Designer peptide cyclic loops for nanopore sequencing

Abstract

In one aspect, the disclosed technology relates to nanopore sequencing with a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a cleavable cyclic loop between two positions of the nucleotide, wherein the cyclic loop comprises reporter moiety corresponding to the identity of the nucleotide. The reporter may be comprised of an amino acid sequence. The cyclic loop may further comprise modifications or arresting constructs for slowing or halting the polynucleotide translocation through a nanopore. Also disclosed are methods of detecting nucleotides and a kit for determining the sequence of nucleotides.

Description

DESIGNER PEPTIDE CYCLIC LOOPS FOR NANOPORE SEQUENCINGINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 560,338, filed March 1, 2024, the entire disclosure of which is incorporated herein by reference in its entirety.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 at a time, giving rise to at least 4A4= 256 different signals that need to be deconvoluted and resolved.

[0005] In one aspect, the disclosed technology provides a method that instead of directly sequencing the sample DNA, a daughter strand is synthesized using cyclic loopnucleotides. In some embodiments, each cyclic loop nucleotide contains a unique barcoding / reporter region that is specific to the original bases (e.g., A, T or U, C, or G) and a cleavable site. The daughter strand is then “elongated” by cutting the cleavable sites. Consequently, when sequencing the elongated daughter strand, the nanopore can “read” the barcoding / reporter region to identify the base that is being encoded by the strand. The linker and barcoding construct that is introduced in the daughter strand via polymerization is designed to occupy the readhead of the nanopore entirely, hence reducing the number of signals to just four, i.e., one per nucleobase. Thus, the disclosed technology allows barcode-based decoding of individual bases. In some embodiments, the cyclic loops contain linker groups, spacers, arresting constructs, and modifications which allow the daughter strand to be read with high fidelity after elongation by cutting the cleavable sites. Thus, the disclosed technology allows improved resolution of the recorded signal. In some embodiments, the cyclic loop may contain both barcoding / reporter regions and non-barcoding regions.

[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. 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 herein 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] Additional details of exemplary nanopore sequencing devices which can be used with the disclosed technology, and methods of operating the devices, can be found in PCTApplication Numbers PCT / US2021 / 038125 and PCT7US2022 / 020395, the entirety of each of the disclosures is incorporated herein by reference.

[0009] Disclosed herein includes a compound having one of the following structures:X is -O-, -=N-, -CH2-, or -NH-; Y is -O-, -S-, or -NH-; Base is a modified or unmodified nucleobase; Li is a first linking group; L2 is a second linking group; and SP comprises a spacer and a reporter, wherein the spacer and the reporter each comprises one or more of the following moieties: 1) polypeptides having 10 to 100 repeating units; 2) pseudopeptides having 10 to 100 repeating units; 3) hydrophilic polymers having 10 to 100 repeating units; and 4) hydrophobic polymers having 10 to 100 repeating units.

[0010] In some aspects, the techniques described herein relate to a compound, wherein the reporter comprises one or more of the following: a homopolymer; an alternating polymer; a random-mer; or an epitope tag.

[0011] In some aspects, the techniques described herein relate to a compound, wherein each of Li and L2 independently includes a conjugating moiety selected from the group consisting of amine-NHS ester, amine- imidoester, amine-pentofluorophenyl 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, azide-norbornene, Cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne- tetrachlorocyclopentadienone ethylene ketal.

[0012] In some aspects, the techniques described herein relate to a compound, wherein each of Li and L2 independently further includes a first linker between the conjugating moiety and X, and a second linker between the conjugating moiety and SP.

[0013] In some aspects, the techniques described herein relate to a compound, wherein the first linker and the second linker are independently selected from the group consisting of 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 including polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, or polyethyleneimine, hydrophobic polymers having 10 to 100 repeating units including poly lactic acid, polymethymethacrylate, or polystyrene, and combinations thereof.

[0014] In some aspects, the techniques described herein relate to a compound, wherein one or both of the spacer and the reporter include peptides or pseudopeptides, with pendant nucleobases attached.

[0015] In some aspects, the techniques described herein relate to a compound, wherein the nucleobase is a modified nucleobase.

[0016] In some aspects, the techniques described herein relate to a compound, wherein the modification is a linear, a branched or a cyclic polymer attached to the nucleobase.

[0017] In some aspects, the techniques described herein relate to a compound, wherein the modification includes a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof.

[0018] In some aspects, the techniques described herein relate to an oligonucleotide including one of the following structures:wherein: X is -0-, -=N-, -CH2-, or -NH-; Y is -O-, -S-, or -NH-; one of Rl, R2, and R3 is allyl, while the others are H, LI is a first linking group; L2 is a second linking group; and SP is a spacer, wherein the spacer includes one or more of the following moieties: 1) polypeptides having 10 to 100 repeating units; 2) pseudopeptides having 10 to 100 repeating units; 3) hydrophilic polymers having 10 to 100 repeating units; 4) hydrophobic polymers having 10 to 100 repeating units.

[0019] In some aspects, the techniques described herein relate to an oligonucleotide, wherein structures (lb) to (XI) further include one or more modifications in the spacer region configured to slow, pause, or halt translocation of the oligonucleotide through a nanopore.

[0020] In some aspects, the techniques described herein relate to an oligonucleotide, wherein the one or more modifications include: (a) alternating positively and negatively charged subregions of polypeptides; (b) bulk moieties to increase steric hinderanceduring translocation; (c) beta sheet or kink-inducing amino acid moieties; (d) hydrophobic subregions configured to non-covalently interact with surfactants.

[0021] In some aspects, the techniques described herein relate to an oligonucleotide, wherein the spacer is a reporter that identifies one or more bases in the oligonucleotide.

[0022] In some aspects, the techniques described herein relate to an oligonucleotide, wherein the reporter is one or more of the following: a homopolymer; an alternating polymer; a random-mer; or an epitope tag.

[0023] In some aspects, the techniques described herein relate to an oligonucleotide, wherein each of Li and L2 independently includes a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine- pentofluorophenyl 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, azidenorbornene, and Cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne-tetrachlorocyclopentadienone ethylene ketal.

[0024] In some aspects, the techniques described herein relate to an oligonucleotide, wherein each of Li and L2 independently further includes a first linker between the conjugating moiety and X, and a second linker between the conjugating moiety and SP.

[0025] In some aspects, the techniques described herein relate to an oligonucleotide, wherein 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 including polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, or polyethyleneimine, hydrophobic polymers having 10 to 100 repeating units including poly lactic acid, polymethymethacrylate, or polystyrene, and combinations thereof.

[0026] In some aspects, the techniques described herein relate to an oligonucleotide, wherein SP further includes a modification.

[0027] In some aspects, the techniques described herein relate to an oligonucleotide, wherein the Base further includes a modification.

[0028] In some aspects, the techniques described herein relate to an oligonucleotide, wherein the modification is a linear, a branched or a cyclic polymer.

[0029] In some aspects, the techniques described herein relate to an oligonucleotide, wherein the modification includes a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof.

[0030] In some aspects, the techniques described herein relate to a method for determining a sequence of a polynucleotide in a nanopore-based sequencing system, the method including: providing a polynucleotide including a plurality of nucleotides, wherein each nucleotide includes a cyclic loop, the cyclic loop having a first end attached to the first position of the nucleotide and a second end attached to the second position of the nucleotide, wherein the cyclic loop contains one or more sequences of polypeptides; cleaving a cleavable bond on each of the plurality of nucleotide between the first and the second positions, thereby elongating the polynucleotide to form an elongated polymer; applying a voltage to cause the elongated polymer to insert into and translocate through a nanopore; and (i) detecting and identifying the one or more sequences of polypeptides when the cyclic loop passes through the nanopore; or (ii) detecting and identifying a base on the nucleotide when the nucleotide passes through the nanopore.

