Patent Application
20230175059
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 28, 2021, is named IP-1955-PCT_SL.txt and is 1,503 bytes in size.
A significant amount of academic and corporate time and energy has been invested into sequencing polynucleotides, such as DNA. Some sequencing systems use “sequencing by synthesis” (SBS) technology and fluorescence-based detection. However, fluorescence-based detection may require optical components such as excitation light sources, imaging devices, and the like, which may be complex, time-consuming to operate, and costly.
Examples provided herein are related to electronically sequencing polynucleotides using partially double-stranded polymer bridges. Compositions and methods for performing such electronic sequencing are disclosed.
In some examples, the bridges may span the space between first and second electrodes. A plurality of nucleotides may be coupled to corresponding labels. A polymerase may be coupled to, or in proximity to, the bridge and may add nucleotides to a first polynucleotide using at least a sequence of a second polynucleotide. The labels corresponding to those nucleotides respectively may hybridize to a portion of the bridge that is not double-stranded. Detection circuitry may detect a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in an electrical signal, for example current or voltage, through the bridge, the changes being responsive to the respective hybridizations between the non-double stranded portion of the bridge and the labels corresponding to those nucleotides.
Provided in some examples herein is a composition that includes first and second electrodes separated from one another by a space, and a bridge spanning the space between the first and second electrodes. The bridge may include first and second polymer chains hybridized to one another. The first polymer chain may have a first length, and the second polymer chain may have a second length shorter than the first length, such that a gap region of the first polymer chain is not hybridized to the second polymer chain. The gap region may include first and second universal monomers. The composition further may include first and second polynucleotides. The composition further may include a plurality of nucleotides, each nucleotide coupled to a corresponding label. The composition further may include a polymerase to add nucleotides from the plurality of nucleotides to the first polynucleotide using at least a sequence of the second polynucleotide. The labels corresponding to those nucleotides respectively may hybridize to the first and second universal monomers. The composition further may include detection circuitry to detect a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in an electrical signal through the bridge, the changes being responsive to the respective hybridizations between the first and second universal monomers and the labels corresponding to those nucleotides.
In some examples, the first and second polymer chains respectively include third and fourth polynucleotides. In some examples, the labels may include respective oligonucleotides having different sequences than one another. In some examples, the first and second universal monomers respectively may include first and second universal bases. In some examples, hybridization between the oligonucleotides and the first and second universal bases changes the electrical signal through the bridge. In some examples, the first and second universal bases independently are selected from the group consisting of inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives. In some examples, the third and fourth polynucleotides and the oligonucleotides of the labels include non-naturally occurring DNA. In some examples, the non-naturally occurring DNA includes enantiomeric DNA.
In some examples, the gap region further includes a stabilization region. The labels further may hybridize to the stabilization region. The stabilization region may stabilize hybridizing of the labels to the first and second universal monomers.
In some examples, the gap region is located at a terminal end of the first polymer chain.
Provided in some examples herein is a method for sequencing. The method may include adding, by a polymerase, nucleotides to a first polynucleotide using at least a sequence of a second polynucleotide. The method may include hybridizing labels respectively coupled to the nucleotides to a gap region of a polymer chain of a bridge spanning a space between first and second electrodes, the gap region including first and second universal monomers. The method may include detecting a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in an electrical signal through the bridge that are responsive to respective hybridizations between the universal monomers and the labels corresponding to those nucleotides.
In some examples, the polymer chain includes a polynucleotide. In some examples, the labels include respective oligonucleotides having different sequences than one another. In some examples, the first and second universal monomers respectively include first and second universal bases. In some examples, hybridization between the oligonucleotides and the first and second universal bases changes the electrical signal through the bridge. In some examples, the first and second universal bases independently are selected from the group consisting of inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives. In some examples, the third polynucleotide and the oligonucleotides of the labels include non-naturally occurring DNA. In some examples, the non-naturally occurring DNA includes enantiomeric DNA.
In some examples, the gap region further includes a stabilization region, and the method further includes stabilizing, by the stabilization region, hybridization of the respective labels to the first and second universal monomers.
In some examples, the gap region is located at a terminal end of the polymer chain.
