Patent Application
20230235388
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 sequencing using at least altering electrical characteristics of bridges between electrodes. Compositions and methods for performing such sequencing are disclosed.
In some examples, a composition provided herein 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 a single-stranded conjugated polymer chain. The composition further may include first and second polynucleotides, and a plurality of nucleotides, each nucleotide coupled to a corresponding label. The composition further may include a polymerase to 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 alter an electrical characteristic of the single-stranded conjugated polymer chain. 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 alteration of the electrical characteristic using the labels corresponding to those nucleotides.
In some examples, the single-stranded conjugated polymer chain includes a first delocalized set of orbitals, and each of the labels includes a respective delocalized set of orbitals that shares electrons with the first delocalized set of orbitals when the label is associated with the single-stranded polymer chain. In some examples, each of the labels and the single-stranded conjugated polymer chain forms a donor:acceptor complex. In some examples, the labels include respective acceptors, the single-stranded conjugated polymer chain includes a donor, and electrons transfer from the single-stranded polymer chain to each of the labels via π-π interactions. In some examples, the labels include different molecular orbitals and detectably change electrical conductivity of the single-stranded conjugated polymer chain differently.
In some examples, the single-stranded polymer chain includes a homopolymer. In some examples, the homopolymer includes repeating monomer units M1, each of the monomer units M1 being coupled to a solubilizing group R.
In other examples, the single-stranded polymer chain includes a copolymer including first monomer units M1 and second monomer units M2, each of the first monomer units M1 being coupled to a solubilizing group R. In some examples, the copolymer includes repeating M1-M2 units.
In still other examples, the single-stranded polymer chain includes a copolymer including first monomer units M1 and second monomer units M3, wherein (i) each of the first monomer units M1, or (ii) each of the second monomer units M3, or (iii) each of the first monomer units M1 and each of the second monomer units M3, is coupled to a solubilizing group R. In some examples, the copolymer includes repeating M1-M3 units.
In other examples, the single-stranded polymer chain includes a copolymer including first monomer units M3 and second monomer units M2, each of the first monomer units M3 being coupled to a solubilizing group R. In some examples, the copolymer includes repeating M3-M2 units.
In some examples, the single-stranded polymer chain includes a copolymer including first monomer units M1, second monomer units M2, and third monomer units M3, wherein (i) each of the first monomer units M1, or (ii) each of the second monomer units M3, or (iii) each of the first monomer units M1 and each of the second monomer units M3, is coupled to a solubilizing group R. In some examples, the copolymer includes repeating M1-M2 units. In some examples, the copolymer includes repeating M3-M2 units.
In some examples, M1 is an electron-donating or electron-neutral monomer unit that includes the solubilizing group R, and wherein the electron-donating or electron-neutral monomer unit that includes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, R is a cationic solubilizing group selected from the group consisting of: (N,N,N-Trimethyl ammonium) alkyl, (N,N,N-Tributyl ammonium)alkyl, 1-methyl-3-(alkyl)-Imidazolium, and 1-(alkyl)Pyridinium.
In some examples, R is an anionic solubilizing group selected from the group consisting of: alkyl sulfonate, alkyl carboxylate, and alkyl phosphonate.
In some examples, M2 is an electron-donating monomer unit that excludes the solubilizing group R, and wherein the electron-donating monomer unit that excludes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, M3 is an electron-withdrawing monomer unit that excludes the solubilizing group R, and wherein the electron-withdrawing monomer unit that excludes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, M3 is an electron-withdrawing monomer unit that includes the solubilizing group R, and wherein the electron-withdrawing monomer unit that includes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, the composition further includes a first end group coupling the single-stranded conjugated polymer chain to the first electrode, and a second end group coupling the single-stranded conjugated polymer to the second electrode. In some examples, each of the first and second end groups is selected from the group consisting of: Benzenethiol, Phenylacetylene, Benzenediazonium, Benzylamine, Methyl benzyl sulfide, and Methyl hexyl sulfide.
In some examples, the labels are acceptors selected from the group consisting of:
in which L indicates a linker and the wavy line represents coupling of linker L to the nucleotide.
A method is provided in some examples herein. The method includes adding, using a polymerase, nucleotides to a first polynucleotide using at least a sequence of a second polynucleotide. The method includes altering, using labels respectively coupled to the nucleotides, an electrical characteristic of a single-stranded conjugated polymer chain of a bridge spanning a space between first and second electrodes. The method includes detecting a sequence in which the polymerase adds the nucleotides to the first polynucleotide using at least changes in electrical signal through the bridge that are responsive to respective alterations of the electrical characteristic using the labels corresponding to those nucleotides.
In some examples, the single-stranded conjugated polymer chain includes a first delocalized set of orbitals, and each of the labels includes a respective delocalized set of orbitals that shares electrons with the first delocalized set of orbitals when the label is associated with the single-stranded polymer chain. In some examples, each of the labels and the single-stranded conjugated polymer chain forms a donor:acceptor complex. In some examples, the labels include respective acceptors, the single-stranded conjugated polymer chain includes a donor, and electrons transfer from the single-stranded polymer chain to each of the labels via π-π interactions. In some examples, the labels include different molecular orbitals and detectably change electrical conductivity of the single-stranded conjugated polymer chain differently.