[0031] In some aspects, the techniques described herein relate to a method, wherein the cyclic loop includes a first linking group, a second linking group, and a spacer between the first and the second linking groups.

[0032] In some aspects, the techniques described herein relate to a method, wherein the spacer includes a polypeptide having 10 to 100 repeating units, alkyl chains having 10 to 200 carbons, hydrophilic polymers having 10 to 100 repeating units selected form the group consisting of polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine, hydrophobic polymers having 10 to 100 repeating units selected from the group consisting of polylactic acid, polymethymethacrylate, and polystyrene, and combinations thereof.

[0033] In some aspects, the techniques described herein relate to a method, wherein the one or more sequences of polypeptides is a reporter barcode, wherein the reporter barcode corresponds to and identifies a nucleotide when passing through the nanopore.

[0034] In some aspects, the techniques described herein relate to a method, wherein each of the first and the second linking groups independently includes a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine- pentofluorophenyl 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, Cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazineisocyanide, and cyclooctyne-tetrachlorocyclopentadienone ethylene ketal.

[0035] In some aspects, the techniques described herein relate to a method, wherein the elongated polymer further includes an arresting construct attached to each nucleobase or each cyclic loop, wherein the arresting construct is configured to slow, pause, or halt the translocation.

[0036] In some aspects, the techniques described herein relate to a method, wherein the arresting construct is a linear, a branched or a cyclic polymer.

[0037] In some aspects, the techniques described herein relate to a method, wherein the arresting construct includes a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof

[0038] In some aspects, the techniques described herein relate to a method, wherein the nanopore includes a constriction having an opening with an inner diameter from about 0.6 nm to about 1.2 nm.

[0039] In some aspects, the techniques described herein relate to a method, wherein the polynucleotide includes one of the oligonucleotides disclosed herein.

[0040] In some aspects, the techniques described herein relate to a plurality of nucleotides selected from the compounds disclosed herein.

[0041] In some aspects, the techniques described herein relate to a kit for performing a method for determining a sequence of a polynucleotide in a nanopore-based sequencing system, the kit comprising the compounds disclosed herein.

[0042] In some aspects, the techniques described herein relate to a system for determining a sequence of a polynucleotide, the system configured to perform a method according to any of the methods disclosed herein.

[0043] In some aspects, the techniques described herein relate to a system for performing a method for determining a sequence of a polynucleotide comprising a plurality of nucleotides, wherein the nucleotides are selected from any of the compounds disclosed herein.

[0044] 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

[0045] 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.

[0046] FIG. 1 schematically illustrates an example of sequencing an elongated polynucleotide with nucleotides containing elongated cyclic loops.

[0047] FIG. 2 schematically illustrates an example of elongating a polynucleotide with nucleotides having cleavable cyclic loops.

[0048] FIG. 3 illustrates a sample nucleotide having a cyclic loop with a peptide backbone for a peptide sequence.

[0049] FIG. 4 illustrates various sequences of peptide backbones.

[0050] FIG. 5 illustrates peptides with varying directionality for nanopore sequencing.

[0051] FIG. 6 illustrates experimental data for directional peptide reporters.

[0052] FIGS. 7(a) to 7(e) illustrates various spacer and reporter moieties to slow or halt translocation of the cyclic loop chain.

[0053] FIGS. 8 and 9 include example nucleotides having cyclic loops with spacer regions containing a peptide backbone.

[0054] FIG. 10 illustrates the readout of several unique peptide signatures.DETAILED DESCRIPTION

[0055] 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

[0056] 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.

[0057] 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.

[0058] 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.

[0059] As used herein, the term “modified oligonucleotide” 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).

[0060] 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 thepassage (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 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.

[0061] As used herein, the term “diameter” is intended to mean the 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.

[0062] 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.

[0063] 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.

[0064] As used herein, the term “biological nanopore” is intended to mean a nanopore whose structure 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.

[0065] 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.

[0066] As used herein, a “reporter” is composed of one or more reporter elements or reporter moieties. Reporters include what are known as “tags” and “labels.” The cyclic loop (when including reporter moiety) or nucleobase residue of the elongated polymer can be considered a reporter. Reporters serve to parse the identity of the target nucleic acid. Reporters may include constituent sub-reporters, and multiple reporters may be present on a single nucleotide. When present in the readhead of a nanopore, reporters can provide distinctive and sometimes unique blockage currents at given read voltages.

[0067] 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.

[0068] As used herein, the term “heavy atom” refers to any atom used within a molecular structure that is not hydrogen. Heavy atoms used within a modified oligonucleotide may be bridging (e.g. used to connect multiple oligonucleotides), or non-bridging (e.g. not directly linked to multiple oligonucleotides).

[0069] 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, RNA, 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.

[0070] As used herein, a “peptide” often refers to two or more amino acids joined together by an amide bond (that is, a “peptide bond”). Peptides may, for example, comprise 50, 100, or more 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.

[0071] As used herein, a “protein” generally refers to an amino acid sequence having 51 or more amino acids.

[0072] 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.

[0073] 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.

[0074] The application of the electric potential difference across a nanopore may force the translocation of a nucleic acid through the nanopore. One or more signals are 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 one of a variety of methods. Each signal is unique to the species of nucleotide(s) (or cyclic loops with a reporter moiety region) in the nanopore, such that the resultant signal can be used to determine acharacteristic 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.

[0075] 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.

[0076] 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, nitroindole, LNA, 2’-0Me, 2’-F, 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.

[0077] 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 in manner 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.

[0078] 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.

[0079] 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 may flow when an electric potential difference is applied across the nanopore.

[0080] 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.

[0081] As used herein, the term “arresting construct” is intended to mean a moiety attached to a nucleotide. An arresting construct may provide 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). Arresting constructs can operate as a ratchet or a brake for the polypeptide translocation through a nanopore. An arresting construct can be attached to any part of the nucleotide and can also be attached to the nucleotide at two locations forming a loop.

[0082] The aspects and examples set forth herein and recited in the claims can be understood in view of the above definitions.Overview

[0083] In some embodiments, by using cleavable sites along the DNA backbone while conjoining adjacent nucleobases with a cyclic loop having a barcoding region, the disclosed technology allows the distance between adjacent nucleobases to be increased and negates the need to deconvolute a large number of signals. Once the backbone is cleaved, the reporter portion of the elongated polynucleotide would occupy the entire readhead of the nanopore for highly accurate single molecule sequencing with single base resolution. In some embodiments, the disclosed technology allows having one nucleobase of the elongated polynucleotide to reside in the readhead at any point in time, successfully reducing the diversity of reads to four (A, T, C and G or A, U, C, and G), enabling more accurate sequencing at a lower cost. In some embodiments, the disclosed technology provides high throughput, cheaper and more accurate DNA sequencing.

[0084] Some cyclic loops may be provided with oligonucleotide sequences for encoding the identity of the nucleobase to which the cyclic loop is attached. However, in some synthesis reactions these oligonucleotides can compete with the nucleobase for incorporation of the nucleobase or nucleobase modifications. For example, they may compete with the modified nucleoside triphosphate unit for hybridization sites on the DNA template sequence. One solution to this is to incorporate repeating subunits other than a sequence of bases in a cyclic loop. Peptides are one such repeating subunit that can advantageously provide a unique signal during a nanopore sequencing event without interfering with polymerase incorporation of nucleotides in a daughter strand.