Provided in some examples herein is a composition that includes first and second electrodes separated from one another by a space, and a bridge spanning the space between the first and second electrodes. The bridge may include first and second polymer chains each having a first region in which the first and second polymer chains are not hybridized to one another, and a second region in which the first and second polymer chains are hybridized to one another. The composition also may include first and second polynucleotides. The composition also may include a plurality of nucleotides, each nucleotide coupled to a corresponding label. The composition also may include a polymerase coupled to the first region of the second polymer chain. The polymerase may add nucleotides of the plurality of nucleotides to the first polynucleotide using at least a sequence of the second polynucleotide. The labels corresponding to those nucleotides respectively may hybridize to the first region of the first polymer chain. The composition also may include detection circuitry to detect a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in an electrical signal through the bridge, the changes being responsive to the respective hybridizations between the first region of the first polymer chain and the labels corresponding to those nucleotides.
In some examples, the first and second polymer chains respectively include third and fourth polynucleotides. In some examples, the labels include respective oligonucleotides having different sequences than one another. In some examples, the third polynucleotide further includes first and second universal bases to which the oligonucleotides respectively hybridize. In some examples, hybridization between the oligonucleotides and the first and second universal bases changes the electrical signal through the bridge. In some examples, the first and second universal bases independently are selected from the group consisting of inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives. In some examples, the first region of the second polymer chain includes a polymer that does not hybridize to the oligonucleotides. In some examples, the third and fourth polynucleotides and the oligonucleotides of the labels include non-naturally occurring DNA. In some examples, the non-naturally occurring DNA includes enantiomeric DNA.
In some examples, the first polymer chain further includes first and second universal monomers to which first and second monomers of each label respectively hybridize. In some examples, the first and second universal monomers are located at a terminal end of the first polymer chain.
In some examples, the first region of the second polymer chain is nonconductive.
Provided in some examples herein is a method for sequencing. The method may include adding, by a polymerase, nucleotides to a first polynucleotide using at least a sequence of a second polynucleotide. The method may include hybridizing labels respectively coupled to the nucleotides to a first region of a first polymer chain of a bridge spanning a space between first and second electrodes. The bridge further may include a second polymer chain. The polymerase may be coupled to the first region of the second polymer chain, and a second region of the first polymer chain may be hybridized to a second region of the second polymer chain. The method may include detecting a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in an electrical signal through the bridge that are responsive to respective hybridizations between the first region of the first polymer chain and the labels corresponding to those nucleotides.
In some examples, the first and second polymer chains respectively include third and fourth polynucleotides. In some examples, the labels include respective oligonucleotides having different sequences than one another. In some examples, the third polynucleotide further includes first and second universal bases to which the oligonucleotides respectively hybridize. In some examples, hybridization between the oligonucleotides and the first and second universal bases changes the electrical signal through the bridge. In some examples, the first and second universal bases independently are selected from the group consisting of inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives. In some examples, the first region of the second polymer chain includes a polymer that does not hybridize to the oligonucleotides. In some examples, the third and fourth polynucleotides and the oligonucleotides of the labels include non-naturally occurring DNA. In some examples, the non-naturally occurring DNA includes enantiomeric DNA.
In some examples, the first polymer chain further includes first and second universal monomers to which first and second monomers of each label respectively hybridize. In some examples, the first and second universal monomers are located at a terminal end of the first polymer chain.
In some examples, the first region of the second polymer chain is nonconductive.
It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.
Examples provided herein are related to electronically sequencing using partially double-stranded polymer bridges. Compositions and methods for performing such electronic sequencing are disclosed.
More specifically, the present compositions and methods suitably may be used to sequence polynucleotides in a manner that is robust, reproducible, sensitive, and has high throughput. For example, the present compositions can include first and second electrodes and a bridge that spans the space between the electrodes. The bridge can include partially double-stranded polymers, e.g., can include first and second polymer chains that are at least partially hybridized to one another in such a manner as to leave available a region to which the label of a labeled nucleotide may be hybridized during a sequencing process. The hybridization of the label to the region may modulate the electrical characteristics of the bridge, for example the conductivity or impedance of the bridge, and using at least such modulation the nucleotide may be identified. In some examples, the region to which the label may be hybridized includes a gap in the bridge where the first and second polymer chains are not hybridized to one another, e.g., where the second polymer chain is shorter than the first polymer chain. In some examples, the gap may include one or more universal bases that may enhance modulation of the bridge's electrical conductivity or impedance when the label hybridizes within the gap, and thus may enhance accuracy, speed or reliability of identifying the nucleotide attached to that label.