In some examples, the single-stranded polymer chain includes a homopolymer. In some examples, the homopolymer includes repeating monomer units M1, each of the monomer units M1 being coupled to a solubilizing group R.
In other examples, the single-stranded polymer chain includes a copolymer including first monomer units M1 and second monomer units M2, each of the first monomer units M1 being coupled to a solubilizing group R. In some examples, the copolymer includes repeating M1-M2 units.
In still other examples, the single-stranded polymer chain includes a copolymer including first monomer units M1 and second monomer units M3, wherein (i) each of the first monomer units M1, or (ii) each of the second monomer units M3, or (iii) each of the first monomer units M1 and each of the second monomer units M3, is coupled to a solubilizing group R. In some examples, the copolymer includes repeating M1-M3 units.
In other examples, the single-stranded polymer chain includes a copolymer including first monomer units M3 and second monomer units M2, each of the first monomer units M3 being coupled to a solubilizing group R. In some examples, the copolymer includes repeating M3-M2 units.
In some examples, the single-stranded polymer chain includes a copolymer including first monomer units M1, second monomer units M2, and third monomer units M3, wherein (i) each of the first monomer units M1, or (ii) each of the second monomer units M3, or (iii) each of the first monomer units M1 and each of the second monomer units M3, is coupled to a solubilizing group R. In some examples, the copolymer includes repeating M1-M2 units. In some examples, the copolymer includes repeating M3-M2 units.
In some examples, M1 is an electron-donating or electron-neutral monomer unit that includes the solubilizing group R, and wherein the electron-donating or electron-neutral monomer unit that includes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, R is a cationic solubilizing group selected from the group consisting of: (N,N,N-Trimethyl ammonium) alkyl, (N,N,N-Tributyl ammonium)alkyl, 1-methyl-3-(alkyl)-Imidazolium, and 1-(alkyl)Pyridinium.
In some examples, R is an anionic solubilizing group selected from the group consisting of: alkyl sulfonate, alkyl carboxylate, and alkyl phosphonate.
In some examples, M2 is an electron-donating monomer unit that excludes the solubilizing group R, and wherein the electron-donating monomer unit that excludes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, M3 is an electron-withdrawing monomer unit that excludes the solubilizing group R, and wherein the electron-withdrawing monomer unit that excludes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, M3 is an electron-withdrawing monomer unit that includes the solubilizing group R, and wherein the electron-withdrawing monomer unit that includes the solubilizing group R is selected from the group consisting of:
where * indicates coupling to an end group or to another monomer unit.
In some examples, the composition further includes a first end group coupling the single-stranded conjugated polymer chain to the first electrode, and a second end group coupling the single-stranded conjugated polymer to the second electrode. In some examples, each of the first and second end groups is selected from the group consisting of: Benzenethiol, Phenylacetylene, Benzenediazonium, Benzylamine, Methyl benzyl sulfide, and Methyl hexyl sulfide.
In some examples, the labels are acceptors selected from the group consisting of:
in which L indicates a linker and the wavy line represents coupling of linker L to the nucleotide.
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 sequencing using at least altering electrical characteristics of polymer chains. Compositions and methods for performing such sequencing are disclosed.
More specifically, the present compositions and methods suitably may have the benefits of being used to sequence polynucleotides in a manner that is robust, reproducible, sensitive, accurate, works in real time, detects single molecules, 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 a single-stranded conjugated polymer chain that allows electrical current to flow from one electrode to another through the bridge. Labels, which may be coupled to respective nucleotides, may alter one or more electrical characteristics of the bridge, for example the electrical conductivity or electrical impedance of the bridge, and using at least such alteration the respective nucleotide may be identified.
First, some terms used herein will be briefly explained. Then, some example compositions and example methods for electronically sequencing polynucleotides 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 need to be directly attached to both structures. However, some bridges directly attach to both structures. For example, in a bridge that includes a polymer chain spanning the space between two electrodes, one end of the polymer chain may attach directly to one of the electrodes, and the other end of the polymer chain may attach directly to the other electrode. 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, the term “single-stranded,” when referring to a bridge, is intended to mean that the bridge includes a single polymer strand.
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. Some polymers are “conjugated,” which is intended to mean that the monomers constituting such polymers include a sufficient number of sp2 hybridized atomic centers that sufficiently align with one another as to provide a delocalized set of orbitals via which electrons may flow along the length of the polymer. Nonlimiting examples of conjugated polymers, and their constituent monomers, are provided further below.
As used herein, a “donor” is intended to mean a molecule that is electron-rich, while an “acceptor” is intended to mean a molecule that is electron deficient. An “acceptor” may associate with (e.g., form a charge-transfer complex with) a “donor.” For example, an “acceptor” molecule that associates with a “donor” conjugated polymer in such a manner, forms a complex by withdrawing an electron from the polymer. This leads to an increase in free charge carrier concentration in the donor polymer and consequently an increase conductivity.