[0085] The amino acid sequence may be tailored to the specific requirements of the nanopore or the nanopore sequencing procedure. In some embodiments the cyclic loop may contain a neutral backbone (such as a repeating polymeric sequence) with amino acid side chains. The amino acid side chains may be positive, negative, neutral, or polar in nature, and the chemical characteristics of the sequence can be tuned to provide a characteristic signal or desired nanopore interaction.

[0086] The incorporation of DNA bases in a cyclic loop may be less desirable from a tunability perspective. For example, DNA bases have a negative backbone, which can introduce a negative charge or a net negative charge to the cyclic loop. On the other hand, amino acids may be incorporated into a cyclic loop with a neutral backbone, increasing the variability in the charge of the cyclic loop. This can be advantageous, as the translocation of the strand translocating through the nanopore is largely influenced by its charge. Furthermore, since there are only four DNA bases and four RNA bases (where the strand is comprised of either DNA or RNA), the tunability of the cyclic loop can be somewhat limited where the reporter region of the cyclic loop only contains DNA bases. On the other hand, there are more than twenty different natural amino acid structures and many additional synthetic amino acid structures, which allows at least an additional order of magnitude difference in the variability of the cyclic loop sequence. Thus, an amino acid sequence can provide unparalleled structural diversity and tunability to the cyclic loop and the encoded signal provided therein.

[0087] The incorporation of amino acids in a cyclic loop may be more desirable than DNA bases from a synthesis and cost perspective. For example, natural amidites can be an order of magnitude more expensive to obtain than a natural amino acid sequence. In a typical synthesis, incorporation of a DNA oligo sequence may take more than four hours of hands-on time. On the other hand, incorporation of a neutral amino acid sequence can take less than 0.5 hours of hands-on time. Some sequencing technologies have a DNA oligo sequence coupling cycle limit of 100, while some sequencing technologies may have a peptide coupling cycle limit of 40, with dimers available. The coupling limit may also depend upon the model of the automated synthesizer used. For example, a synthesizer may be capable of synthesizing 200- mer oligos and 94-mer peptides. Thus, a peptide sequence may be more advantageous than a nucleotide sequence, as it reduces costs and lowers synthetic complexity.System and Method

[0088] FIG. 1 schematically illustrates an example of sequencing an elongated polynucleotide. A protein nanopore 101 is deposited in a lipid bilayer 102. An elongated polynucleotide 103 translocates through the nanopore 101. The polynucleotide 103 includes cyclic loop regions 117 between successive nucleotides. By introducing a cyclic loop 117 between successive nucleotides, the k-mer length can be reduced to one, resulting in just four signals (each for A, T, C and G, or A, U, C, and G), reducing the complexity of base calling. Additionally, a characteristic linker / barcode may be assigned to each of the four individual bases to achieve base recognition. For example, the signal unit or moiety 105 includes an “A” nucleotide and a corresponding cyclic loop 117 which may contain one or more reporters that serve as the barcode for nucleotide A. Diversity of reads is reduced to four with a single barcode characteristic of each nucleobase residing or interacting in the nanopore readhead.

[0089] The cyclic loop may also contain one or more modifications or “arresting” constructs (ARC), which may operate to stop or slow down the translocation of the daughter sequence through the nanopore. With the incorporation of arresting constructs the nucleotide sequence may be advanced through the nanopore in a “ratcheted” manner. The advancement of the nucleotide sequence through the nanopore may be performed either in a faster “autoratchet” mode (with stochastic and fairly unpredictable advancement of the nucleotide sequence) or in a slower “pulsed-ratchet” mode which promotes the advancement of nucleotides (along with their arresting constructs) in fairly pre-defined time intervals.

[0090] By “translocation,” it is meant that an analyte (e.g., a polynucleotide, such as RNA or DNA) enters one side of an opening of a nanopore and moves to the other side 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 or modified analyte. By “interacts,” it is meant that the analyte 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. 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 a magnetic force causes the modified analyte to interactwith, 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.

[0091] In some embodiments, the nanopore may comprise a solid-state material, such as silicon nitride, modified silicon nitride, silicon, silicon oxide, 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 comprised in a lipid bilayer. In some embodiments, the nanopore is comprised in an artificial membrane comprising a mycolic acid. The nanopore may be a Mycobacterium 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 wild-type 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.

[0092] 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 isabout, 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.

[0093] 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 the tunnel as well as the movement of an analyte with respect to the tunnel, either temporarily or permanently.

[0094] 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.

[0095] As illustrated in FIG. 2, an elongated polynucleotide may be formed from a polynucleotide having modified nucleotides, each modified nucleotide comprises a cyclic loop modification (dashed line). A daughter strand polynucleotide can be synthesized bypolymerase from a template DNA using modified nucleotides (e.g., modified dNTPs). In the polymerization process, the modified dNTPs with a cyclic loop is incorporated into a growing daughter strand. Once the daughter strand is made, the polynucleotide backbone is cleaved at cleavable sites, allowing the cyclic loop modification to open and result in elongation of the daughter strand polynucleotide. The cyclic loop modifications on the modified dNTPs become the cyclic loops in the elongated polynucleotide that create distance between adjacent nucleotides.

[0096] The polymerase used is 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, RepliPHI Phi29 Polymerase, Tii DNA polymerase, eukaryotic DNA polymerase beta, telomerase, Therminator™ polymerase (New England Biolabs), KOD HiFi™ DNA polymerase (Novagen), KOD1 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 refers to transferring the genetic information of target template base sequence into an arrangement of reporters.

[0097] After the polymerization process is completed, cleavage at predetermined locations opens the loops and increases the distances between adjacent nucleotides. Cleavage of the daughter 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 cyclic loop is attached to the nucleotide structure. Cleavage of the daughter strand along the backbone opens the loops andelongates the daughter strand, leaving the opened cyclic loop conjoining the backbone phosphate and the sugar. In embodiments where the cyclic loop contains one or more arresting constructs configured to interact with the nanopore, the arresting constructs may slow or halt the translocation of the elongated polymer and allow the nucleotides to be read by the nanopore one at a time.

[0098] In some embodiments a reporter moiety (such as a reporter barcode) is a part of a cyclic loop. The cleaved product, which may take the form of an elongated polymer that is substantially linear, exposes a series of reporter moieties, each of which reports the identity of the base to which it corresponds. In embodiments where the cyclic loop also contains an arresting construct configured to interact with the nanopore, the elongated polymer can be sequenced in the nanopore one barcode at a time.Peptide-Based Cyclic Loops

[0099] As discussed herein, in some embodiments the cyclic loop may include a peptide sequence in the cyclic loop that corresponds with the identity of one or more nucleobases. For example, the cyclic loop, and sequence of peptides therein, may produce a signal that identifies the nucleobase to which the cyclic loop is attached. The cyclic loop may be attached to one portion of the nucleotide and the other end of the cyclic loop may be attached to a different portion of the nucleotide or to an adjacent nucleotide. In some embodiments, the cyclic loop may be attached to the nucleotide via the non-bridging position of the backbone phosphorous atom, and the other end of the cyclic loop may be connected to the base or the 5’- position of the nucleotide.

[0100] FIG. 3 shows a non-limiting example of a nucleotide with a cyclic loop containing a reporter region 302, a spacer region 306, and an arresting construct 304. The arresting construct 304 may be attached to the nucleobase or it may be provided in the chain of the cyclic loop or as a branch off the cyclic loop. In some embodiments the reporter region 302 is a subregion within the spacer region 306, thus compounds shown herein may have reporter subregions within the spacer (SP). The nucleotide in FIG. 3 also contains linker regions which connect the cyclic loop to a first position and a second position of the nucleotide, such as at a first position and a second position on the phosphate backbone. FIG. 3 provides a peptide backbone including amides to which the amino acids may be attached. The positions for the attachment of amino acids are depicted as “X” in FIG. 3. The amino acids may benatural or unnatural or synthetic amino acids, and the amino acids may be provided in a sequence that provides a characteristic signal when the amino acids are translocated through a nanopore. As discussed further herein, other moieties may be provided in the cyclic loop in conjunction with amino acids, such as polymers, bases, etc.