In other examples, the region to which the label may be hybridized includes a portion of a bifurcated bridge in which the first and second polymer chains are partially hybridized to each other in one region, and are not hybridized to each other in another region. The polymerase may be coupled to one of the polymer chains in the non-hybridized region, and the label may hybridize with the other polymer chain in the non-hybridized region so as to modulate conductivity or impedance of that portion of the bridge. Such a bifurcated arrangement may reduce or inhibit the polymerase from applying forces to the portion of the polymer chain to which the label hybridizes, and such forces otherwise may themselves modulate conductivity or impedance in such a manner as to at least partially obscure conductivity or impedance changes resulting from the label. Alternatively, in other embodiments, such forces may be advantageous. Forces exerted by the polymerase that result in a modulation of conductivity or impedance that is detectable through the bridge may carry beneficial information that enhances the accuracy, speed or reliability of identifying the nucleotide in the polymerase active site.
First, some terms used herein will be briefly explained. Then, some example compositions and example methods for electronically sequencing will be described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
The terms “substantially”, “approximately”, and “about” used throughout this Specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.
As used herein, the term “electrode” is intended to mean a solid structure that conducts electricity. Electrodes may include any suitable electrically conductive material, such as gold, palladium, or platinum, or combinations thereof.
As used herein, the term “bridge” is intended to mean a structure that extends between, and attaches to, two other structures. A bridge may span a space between other structures, such as between two electrodes. Not all elements of a bridge necessarily need to attach to both structures. For example, in a bridge that includes first and second polymer chains associated with one another and spanning the space between two electrodes, at least one end of one of the polymer chains attaches to one of the electrodes, and at least one end of one of the polymer chains attaches to the other electrode. However, both polymer chains need not connect to both of the electrodes, and indeed one of the polymer chains need not contact either of the electrodes. A bridge may include multiple components which are attached to one another in such a manner as to extend between, and collectively connect to, other structures. A bridge may be attached to another structure, such as an electrode, via a chemical bond, e.g., via a covalent bond, hydrogen bond, ionic bond, dipole-dipole bond, London dispersion forces, or any suitable combination thereof.
As used herein, a “polymer” refers to a molecule including a chain of many subunits, that may be referred to as monomers, that are coupled to one another. The subunits may repeat, or may differ from one another. Polymers and their subunits can be biological or synthetic. Example biological polymers that suitably can be included within a bridge or a label include polynucleotides (made from nucleotide subunits), polypeptides (made from amino acid subunits), polysaccharides, polynucleotide analogs, and polypeptide analogs. Example polynucleotides and polynucleotide analogs suitable for use in a bridge or a label include DNA, enantiomeric DNA, RNA, PNA (peptide-nucleic acid), morpholinos, and LNA (locked nucleic acid). Polymers may include spacer subunits, derived from phosphoramidites, which may be coupled to polynucleotides, but which lack nucleobases, such as commercially available from Glen Research (Sterling, Va.), for example Spacer Phosphoramidite 18 (18-O-Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite). Example synthetic polypeptides can include all natural amino acids, such as charged amino acids, hydrophilic, hydrophobic and neutral amino acid residues. Example synthetic polymers that suitably can be included within a bridge or label include PEG (polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinyl alcohol), PE (polyethylene), LDPE (low density polyethylene), HDPE (high density polyethylene), polypropylene, PVC (polyvinyl chloride), PS (polystyrene), NYLON (aliphatic polyamides), TEFLON® (tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes, polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters), poly(alkyl methacrylates), and other polymeric chemical and biological linkers such as described in Hermanson, Bioconjugate Techniques, third edition, Academic Press, London (2013).
As used herein, “hybridize” is intended to mean noncovalently associating a first polymer to a second polymer along the lengths of those polymers. For instance, two DNA polynucleotide strands may associate through complementary base pairing. The strength of the association between the first and second polymers increases with the complementarity between the sequences of monomer units within those polymers. For example, the strength of the association between a first polynucleotide and a second polynucleotide increases with the complementarity between the sequences of nucleotides within those polynucleotides.
As used herein, the term “stabilization region” is intended to mean a portion of a polymer that enhances the strength of attachment between a first polymer and a second polymer. Stabilization can be accomplished by various means, including, but not limited to, regions of complementarity between a first and second polymers, and pi stacking of bases at a nick in a polymer.
As used herein, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase. A nucleotide that lacks a nucleobase can be referred to as “abasic.” Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).
