. The method of any one of Claims 13 to 24, wherein only one nucleotide resides in the readhead during reading.

26. The method of any one of Claims 13 to 25, wherein the nanopore further comprises an adapter inside of the nanopore, and the adapter is configured to interact with the separated nucleotide to arrest the separated nucleotide relative to the nanopore during translocation.

27. The method of Claims 1 to 26, wherein the nanopore is a Mycobacterium smegmatis porin A (MspA).

28. The method of Claims 1 to 26, wherein the nanopore is an Alpha hemolysin(aHL).

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