[0101] FIG. 4 illustrates non-limiting sequences for peptide reporters or spacers. Sequence 402 is a homopolymer sequence including a single repeating amino acid moiety. The homopolymer may be a repeating sequence of natural or unnatural amino acids or “non-amino” acids. For example, the non-amino acid may have an amino-type structure of a reactive amine group at one terminus and a carboxylic acid group at the other terminus. Thus, natural amino acids, unnatural amino acids, and amino-type non-amino acids may be used as an identifier in a homopolymer sequence.

[0102] In some embodiments, the homopolymer sequence may be a repeating sequence of positively charged amino acids to identify a particular nucleobase. In some embodiments, the homopolymer sequence may be a repeating sequence of negatively charged amino acids or non-amino acids to identify a particular nucleobase. In some embodiments, consecutive cyclic loops may contain a homopolymer of positively charged amino acids or non-amino acids, negatively charged amino acids or non-amino acids, neutral amino acids or non-amino acids, or zwitterionic amino acids or non-amino acids. In some embodiments, four different amino acids or non-amino acids may each correspond with a single base (A, T or U, C, and G) such that a cyclic loop with a single amino acid may provide a distinct signal indicative of a single nucleobase (i.e. A = Ala, T= GLY, etc.).

[0103] Sequence 404 illustrates an alternating polymer sequence including two alternating amino acid or non-amino acid moieties. In some embodiments, two alternating amino acid sequences may embed a binary signal. In some embodiments, the alternating amino acid sequence may comprise alternating individual amino acids (i.e. GLY-ALA-GLY-ALA... ) or the alternating amino acid sequence may comprise alternating groups of amino acids (i.e. a group of GLY followed by a group of ALA repeating at least one time). In some embodiments, the alternating sequence may be an alternating sequence of positively charged and negatively charged amino acids in a single cyclic loop. The groups of positively charged and negatively charged amino acids may be configured to slow translocation of the polynucleotide through the nanopore, as discussed further herein.

[0104] Sequence 406 illustrates a random-mer sequence containing more than two amino acids or non-amino acids in a single cyclic loop. The sequence 406 may be a predetermined sequence such as repeating units of at least two, three, four, five, six or more amino acids or non-amino acids. In some embodiments the random-mer may provide a predetermined sequence of positive or negative amino acids, as discussed further herein. In some embodiments the random-mer may be a sequence of amino acids such that a predetermined sequence of four amino acids substantially correspond with a predetermined nucleobase. In some embodiments the random-mer may contain double redundancy in order to prevent errors in the readout of the polynucleotide sequence.

[0105] The random-mer may be further tailored to include epitope tags in conjunction with the amino acids, such as shown in sequence 408. In some embodiments a single epitope tag may be associated with a single amino acid, a group of amino acids, or a sequence of amino acids. The epitope tag may be provided to ensure accurate readout and can provide a duplicative signal, in conjunction with the amino acid. The epitope tag may be used as a secondary signal or as an alternative to the amino acid signal. The epitope tags may be any commercially available epitope tags known in the art.

[0106] As discussed herein the backbone of the amino acid sequence may be an unmodified peptide backbone, such as backbone 410, with spheres representing amino acids. However, in some embodiments the backbone may be a modified peptide backbone, such as pseudopeptide backbone 412. Pseudopeptide backbone 412 may contain various bases shown as pendant groups off of the peptide backbone (represented as spheres). Thus, the bases (such as A, T or C, C, and G) may be provided in conjunction with amino acids in the reporter region of the cyclic loop. In some embodiments the peptide backbone contains alternating sequences of amino acids and bases. In some embodiments a single base and a single amino acid may be associated with a nucleobase in the polynucleotide, or a sequence of bases and a sequence of amino acids may be associated with a nucleobase in the polynucleotide. Other backbones may be envisioned and could include peptoids, N-methyl amino acids, and azapeptides.

[0107] In the sequences depicted in FIG. 4, the readout may measure an average of the signals of the different combinations of amino acids in the nanopore and a peptide map of estimated ion current levels can be generated and used as a reference to interpret reads of previously unsequenced DNA strands using the nanopore, such as mutant MspA.Peptide Directionality

[0108] FIG. 5 illustrates the influence of peptide directionality on the readout of a polynucleotide containing cyclic loops and whether modifying directionality alone can produce a resolvable current signature. In this experiment a GlulO peptide reporter was immobilized inside a nanopore via a biotin / traptavidin protein lock under a positive applied potential. The peptide shown in FIG. 5 is a homopolymeric peptide, but other peptides are envisioned such as alternating peptides, random-mers, and peptides with epitope tags. Peptide11 contains the reporter barcode near the c-terminus of the polymeric chain whereas peptide12 contains the reporter barcode near the n-terminus of the polymeric chain. As shown in FIG. 5, the polymeric chain can be configured to occupy the entirety of the nanopore vestibule and constriction zone, which can be on the order of ~10 nm.

[0109] FIG. 6 shows the current readout signal associated with polymeric chains from FIG. 5. As shown in the figure, peptide 11 displays a signal level that is distinct from peptide 12 at both 70 and 100 mV read voltage levels. Thus, this shows that the peptide reporter may provide a resolvable signal that is distinguishable based on the location of the peptide reporter in the nanopore. This is true even if the peptide reporter is a single homopolymer, such as shown in FIG. 5. Peptide 11 exhibits extremely low docking rates over 406 cycles and Peptide 12 additionally exhibits low docking rates over 380 cycles. For comparison, the docking rates of Peptides 11 and 12 are 1000-fold and 100-fold lower than oligo-based reporters, respectively. These docking rates are compared to a polyT and polyC control. The lower docking rates may be due to peptides 11 and 12 having a neutral capping group and may be mitigated by the introduction of negatively charged leader strand. Additional peptide reporter measurements are included later with reference to Figure 10.Arresting Constructs

[0110] The cyclic loops embodiments herein may be provided with one or more arresting constructs. An arresting construct is generally configured to slow, pause, or halt the translocation of the polynucleotide through the nanopore. The slowing, pausing, or halting the translocation of the elongated polymer through the nanopore may allow the nucleotides to be read by the nanopore one at a time. In some embodiments the cyclic loop halts the translocation of the polynucleotide through the nanopore until a forward voltage pulse is applied to advance the polynucleotide to the next reporting region or arresting construct.

[0111] The arresting construct can be constructed of one or more durable, aqueous- or solvent-soluble polymers including, but not limited to, the following segment or structures: polyethylene glycols, polyglycols, polypyridines, polyisocyanides, polyisocyanates, poly(triarylmethyl) methacrylates, polyaldehydes, polypyrrolinones, polyureas, polyglycol phosphodiesters, polyacrylates, polymethacrylates, polyacrylamides, polyvinyl esters, polystyrenes, polyamides, polyurethanes, polycarbonates, polybutyrates, polybutadienes, polybutyrolactones, polypyrrolidinones, polyvinylphosphonates, polyacetamides, polysaccharides, polyhyaluranates, polyamides, polyimides, polyesters, polyethylenes, polypropylenes, polystyrenes, polycarbonates, polyterephthalates, polysilanes, polyurethanes, polyethers, polyamino acids, polyglycines, polyprolines, N-substituted polylysine, polypeptides, side-chain N-substituted peptides, poly -N-substituted glycine, peptoids, sidechain carboxyl-substituted peptides, homopeptides, oligonucleotides, ribonucleic acid oligonucleotides, deoxynucleic acid oligonucleotides, oligonucleotides modified to prevent Watson-Crick base pairing, oligonucleotide analogs, polycytidylic acid, polyadenylic acid, polyuridylic acid, polythymidine, polyphosphate, polynucleotides, polyribonucleotides, polyethylene glycol-phosphodiesters, peptide polynucleotide analogues, threosyl- polynucleotide analogues, glycol-polynucleotide analogues, morpholino-polynucleotide analogues, locked nucleotide oligomer analogues, polypeptide analogues, branched polymers, comb polymers, star polymers, dendritic polymers, random, gradient and block copolymers, anionic polymers, cationic polymers, polymers forming stem-loops, rigid segments and flexible segments.