As used herein, the term “nucleotide” also is intended to encompass any nucleotide analogue which is a type of nucleotide that includes a modified nucleobase, sugar and/or phosphate moiety compared to naturally occurring nucleotides. Example modified nucleobases include inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5′-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates.
As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. A polynucleotide is one nonlimiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof. A polynucleotide can be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, or can include a mixture of a single stranded and double stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa. Polynucleotides can include non-naturally occurring DNA, such as enantiomeric DNA. The precise sequence of nucleotides in a polynucleotide can be known or unknown. The following are example examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing.
As used herein, a “universal monomer” refers to a monomer unit of a polymer that may hybridize with more than one unit of such a polymer. In some examples, a universal monomer may hybridize with any other monomer unit of such a polymer. An example of a “universal monomer” of a polynucleotide is a “universal base” which refers to a nucleobase that may hybridize with more than one base type, and in some examples may hybridize with any other nucleobase. Examples of universal bases include modified nucleobases such as inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives. For further details regarding universal bases, see Loakes, “The applications of universal DNA base analogues,” Nucleic Acids Research 29(12): 2437-2447 (2001).
As used herein, a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind a primed single stranded polynucleotide template, and can sequentially add nucleotides to the growing primer to form a polynucleotide having a sequence that is complementary to that of the template.
As used herein, the term “primer” is defined as a polynucleotide to which nucleotides are added via a free 3′ OH group. A primer may have a 3′ block preventing polymerization until the block is removed. A primer can also have a modification at the 5′ terminus to allow a coupling reaction or to couple the primer to another moiety. The primer length can be any number of bases long and can include a variety of non-natural nucleotides.
As used herein, the term “label” is intended to mean a structure that attaches to a bridge in such a manner as to cause a change in the electrical characteristics of the bridge, such as impedance or conductivity, and based upon which change the nucleotide may be identified. For example, a label may hybridize to a polymer chain within such a bridge, and the hybridization may cause a conductivity or impedance change of the bridge. In examples provided herein, labels can be attached to nucleotides.
As used herein, the term “substrate” refers to a material used as a support for compositions described herein. Example substrate materials may include glass, silica, plastic, quartz, metal, metal oxide, organo-silicate (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated by reference in its entirety. In some examples, substrates used in the present application include silica-based substrates, such as glass, fused silica, or other silica-containing material. In some examples, substrates can include silicon, silicon nitride, or silicone hydride. In some examples, substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, and poly(methyl methacrylate). Example plastics materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or plastic material or a combination thereof. In particular examples, the substrate has at least one surface comprising glass or a silicon-based polymer. In some examples, the substrates can include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface comprising a metal oxide. In one example, the surface comprises a tantalum oxide or tin oxide. Acrylamides, enones, or acrylates may also be utilized as a substrate material or component. Other substrate materials can include, but are not limited to gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers and copolymers. In some examples, the substrate and/or the substrate surface can be, or include, quartz. In some other examples, the substrate and/or the substrate surface can be, or include, semiconductor, such as GaAs or ITO. The foregoing lists are intended to be illustrative of, but not limiting to the present application. Substrates can comprise a single material or a plurality of different materials. Substrates can be composites or laminates. In some examples, the substrate comprises an organo-silicate material.
Substrates can be flat, round, spherical, rod-shaped, or any other suitable shape. Substrates may be rigid or flexible. In some examples, a substrate is a bead or a flow cell.
Substrates can be non-patterned, textured, or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may comprise posts, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. Patterns may be regular or irregular across the surface of the substrate. Patterns can be formed, for example, by nanoimprint lithography or by use of metal pads that form features on non-metallic surfaces, for example.
In some examples, a substrate described herein forms at least part of a flow cell or is located in or coupled to a flow cell. Flow cells may include a flow chamber that is divided into a plurality of lanes or a plurality of sectors. Example flow cells and substrates for manufacture of flow cells that can be used in methods and compositions set forth herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, Calif.).