[0112] In some embodiments the arresting construct is a branch off of the chain of the cyclic loop of the nucleotide. In elongation of the cyclic loop with the arresting construct branch, the cyclic loop may still be considered linear or substantially linear, as the longest chain of the cyclic loop continues in a linear or substantially linear connection between two nucleotides. In some embodiments the arresting construct is sequentially provided in the direct sequence (e.g. longest chain) of the modified cyclic loop. In some embodiments the width of the arresting construct is larger than the width of the linker, spacer, reporter, or barcode on the cyclic loop, where the length of the molecule or group of molecules is a part of the sequence of the modified cyclic loop. In some embodiments the width of the arresting construct is at least 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, atleast about 75%, at least about 100% larger than the width of the linker, spacer, reporter or barcode. In some embodiments the arresting construct is at least 50% of the width of the nanopore.

[0113] In some embodiments each reporter or spacer is adjacent to an arresting construct. In some embodiments the arresting construct is before or after each reporter or spacer in the cyclic loop. In some embodiments the spacer is provided with two reporter regions flanking an arresting construct in the middle of the two reporter regions.

[0114] In some embodiments the cyclic loop may further include one or more nonreporting spacer regions which operate to increase the distances between one or more reporting elements. In the case where a voltage pulse is applied, the non-reporting spacer permits the force on the arresting construct due to the voltage pulse to fade away before the next arresting construct arrives in the pore to avoid skipping. In the alternative, the translocation of the sequence may operate via a constant voltage (“auto ratchet” mode) that does not rely on a voltage pulse for translocation, but rather automatically advances at a slow rate during the application of a constant voltage. In the auto ratchet mode, the spacer also provides a means to control translocation rates and allow distance between one or more reporters.Configurations to Slow Translocation

[0115] FIGS. 7a-7e illustrates various spacer and reporter moieties to slow or halt translocation of the polynucleotide when the cyclic loop is opened to an extended daughter strand. FIG. 7(a) illustrates a designer cyclic loop with various negatively charged residues 702 and positively charged residues 704. The negatively and positively charged residues may interact with the interior or sides of the nanopore to slow or halt translocation through the nanopore. Electrophoresis is the main driving force for DNA translocation through nanopore. Thus, under a positive applied potential, negatively charged amino acids are driven through the pore. Positively charged amino acids on the other hand are pulled towards the cis chamber. By leveraging this “tug of war” effect and appropriately placing charges within the spacer domain, the residence time of the DNA construct in the pore may be increased. The charges of the positive and negative charges may be net negative charges of a region or polarization of charges in a portion of the sequence.

[0116] FIG. 7(b) illustrates various designer knots or bulky groups that can be included in the spacer or reporter regions of the cyclic loop in order to slow or halttranslocation. The designer knots can be engineered to be of the right size to obstruct the pore and can be assembled via the attachment of bulky substituents to reactive amino acid side chains. In some embodiments the designer knots are arresting constructs. In some embodiments the width of the bulky groups at least 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 100% larger than the width of the linker, spacer, reporter or barcode. As a non-limiting example the bulky groups may take the form of crown ethers, supramolecular structures, adamantyl, camphor, attached to various nitrogens of the cyclic loop or amino acids:

[0117] FIG. 7(c) illustrates various designer kinks that may be included in the spacer or reporter regions of the cyclic loop in order to slow translocation. Translocation speed may be increased via a straight cyclic loop that is generally flexible. Thus, providing inflexible kinks in the cyclic loop may slow the translocation. For example, trans double bonds may be incorporated into the cyclic loop, providing a trans configuration for the longest carbon chain that introduces kinks into the loop. Other configurations are possible such as amino acids that induce the formation of kinks and secondary structures such as beta-sheets (shown in FIG. 7(d)) that can reduce peptide flexibility and increase collision interactions with the walls of the constriction zone, slowing down translocation.

[0118] FIG. 7(e) illustrates a cyclic loop having various hydrophobic sections or residues 706 to which surfactants 708 may be attached. The hydrophobic residues 706 may be amino acid side chains or groups. The cyclic loop may be configured such that the surfactants preferentially align their hydrophobic tails with the hydrophobic amino acid side chains, forming a peptide-surfactant complex with an apparent steric bulk that’s larger than the diameter of the constriction zone of the nanopore. This increases the energy barrier of the nanopore and slows the translocation time. In some embodiments the surfactants are removed from the hydrophobic residues 706 prior to translocation. In some embodiments the nanopore may be configured to allow the translocation of surfactants with the hydrophobic residues and may be used as a signal in the translocation readout.Example Nucleotides having Cyclic Loops

[0119] FIG. 8 illustrates an example of a nucleotide having a cyclic loop. The cyclic loop may be linked from the alpha phosphate to the nucleobase, as shown in the figure. The loop may be provided with one or more linking groups 802 having conjugating chemistry. The linking groups may facilitate the connection of the cyclic loop to the nucleotide or the cyclic loop to modifications that are present on the nucleobase and / or phosphate backbone. In some embodiments, the cyclic loop may be attached to the nucleotide through click chemistry. In some embodiments, the linking group includes an “X” linking group attached to the phosphate backbone, as shown in the figure. The X attached to the phosphate backbone may be -O-, -NH- , -=N-, or -CH2-. The cyclic loop may also contain one or more spacer regions 804 containing one or more amino acids connected to an amino acid backbone. The figure generally shows a peptide backbone with “X” amino acid substituents that may be configured as a sequence to encode one or more nucleobases. The spacer region 804 may include encoding subregions and non-encoding subregions, as discussed earlier. The spacer region 804 may also be provided with an arresting construct 806, which can be configured as a bulky or steric group to decrease, slow, or halt the translocation of the cyclic loop through the nanopore. Although a single arresting construct is shown here bisecting the spacer region 804, two, three, or several arresting constructs may be provided in a single cyclic loop. The arresting construct 806 may symmetrically bisect the cyclic loop or in some embodiments it may asymmetrically be positioned on the cyclic loop or attached to the nucleobase.