Substrate 101 may support first electrode 102 and second electrode 103. First electrode 102 and second electrode 103 may be separated from one another by a space, e.g., a space of length L as indicated in
Bridge 110 may span the space between first electrode 102 and second electrode 103, and may include first polymer chain 111 and second polymer chain 112 hybridized to one another (the circles within the respective polymer chains being intended to suggest monomer units that are coupled to one another along the lengths of the polymer chains). First polymer chain 111 and second polymer chain 112 may include the same type of polymer as one another, although the sequence of monomer units in the respective polymer chains may be different than one another. Indeed, in the nonlimiting example shown in
As explained in greater detail below with reference to
Composition 100 illustrated in
Composition 100 illustrated in
At the particular time illustrated in
In comparison,
In the example illustrated in
In one nonlimiting example, labels 131, 132, 133, 134 include respective oligonucleotides having at least partially different sequences than one another, and gap region 113 includes a polynucleotide which in some examples has the same length as those oligonucleotides, such that hybridization of the labels to gap region 113 provides a fully double-stranded polynucleotide along the length of bridge 110. The label's respective oligonucleotide sequences may hybridize differently with the sequence of polymer chain 111 within gap region 113. For example, first and second signal monomers 171, 172 of label 131 may be nucleotides that are the same as or different from one another. The first and second signal monomers of the other labels may be nucleotides that are different in sequence or in type, or both, from the first and second signal monomers of the other labels, such that each label 131, 132, 133, 134 has a unique sequence of first and signal monomers. The respective hybridization between the first and second signal monomers for each label and the first and second universal bases 114, 115 may provide a particular electrical signal through bridge 110. For example, label 131 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 114, 115 so as to modulate the electrical conductivity or impedance of bridge 110 to a first level; label 132 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 114, 115 so as to modulate the electrical conductivity or impedance of bridge 110 to a second level that is different from the first level; label 133 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 114, 115 so as to modulate the electrical conductivity or impedance of bridge 110 to a third level that is different from the first and second levels; and label 134 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 114, 115 so as to modulate the electrical conductivity or impedance of bridge 110 to a fourth level that is different from the first, second, and levels. Note that in addition to differences in the first and second signal monomers for different labels providing different signals, other monomers in those labels may differ from one another in such a way as to modulate the electrical signal through bridge 110. As one example, such monomers may include one or more base modifications, such as methylation, that do not change the hybridization specificity, but may alter electrical characteristics.
In particular, first and second universal bases 114, 115 may be expected to provide bridge 110 with enhanced conductivity, as well as greater modulations in electrical signal when labels hybridize, than may other types of nucleobases when labels hybridize to gap region 113. For example, in illustrative configurations in which first and second polymer chains 111, 112 respectively include third and fourth polynucleotides and labels 131, 132, 133, 134 respectively include oligonucleotides, nucleotides within the labels respectively hybridize with nucleobases within the gap region 113 of first polymer chain 111. Labels 131, 132, 133, and 134 include different nucleotide sequences than one another so as to provide different electrical conductivities or impedances through bridge 110 than one another, based upon which the corresponding nucleotides to which the labels are coupled may be identified using detection circuitry 160. However, because the labels' sequences are different than one another while the sequence of gap region 113 remains the same, not all nucleotides in the labels' sequences necessarily complement all nucleotides in the gap region. In some examples, any mismatches between base pairs may significantly decrease current flowing through bridge 110, even when hybridization of the labels to gap region 113 provides a fully double-stranded polymer along the length of bridge 110, potentially leading to lower overall current and difficulty in distinguishing different electrical signals from one another. Because universal bases 114, 115 may hybridize with two or more nucleotides in the labels, and in some examples may hybridize with all nucleotides in the labels, the occurrence of mismatches between nucleotides in the labels to nucleotides in gap region 113 may be reduced or avoided, thus increasing current flowing through bridge 110, while still allowing different (and distinguishable) electrical signals through bridge 110 responsive to different ones of labels 131, 132, 133, and 134 being hybridized to the gap region. In one nonlimiting example, first and second universal bases 114, 115 independently are selected from the group consisting of inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives.