[0120] FIG. 9 illustrates an additional example of a cyclic loop having one or more linking groups 902, spacer regions 904, and arresting constructs 906. The spacer region 904 of cyclic loop may be provided with a peptide backbone, as shown, and various amino acids (“X”) may be attached to the backbone and configured as a sequence to encode one or more nucleobases. As with FIG. 8, the linking group (X) attached to the phosphate backbone may be -O-, -NH-, -=N- or -CH2-. FIG. 9 generally differs from FIG. 8 in the attachment location of the cyclic loop. In FIG. 9 the cyclic loop is attached at two different portions of the phosphate backbone. For example, in FIG. 9 the cyclic loop may be attached to the nucleotide through allyl chemistry.Unique Peptides Signatures

[0121] FIG. 10 illustrates the readout of several unique peptide signatures. These peptide signatures may be used as reporters that identify a nucleobase, as discussed herein. In this experiment, repeat polymeric peptides were conjugated to a negatively charged oligo leading strand at one end and a biotin / traptavidin protein cap on the other end. The repeating peptides consisted of a negatively charged residue (Glu or Gia) followed by a string of natural amino acids and / or non-amino acids (PEG2 or PEG4). The peptide-oligo construct was then immobilized inside a nanopore, for example with the protein cap, and the current signals measured at 70 mV read voltage level. Under these conditions, all peptide reporters showed a unique current blockade that is distinguishable from one another. Specifically, a unique signal was obtained for each of the unique peptide-oligo constructs comprising Glu-PEG2, Glu- PEG4, Glu-PEG2-Gly-Ser, Glu-PEG2-Tyr-Tyr, Glu-PEG2-Ser-Tyr, Gla-PEG2. Thus, amino acids may be used in combination with peptides or non-amino acids to produce a unique current as a reporter in a cyclic loop to identify a nucleobase. This may also be extended to non-natural amino acids in combination with peptides or other polymeric units.Cleavable Cyclic Loop Nucleotides

[0122] Cleavable cyclic loop nucleotides are nucleotides / nucleotide analogs that are modified to include a cyclic loop attached to two positions of the nucleotide / nucleotide analog structure. Although “cleavable cyclic loop nucleotide” is used, it includes both modified natural nucleotide and modified nucleotide analogs.

[0123] Depending on the structure and composition of a cyclic loop, several functions or structures may be present, including one or more of the following: conjugating moieties, linkers, spacers, reporters (reporter elements, barcodes), and arresting constructs.

[0124] Conjugating moieties in a cyclic loop modification is formed by the conjugation of the spacer moiety to one or more nucleotides or the conjugation of additional modifications (such as arresting constructs) to the cyclic loop. A reactive group at each end of the spacer moiety reacts with the reactive groups on the bifunctional nucleotide to form the conjugating moieties. In some embodiments, one or more arresting constructs may be attached to the cyclic loop through the conjugating moiety also. Arresting constructs are moieties configured to slow the translocation of the polynucleotide so one or more reporter elements could have a longer dwell time within the nanopore read head in the presence of a drivingvoltage, permitting identification of the reporter, thus corresponding base. Spacers (SP) distance successive arresting constructs to allow for sufficient decay of an applied pulse voltage before the next arresting construct. Spacers may also serve to elongate any polynucleotide once particular backbone elements are cleaved. Therefore, a cyclic loop is comprised of any number of sub-elements which may serve to affect and attenuate sequencing.In some embodiments, the strength of the background electric field, the type of nanopore, and the properties of an elongated polynucleotide affect translocation speed, efficiency, and accuracy.

[0125] Examples of the cleavable cyclic loop nucleotides include:

[0126] X is -O-, -CH2-, -=N- or -NH-; Y is -O-, -S-, or -NH-; Base is a modified or unmodified nucleobase; Li is a first linking group; L2 is a second linking group; SP comprises at least one of a spacer and reporter, and Base is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil.

[0127] 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:X is -0-, -CH2-, -=N- or -NH-; Y is -O-, -S-, or -NH-; Base is a modified or unmodified nucleobase; Li is a first linking group; L2 is a second linking group; and SP comprises at least one of a spacer and reporter.

[0128] 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, azide-norbornene, cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne- tetrachlorocyclopentadienone ethylene ketal. Li and L2 may or may not be the same.

[0129] In some embodiments, each of the first linking group Li and the second linking group L2 may independently further comprises a linker. A first linker may be present between the conjugating moiety and X / 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 (e.g., polyethylene glycol,polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine), hydrophobic polymers (e.g., 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 comprise peptides, polypeptides, alkyl chains, polyethylene glycol, or combinations thereof. In some embodiments, one or more linkers may be absent.

[0130] In some embodiments, SP comprises a spacer and a reporter, wherein the spacer and the reporter each comprises one or more of the following moieties: 1) polypeptides having 10 to 100 repeating units; 2) pseudopeptides having 10 to 100 repeating units; 3) hydrophilic polymers having 10 to 100 repeating units; 4) hydrophobic polymers having 10 to 100 repeating units. In some embodiments the SP additionally or alternatively comprises one or more of the following moieties: (1) simple aliphatic chains, such as alkyl chains having 5 to 50 carbons, and 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), (2) oligonucleotides, modified oligonucleotides or polyphosphates having 1 to 100 repeating units, (3) polypeptides having 1 to 100 repeating units, (4) hydrophilic polymers having 1 to 100 repeating units, examples include polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine, (5) hydrophobic polymers having 1 to 100 repeating units, examples include polylactic acid, polymethylmethacrylate, and polystyrene, and (6) pseudopeptides having 10 to 100 repeating units. In some embodiments, the alkyl chains may be substituted or unsubstituted. In some embodiment, the number of repeating units (monomers) in SP may range from, for example 1 - 5, 6-10, 11-15, 16-20, 20-25, 26-50, or 50-100, or a combination of any of the foregoing ranges. In some embodiments, the total number of repeating units in SP may be 5-100, 10-100, 10-80, 10-70, 5-60 or 5-50.

[0131] The number of the repeating units and the length of the spacer SP may depend on the following factors: (1) the choice of repeating units / monomers - a monomer that is shorter / smaller would likely require more repeats to make up a similar length as compared to a longer monomer; (2) the steric bulk of the spacer - a larger spacer monomer would likely result in steric clash with the nanopore readhead and consequently, a slower translocation speed as compared to a less bulky monomer; (3) the interactions of the spacer with thenanopore - a spacer monomer that is capable of forming stronger interactions (e.g., electrostatic interactions, H-bonding) with nanopore residues is likely to experience slower translocation speed as compared to a monomer that forms weaker interactions (e.g., non-polar interactions); (4) the charge of the selected modifications - a loop with higher net negative charge would experience a higher translocation rate (compared to a lower net negative charged loop) in the presence of an applied voltage.

[0132] In some embodiments the cyclic loop may contain polypeptides and the polypeptides may be homopolypeptides or heteropolypeptides.Homopolypeptide heteropolypeptide wherein and a is 1-100. In some embodiments, a is 1-5, 6-10, 11-15, 16-20, 20-25, 26-50, or 50-100, or a combination of any of the foregoing ranges. Polypeptides can comprise both natural and unnatural amino acid residues, including non-exhaustive examples of residues selected from the following:L / D-Natural amino acids

[0133] In some embodiments, modified oligonucleotides in SP 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

[0134] In some embodiments, polyamide compounds may be homopolyamide or heteropolyamides.Homopolyamide heteropolyamide

[0135] Polyamide compounds can include one or more of the following residues:

[0136] In some embodiments, the spacer may comprise a reporter moiety that correspond to the specific nucleobase. In other embodiments, the spacer may contain subregions that are configured to provide spacing between reporter subregions of the cyclic loop.

[0137] In some embodiments, the cleavable cyclic loop nucleotide may further comprise an arresting construct configured to interact with the nanopore. The arresting construct 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 arresting construct may be a side branch attached to the cleavable cyclic loop nucleotide. In some embodiments, the arresting construct may be a side branch attached to the nucleobase of the nucleotide / nucleotide analog. In some embodiments, the arresting construct may be a side branch attached to the cyclic loop - on either the spacer SP or the linking group Li or L2. In some embodiments, the arresting construct may be integrated into the cyclic loop structure or is a part of the cyclic loop structure. In some embodiments, the arresting construct may be adjacent to the reporter in the cyclic loop structure.