However, the strength of hybridization between first and second universal bases 114, 115 and corresponding bases within labels 131, 132, 133, 134 may not, by itself, be sufficiently strong to maintain those labels for a sufficient amount of time for detection circuitry 160 to reliably detect the resulting modulation in electrical signal. Optional stabilization region 116 may help to stabilize the respective hybridization of labels 131, 132, 133, and 134 to gap region 113. For example, the stabilization complement regions of the labels may hybridize relatively strongly to stabilization region 116 so as to maintain hybridization of the labels to gap region 113 for a sufficient amount of time for detection circuitry 160 to reliably detect the resulting modulation in electrical signal. The length of stabilization region 116 (e.g., the number of monomer units providing stabilization region 116) may be selected so as to provide sufficient strength of hybridization between the labels and gap region 113 that the labels may remain hybridized to gap region 113 while polymerase 105 is adding the corresponding nucleotides to first polynucleotide 140, and may dehybridize from gap region 113 thereafter so that the label of the next nucleotide in the sequence then may hybridize to the gap region. In examples in which the first and second polymer chains include oligonucleotides, stabilization additionally or alternatively may be provided by base stacking between the 3′ nucleotide of the second polymer chain (the nucleotide to which arrow 112 is pointing in
It will be appreciated that gap region 113 may include any suitable combination, order, and type of monomer units (e.g., nucleotides) to allow electrical signals from different labels to be detected and distinguished from one another, while sufficiently stabilizing hybridization between the labels and the gap region. For example, the gap region may include any suitable number of universal monomers (e.g., universal bases), e.g., one, two, three, four, or more than four universal monomers. The universal monomers may be, but need not necessarily be, located adjacent to one another. For example, the universal monomers may be spaced apart from one another by one or more monomers that are not universal. The gap region in some examples also may include any suitable number of monomers that sufficiently stabilize hybridization between the labels and the universal monomers. For example, the gap region may include any suitable number of stabilizing monomers (e.g., nucleotides), e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten stabilizing monomers. The stabilizing monomers may be, but need not necessarily be, located adjacent to one another. For example, the stabilizing monomers may be spaced apart from one another by one or more universal monomers. In some examples, the sequence in the gap region may include regions that are relatively GC-rich so as enhance stability as compared to AT-rich regions. Additionally, or alternatively, the optional stabilization region may include modified nucleotides that are known to increase stability, such as PNA, LNA, 2,6-Diaminopurine (2-Amino-dA), or 5-hydroxybutynl-2′-deoxyuridine.
Similarly, the labels 131, 132, 133, and 134 respectively may include any suitable combination, order, and type of monomer units (e.g., nucleotides) to allow electrical signals from different labels to be detected and distinguished from one another, while sufficiently stabilizing hybridization between the labels and gap region 113. For example, the labels may include any suitable number of monomers that respectively may hybridize with universal monomers (e.g., universal bases) of the gap region, e.g., one, two, three, four, or more than four universal monomers. These monomers may be, but need not necessarily be, located adjacent to one another. For example, these monomers may be spaced apart from one another by one or more monomers that may not hybridize with the universal monomers. The labels in some examples also may include any suitable number of monomers that sufficiently stabilize hybridization between the labels and the universal monomers. For example, the labels may include any suitable number of stabilizing complement monomers (e.g., nucleotides), e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten stabilizing complement monomers. The stabilizing complement monomers may be, but need not necessarily be, located adjacent to one another. For example, the stabilizing complement monomers may be spaced apart from one another by one or more monomers that respectively may hybridize with universal monomers. The number of monomer units (e.g., nucleotides) within each label in some examples is the same, or approximately the same, as the number of monomer units within gap region 113.
Illustratively,
In the example illustrated in
The polymers included within the bridge between the electrodes and within the labels coupled to the nucleotides may include any suitable material such as exemplified herein. In certain examples, these polymers respectively include polynucleotides.
In the example illustrated in
Compositions such as described with reference to
Method 400 illustrated in
Referring again to
Referring again to
It will be appreciated that uses of partially double-stranded bridges for electronically sequencing are not limited to the specific examples described with reference to
Substrate 501 may support first electrode 502 and second electrode 503. First electrode 502 and second electrode 503 may be separated from one another by a space, e.g., a space of length L as indicated in
First polymer chain 511 and second polymer chain 512 may include the same type of polymer as one another, although the sequence of monomer units in the respective polymer chains may be different than one another. In the nonlimiting example shown in
As explained in greater detail below with reference to
Composition 500 illustrated in
Composition 500 illustrated in
In comparison,
In the example illustrated in
In one nonlimiting example, labels 531, 532, 533, 534 include respective oligonucleotides having at least partially different sequences than one another, and first region 518 of first polymer chain 511 includes a third polynucleotide which in some examples has the same length as those oligonucleotides, such that hybridization of the labels to first region 518 of first polymer chain 511 provides a fully double-stranded polynucleotide along the length of bridge 510. The label's respective oligonucleotide sequences may hybridize differently with the sequence of polymer chain 511 within first region 518. For example, first and second signal monomers 571, 572 of label 531 may be nucleotides that are the same as or different from one another. The first and second signal monomers of the other labels may be nucleotides that are different in sequence or in type, or both, from the first and second signal monomers of the other labels, such that each label 531, 532, 533, 534 has a unique sequence of first and signal monomers. The respective hybridization between the first and second signal monomers for each label and the first and second universal bases 514, 515 may provide a particular electrical signal through bridge 510. For example, label 531 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 514, 515 so as to modulate the electrical conductivity or impedance of bridge 510 to a first level; label 532 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 514, 515 so as to modulate the electrical conductivity or impedance of bridge 510 to a second level that is different from the first level; label 533 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 514, 515 so as to modulate the electrical conductivity or impedance of bridge 510 to a third level that is different from the first and second levels; and label 534 may have a sequence with a particular pair of bases that hybridizes with first and second universal bases 514, 515 so as to modulate the electrical conductivity or impedance of bridge 510 to a fourth level that is different from the first, second, and levels.
In particular, first and second universal bases 514, 515 may be expected to provide bridge 510 with enhanced conductivity, as well as greater modulations in electrical signal when labels hybridize, than would other types of nucleobases when labels hybridize to first region 518 of first polymer chain 511. For example, in illustrative configurations in which first polymer chain 511 includes a third polynucleotide and labels 531, 532, 533, 534 respectively include oligonucleotides, nucleotides within the labels respectively hybridize with nucleobases within first region 518 of first polymer chain 511. Labels 531, 532, 533, and 534 include different nucleotide sequences than one another so as to provide different electrical conductivities or impedances through bridge 510 than one another, based upon which the corresponding nucleotides to which the labels are coupled may be identified using detection circuitry 560. However, because the labels' sequences are different than one another while the sequence of first region 518 of first polymer chain 511 remains the same, not all nucleotides in the labels' sequences necessarily complement all nucleotides in first region 518 of first polymer chain 511. Any mismatches between base pairs may significantly decrease current flowing through bridge 510, even when hybridization of the labels to first region 518 of first polymer chain 511 provides a fully double-stranded polymer along the length of bridge 510, potentially leading to lower overall current and difficulty in distinguishing different electrical signals from one another. Because universal bases 514, 515 may hybridize with two or more nucleotides in the labels, and in some examples may hybridize with any and all nucleotides in the labels, the occurrence of mismatches between nucleotides in the labels to nucleotides in first region 518 of first polymer chain 511 may be reduced or avoided, thus increasing current flowing through bridge 510, while still allowing different (and distinguishable) electrical signals through bridge 510 responsive to different ones of labels 531, 532, 533, and 534 being hybridized first region 518 of first polymer chain 511. In one nonlimiting example, first and second universal bases 514, 515 independently are selected from the group consisting of inosine, nitroindole, nitropyrrole, benzimidazole, 5-fluoroindole, indole nucleoside derivatives, and isocarbostyril nucleoside derivatives.
Universal bases 514, 515 in some examples may be located at a terminal end of first polymer chain 511, as opposed to an internal position within the first polymer chain (similar to as illustrated in the example shown in
Providing polymerase 505 coupled to first region 518 of second polymer chain 512 may further stabilize the current flowing through bridge 510, and thus may further improve the ability to distinguish different electrical signals from one another. For example, as polymerase 505 incorporates nucleotides 521, 522, 523, and 524 into first polynucleotide 540, the polymerase undergoes conformational changes that otherwise may affect the conductivity or impedance of bridge 510 and thus may add signal components (e.g., noise) to measurements of changes to the signal through bridge 510. Providing polymerase 505 coupled to first region 518 of second polymer chain 512 may at least partially decouple the polymerase from the portion of bridge 510 through which current is flowing, namely first polymer chain 511, and thus at least partially inhibit signal components that otherwise would result from conformational changes of the polymerase. As a further option, second polymer chain 512 may include a nonconductive polymer that may not hybridize with any of labels 531, 532, 533, and 534, such that no or substantially no current flows through second polymer chain 512 at any time, while current may flow through first polymer chain 511 only or substantially only when one of labels 531, 532, 533, or 534 is hybridized to first region 518 of first polymer chain 511. Illustratively, first polymer chain 511 may include a third polynucleotide and the labels may include oligonucleotides that may hybridize to first portion 518 of the third polynucleotide. First portion 518 of second polymer chain 512 may include a nonconductive polymer to which the oligonucleotide labels will not hybridize, such as a polymer including spacer phosphoramidites, to which polymerase 505 may be coupled, while second portion 519 of second polymer chain 512 may include a polymer to which second portion 519 of first polymer chain 511 may hybridize, such as a polynucleotide. Polymer chains that include both spacer phosphoramidites and polynucleotides, such as may be provided for second polymer chain 512, are commercially available, e.g., from Glen Research (Sterling, Va.).