[0138] When the arresting construct is attached to the spacer SP portion of the cyclic loop, the arresting construct 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. Inembodiments where the arresting construct is attached to the middle of the cyclic loop structure, the cyclic loop may be a symmetrical loop. In embodiments where the arresting construct is attached to other part of the cyclic loop structure, the cyclic loop may be an asymmetrical loop.

[0139] The arresting construct 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.

[0140] In some embodiments the cyclic loop comprises one or more sub-elements, including linkers, conjugating moieties, spacers, arresting constructs, and reporters. The arrangement of sub-elements for a cyclic loop may be asymmetric (i.e. Asymmetric Cyclic Loops - ACLs) or symmetric with respect to the order of sub-elements (i.e. Symmetric Cyclic Loops - SCLs).Daughter Strand

[0141] Compound (la), (Ila), (Illa), (IVa), and (Va) may be used as cyclic loop nucleotides for synthesizing a daughter strand. IN some embodiments the daughter strand comprises at least one of the compounds (I), (II), (III), (IV), 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) together into a polynucleotide or an oligonucleotide. The daughter strand comprises one of the following structures:wherein: X is -0-, -CH2-, -=N- or -NH-;Y is -O-, -S-, or -NH-; one of R1, R2, and R3is allyl, while the others are H; Li is a first linking group; L2 is a second linking group; and SP is a spacer. In some embodiments the adjacent Bases on the daughter strand may be different or the same.

[0142] In some embodiments the spacer includes at least one of a spacer and a reporter wherein the spacer includes one or more of the following moieties: 1) polypeptides having 10 to 100 repeating units; 2) pseudopeptides having 10 to 100 repeating units; 3) hydrophilic polymers having 10 to 100 repeating units; and 4) hydrophobic polymers having 10 to 100 repeating units.

[0143] In some embodiments, structure (VII) can further be represented by the following structures:

[0144] In some embodiments, structure (VIII) can further be represented by the following structures:

[0145] In some embodiments, structure (X) can further be represented by the following structures:Cleavage of Cyclic Loop Nucleotide

[0146] The daughter strand can further be subject to a condition disclosed herein suitable for cleaving the cyclic loop nucleotides, elongating the daughter strand to form an elongated polynucleotide:wherein 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.Cleavable Cyclic Loop 4-mer Oligonucleotide

[0147] In some embodiment, two ends of a linker construct may be conjugated to 2 nucleobases of a 4-mer oligonucleotide to form a cleavable cyclic loop 4-mer oligonucleotide. An allyl cleavable bond can be situated anywhere along the oligonucleotide backbone in between the two points of conjugations and allow the cyclic loop to be opened upon cleavage. In some embodiments, the conjugation points may be the first and the second bases, the first and the third bases, the first and the fourth bases, the second and the third bases, the second and the fourth bases, or the third and the fourth bases. For example, when the conjugation points are the first and the second bases, the allyl cleavage site may be situated on the backbone between the first and the second bases. When the conjugation points are the first and the third bases, the allyl cleavage site may be situated on the backbone between the first and the third bases. When the conjugation points are the first and the fourth bases, the allyl cleavage site may be situated on the backbone between the first and the fourth bases. When the conjugation points are the second and the third bases, the allyl cleavage site may be situated on the backbone between the second and the third bases. When the conjugation points are the second and the fourth bases, the allyl cleavage site may be situated on the backbone between the second and the fourth bases. When the conjugation points are the third and the fourth bases, the allyl cleavage site may be situated on the backbone between the third and the fourth bases.

[0148] In some embodiments, when the conjugation points are the first and the second bases, the allyl group may be located on the Cs’ of the second nucleotide. When the conjugation points are the first and the third bases, the allyl group may be located on the Cs’ of the second or the third nucleotide. When the conjugation points are the first and the fourth bases, the allyl group may be located on the Cs’ of the second, the third, or the fourth nucleotide. When the conjugation points are the second and the third bases, the allyl group may be located on the Cs’ of the third nucleotide. When the conjugation points are the second and the fourth bases, the allyl group may be located on the Cs’ of the third or the fourth nucleotide. When the conjugation points are the third and the fourth bases, the allyl group may be located on the Cs’ of the fourth nucleotide. In some embodiments, heavy atom substituted nucleotides may be used to generate resulting 4-mer oligonucleotides.

[0149] Examples of the cleavable cyclic loop 4-mer oligonucleotides include:wherein Li is a first linking group; L2 is a second linking group; one of R1, R2, and R3is allyl while the others are H; SP is a spacer; and Base is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil.

[0150] The cyclic loops can be designed symmetrically or asymmetrically, depending on the conjugation chemistries 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, 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, azidenorbornene, Cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazineisocyanide, and cyclooctyne-tetrachlorocyclopentadienone ethylene ketal. Li and L2 may or may not be the same.

[0151] In some embodiments, each of the first linking group Li and the second linking group L2 may independently further comprises a linker. A first linker may be present between the conjugating moiety and a base in the 4-mer oligonucleotide, and a second linker may be present between the conjugating moiety and another base in the 4-mer oligonucleotide. 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 comprise peptides, polypeptides, alkyl chains, polyethylene glycol, or combinations thereof. In some embodiments, one or more linkers may be absent.

[0152] In some embodiments, SP (spacer) comprises a polymer. Spacers within a cyclic loop provide buffering distance between successive cyclic loops or successive subelements comprising one or more cyclic loops. In some embodiments, the polymer in the SP 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, modifiedoligonucleotides may include oligonucleotides that do not have a base attached to the sugar,In some embodiments, modified oligonucleotides may includethat can be assembled into a polymer using an 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 (barcode).

[0153] In some embodiments, the cyclic loop may comprise one or more barcodes, or reporters, including reporter moieties, sub-reporters, reporter elements, and sub-reporter elements. Examples include, but not limited to, nucleosidic bases, non-nucleosidic bases, peptides or other synthetic polymers such as polyethyleneglycol, polyvinylalcohol, polyacrylamide, polyvinylpyrrolidone, polyethyleneimine, etc. In some embodiments, the reporter element can comprise of macromolecules such as crown ethers, cucurbiturils, pillararenes or cyclodextrins. Conjugation of these macromolecules to the cyclic loop construct is possible through covalent conjugation chemistries such as amine-NHS ester, amine- imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxylcarbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azidealkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, azide-norbornene, Cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne-tetrachlorocyclopentadienone ethylene ketal. In some embodiments, reporters can be selected from any moiety, including spacers, conjugating moiety, and arresting constructs. In some embodiments, reporters can comprise a plurality of modifications ranging anywhere from 1-10, or 11-15, or 16-20, or 21-25, 26-50, or 50-100units in length, as long as the reporter provides reproducible signals at a given voltage waveform when resident in the readhead of a nanopore.

[0154] In some embodiments, advancement of each nucleotide bearing one or more barcodes or reporter moieties, corresponds to a translocation event. In some embodiments, the reporter moiety comprises one or more sub-reporter moieties, the sub-reporter moieties configured to identify a translocation event and generate a signal when passed through the readhead of a nanopore. In some embodiments, the reporter moiety comprises two or more sub-reporter moieties, wherein each sub-reporter moiety in the two or more sub-reporter moieties are distinguishable, reproducible, and resolvable. In some embodiments, a cyclic loop may comprise a first set of reporter moieties, and a second set of reporter moieties, wherein the first set of reporter moieties are configured to generate signals to identify particular nucleotides passing through the readhead, and wherein the second set of reporter moieties are configured to generate signals to identify the passage of each nucleotide regardless of nucleotide identity.Additional Notes

[0155] 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.

[0156] 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.

[0157] 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 explicitlyrecited. 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.

[0158] 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.

[0159] 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.

[0160] 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 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.

[0161] 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 anysuitable 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.

[0162] 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.

[0163] 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.

[0164] 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, certainfeatures, 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 input or prompting, whether these features, elements, and / or steps are included or are to be performed in any particular example.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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. All references cited herein are incorporated by reference in their entirety.

[0169] 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 thespecification. 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 limiting the 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.

[0170] 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 compound having one of the following structures:X is -O-, -CH2-, -=N- or -NH-;Y is -O-, -S-, or -NH-;Base is a modified or unmodified nucleobase;Li is a first linking group;L2 is a second linking group; andSP comprises a spacer and a reporter, wherein the spacer and the reporter each comprises one or more of the following moieties: a) polypeptides having 10 to 100 repeating units; b) pseudopeptides having 10 to 100 repeating units; c) hydrophilic polymers having 10 to 100 repeating units; d) hydrophobic polymers having 10 to 100 repeating units.

2. The compound of Claim 1, wherein the reporter comprises one or more of the following: a) a homopolymer; b) an alternating polymer;c) a random-mer; and d) an epitope tag.

3. The compound of Claim 1 or 2, wherein each of Li and L2 independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiolthiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxyisocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, azide-norbornene, and Cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne-tetrachlorocyclopentadienone ethylene ketal.

4. The compound of Claim 3, wherein each of Li and L2 independently further comprises a first linker between the conjugating moiety and X, and a second linker between the conjugating moiety and SP.

5. The compound of Claim 4, wherein the first linker and the second linker are independently selected from the group consisting of 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, polymethymethacrylate, or polystyrene, and combinations thereof.

6. The compound of any one of Claims 1 to 5, wherein one or both of the spacer and the reporter comprise peptides or pseudopeptides, with pendant nucleobases attached.

7. The compound of any one of Claims 1 to 6, wherein the nucleobase is a modified nucleobase.

8. The compound of Claim 7, wherein the modification is a linear, a branched or a cyclic polymer attached to the nucleobase.

9. The compound of Claim 8, wherein the modification comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof.

10. An oligonucleotide comprising one of the following structures:X is -0-, -CH2-, -=N- or -NH-;Y is -O-, -S-, or -NH-; one of R1, R2, and R3is allyl, while the others are H,Li is a first linking group;L2 is a second linking group; andSP comprises a spacer, wherein the spacer comprises one or more of the following moieties: a) polypeptides having 10 to 100 repeating units; b) pseudopeptides having 10 to 100 repeating units; c) hydrophilic polymers having 10 to 100 repeating units; d) hydrophobic polymers having 10 to 100 repeating units.

11. The oligonucleotide of Claim 10, wherein structures (lb) to (XI) further comprise one or more modifications in the spacer region configured to slow, pause, or halt translocation of the oligonucleotide through a nanopore.

12. The oligonucleotide of Claim 11, wherein the one or more modifications comprise: a) alternating positively and negatively charged subregions of polypeptides; b) bulk moieties to increase steric hinderance during translocation; c) beta sheet or kink-inducing amino acid moieties; d) hydrophobic subregions configured to non-covalently interact with surfactants.

13. The oligonucleotide of Claim 10, wherein the spacer is a reporter that identifies one or more bases in the oligonucleotide.

14. The oligonucleotide of Claim 13, wherein the reporter is one or more of the following: a) a homopolymer; b) an alternating polymer; c) a random-mer; or d) an epitope tag.

15. The oligonucleotide of any one of Claims 10 to 14, wherein each of Li and L2 independently comprises a conjugating moiety selected from the group consisting of amine- NHS ester, amine- imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxyisocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, azide-norbornene, cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne-tetrachlorocyclopentadienone ethylene ketal.

16. The oligonucleotide of Claim 15, wherein each of Li and L2 independently further comprises a first linker between the conjugating moiety and X, and a second linker between the conjugating moiety and SP.

17. The oligonucleotide of Claim 16, wherein 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, polymethymethacrylate, or polystyrene, and combinations thereof.

18. The oligonucleotide of any one of Claims 10 to 17, wherein SP further comprises a modification.

19. The oligonucleotide of any one of Claims 10 to 17, wherein the Base further comprises a modification.

20. The oligonucleotide of Claim 18 or 19, wherein the modification is a linear, a branched or a cyclic polymer.

21. The oligonucleotide of Claim 20, wherein the modification comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof.

22. A method for determining a sequence of a polynucleotide in a nanopore-based sequencing system, the method comprising: providing a polynucleotide comprising a plurality of nucleotides, wherein each nucleotide comprises a cyclic loop, the cyclic loop having a first end attached to a first position of the nucleotide and a second end attached to a second position of the nucleotide, wherein the cyclic loop contains one or more sequences of polypeptides; cleaving a cleavable bond on each of the plurality of nucleotide between the first and the second positions, thereby elongating the polynucleotide to form an elongated polymer; applying a voltage to cause the elongated polymer to insert into and translocate through a nanopore; and(i) detecting and identifying the one or more sequences of polypeptides when the cyclic loop passes through the nanopore; or(ii) detecting and identifying a base on the nucleotide when the nucleotide passes through the nanopore.

23. The method of claim 22, wherein the cyclic loop comprises a first linking group, a second linking group, and a spacer between the first and the second linking groups.

24. The method of claim 23, wherein the spacer comprises a polypeptide having 10 to 100 repeating units, alkyl chains having 10 to 200 carbons, hydrophilic polymers having 10 to 100 repeating units selected form the group consisting of polyethyleneglycol, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, and polyethyleneimine, hydrophobic polymers having 10 to 100 repeating units selected from the group consisting of polylactic acid, polymethymethacrylate, and polystyrene, and combinations thereof.

25. The method of claim 23, wherein the one or more sequences of polypeptides is a reporter barcode, wherein the reporter barcode corresponds to and identifies a nucleotide when passing through the nanopore.

26. The method of any one of Claims 23 to 25, wherein each of the first and the second linking groups independently comprises a conjugating moiety selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentofluorophenyl 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, azide-norbornene, cyclooctyne-tetrazine, hydroxylamine-potassium acyltrifluoroborate, tetrazine-isocyanide, and cyclooctyne- tetrachlorocyclopentadienone ethylene ketal.

27. The method of any one of claim 22 to 26, wherein the elongated polymer further comprises an arresting construct attached to each nucleobase or each cyclic loop, wherein the arresting construct is configured to slow, pause, or halt the translocation.

28. The method of Claim 27, wherein the arresting construct is a linear, a branched or a cyclic polymer.

29. The method of Claim 28, wherein the arresting construct comprises a synthetic hydrophobic polymer, a synthetic hydrophilic polymer, an oligonucleotide / polynucleotide, a peptide / polypeptide, or combinations thereof.

30. The method of any one of claims 22 to 29, wherein the nanopore comprises a constriction having an opening with an inner diameter from about 0.6 nm to about 1.2 nm.

31. The method of claim 22, wherein the polynucleotide comprises one of the oligonucleotides of any one of Claims 10 to 21.

32. The method of claim 22, wherein the plurality of nucleotides are each selected from the compounds according to any one of claims 1 to 10.

33. A kit for performing a method for determining a sequence of a polynucleotide in a nanopore-based sequencing system, the kit comprising the compound according to any of claims 1 to 10.

34. A system for determining a sequence of a polynucleotide, the system configured to perform a method according to any of claims 22 to 31.

35. A system for performing a method for determining a sequence of a polynucleotide comprising a plurality of nucleotides, wherein the nucleotides are selected from any of the compounds according to claims 1-10.

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