It will be appreciated that first region 518 of first polymer chain 511 may include any suitable combination, order, and type of monomer units (e.g., nucleotides) to allow signals from different labels to be detected and distinguished from one another, while sufficiently stabilizing hybridization between the labels and the first region 518 of first polymer chain 511. For example, the first region 518 of first polymer chain 511 region may include any suitable number of universal monomers (e.g., universal bases), e.g., one, two, three, four, or more than four universal monomers. The universal monomers may be, but need not necessarily be, located adjacent to one another. For example, the universal monomers may be spaced apart from one another by one or more monomers that are not universal. The first region 518 of first polymer chain 511 in some examples also may include any suitable number of monomers that sufficiently stabilize hybridization between the labels and the universal monomers. For example, the first region 518 of first polymer chain 511 may include any suitable number of stabilizing monomers (e.g., nucleotides), e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten stabilizing monomers. The stabilizing monomers may be, but need not necessarily be, located adjacent to one another. For example, the stabilizing monomers may be spaced apart from one another by one or more universal monomers.
Similarly, the labels 531, 532, 533, and 534 respectively may include any suitable combination, order, and type of monomer units (e.g., nucleotides) to allow signals from different labels to be detected and distinguished from one another, while sufficiently stabilizing hybridization between the labels and first region 518 of first polymer 511. For example, the labels may include any suitable number of monomers that may respectively hybridize with universal monomers (e.g., universal bases) of the first polymer, e.g., one, two, three, four, or more than four universal monomers. These monomers may be, but need not necessarily be, located adjacent to one another. For example, these monomers may be spaced apart from one another by one or more monomers that may not hybridize with the universal monomers. The labels in some examples also may include any suitable number of monomers that sufficiently stabilize hybridization between the labels and the universal monomers. For example, the labels may include any suitable number of additional monomers (e.g., nucleotides) that hybridize with other monomers (e.g., nucleotides) of the first polymer chain, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten additional monomers. Such additional monomers may be, but need not necessarily be, located adjacent to one another. For example, the additional monomers may be spaced apart from one another by one or more monomers that may respectively hybridize with universal monomers. The number of monomer units (e.g., nucleotides) within each label in some examples is the same, or approximately the same, as the number of monomer units within first region 518 of first polymer 511.
The polymers included within the bridge between the electrodes and within the labels coupled to the nucleotides may include any suitable material such as exemplified herein. In certain examples, these polymers respectively include polynucleotides.
In the example illustrated in
First region 618 of first polynucleotide chain 611 may include any suitable number of universal bases (illustratively, inosines (I)), e.g., first, second, and third universal bases 614, 615, 616, and any suitable sequence of remaining nucleotide units. Label 631 of nucleotide 621 may include first, second, and third signal nucleotides 671, 672, 673 that respectively hybridize with universal monomers 614, 615, 616 of first polynucleotide chain 611, and any suitable sequence of remaining nucleotide units that hybridize with the remaining nucleotide units of first region 618 of first polynucleotide chain 611 so as to provide a second conductive portion of bridge 610. First, second, and third signal nucleotides 671, 672, 673 are represented as NNN in
Compositions such as described with reference to
Method 700 illustrated in
Referring again to
Any suitable modifications may be made to any of the compositions and methods provided herein. For example, any of compositions 100, 200, 300, 500, or 600 may be modified such that any suitable polymers therein respectively include non-naturally occurring polynucleotides, such as non-naturally occurring DNA, e.g., enantiomeric DNA. Such non-naturally occurring polynucleotides may not hybridize with any naturally occurring polynucleotides in the compositions, for example, the first and second polynucleotides being acted upon by the polymerase, thus inhibiting any interference that otherwise may result from such hybridization.
While various illustrative examples are described above, it will be apparent to one skilled in the art that various changes and modifications may be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.
This applications claims the benefit of U.S. Provisional Patent Application No. 63/019,882, filed May 4, 2020 and entitled “Compositions and Methods for Sequencing Using Polymer Bridges,” the entire contents of which are incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/030453 | 5/3/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63019882 | May 2020 | US |