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 “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, xanthine, hypoxanthine, 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 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 “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 an electrical characteristic of the bridge, such as electrical impedance or electrical conductivity, and based upon which change the nucleotide may be identified. For example, an acceptor-based label may non-covalently associate to a polymer chain within such a bridge, and the association may cause an electrical conductivity or electrical impedance change of the bridge. Or, for example, a polymer-based label may hybridize to a polymer chain within such a bridge, and the hybridization may cause an electrical conductivity or electrical impedance change of the bridge. However, it should be appreciated that a label may alter any suitable electrical characteristic of a polymer chain within a 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.).
Example Compositions and Methods for Sequencing Polynucleotides
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 a single-stranded conjugated polymer chain 111 (the circles within the polymer chain being intended to suggest monomer units that are coupled to one another along the length of the polymer chain). Conjugated polymer chain 111 may have length that is approximately the same as length L of the space between first electrode 102 and second electrode 103 or otherwise permits polymer chain 111 to span the space between first electrode 102 and second electrode 103, e.g., such that polymer chain 111 may be attached directly to each of first electrode 102 and second electrode 103 (e.g., via respective bonds). It should be understood that in some configurations, polymer chain 111 need not necessarily be attached directly to one or both of first electrode 102 or second electrode 103. Instead, polymer chain 111 may be directly attached to one or more other structures that respectively are attached, directly or indirectly, to one or both of first electrode 102 and second electrode 103.
As explained in greater detail herein, labels 131, 132, 133, and 134 respectively may alter an electrical characteristic of single-stranded conjugated polymer chain 111 in such a manner as to modulate the electrical conductivity or impedance of bridge 110, based upon which modulation the identity of the corresponding nucleotides 121, 122, 123, and 124 may be determined. For example, as explained in greater detail with reference to
Composition 100 illustrated in
As described below with reference to
Composition 100 illustrated in
At the particular time illustrated in
In comparison,
Similarly, label 132 may have a property that, when altering an electrical characteristic of polymer chain 111, alters an electrical characteristic, such as electrical conductivity or impedance, of bridge 110 via which detection circuitry 160 may uniquely identify the added nucleotide as 122 (illustratively T) as compared to any of the other nucleotides. Similarly, label 133 may have a property that, when altering an electrical characteristic of polymer chain 111, alters an electrical characteristic, such as electrical conductivity or impedance, of bridge 110 via which detection circuitry 160 may uniquely identify the added nucleotide as 123 (illustratively C) as compared to any of the other nucleotides. Similarly, label 134 may have a property that, when altering an electrical characteristic of polymer chain 111, alters an electrical characteristic, such as electrical conductivity or impedance, of bridge 110 via which detection circuitry 160 may uniquely identify the added nucleotide as 124 (illustratively C) as compared to any of the other nucleotides. It should be appreciated that labels 131, 132, 133, and 134 may have any suitable respective properties based upon which the electrical signal between first electrode 102 and second electrode 103 may vary in such a manner that detection circuitry 160 may identify nucleotides 121, 122, 123, 124 respectively coupled to those labels.
In some examples, the present conjugated polymer chains may provide for extended conjugation of π-electrodes over the polymer backbone. The conjugated polymer chains may include alternating electron-rich and electron-deficient aromatic molecules, leading to overlap of π-orbitals via which electrons may transfer along the polymer chain. The present polymer chains may include at least one building block, such as a monomer unit, that includes a pendant group that can be used to conjugate an electrolyte such as sulfonate, carboxylate, or quaternary amine to the polymer chain. Such groups, which may be referred to as solubilizing groups, may enhance the solubility of the polymer chain in an aqueous medium and may be introduced. Solubilizing groups may be coupled to the polymer chain, for example, using orthogonal click reactions via PEG linkers, or via direct conversion of a pendant group.
For example,
Referring now to
Conjugated polymer 212 illustrated in
Conjugated polymer 213 illustrated in
Conjugated polymer 214 illustrated in
Conjugated polymer 215 illustrated in
In examples such as described with reference to
in which * indicates coupling of the monomer to another element (e.g., to an end group or another monomer unit) and R indicates the solubilizing group.
The solubilizing groups R of the polymer chain may be selected so as to provide suitable solubility of the polymer chain in an aqueous medium. In examples such as described with reference to
where X is a counter ion (X=Cl−, Br−, or I−).
Example structures for such anionic solubilizing groups are shown below:
where X is a counter ion (X=H+, Na+, K+, or (n-C2H9)4N+).
In examples such as described with reference to
in which * indicates coupling of the monomer to another element (e.g., to an end group or another monomer unit) and R indicates the solubilizing group.
In examples such as described with reference to
where * indicates coupling to another monomer unit or to an end group. Example structures for such electron-withdrawing monomers that include one or more solubilizing groups are provided below:
where * indicates coupling to another monomer unit or to an end group.
The end groups of the polymer chain suitably may be selected so as to provide attachment to respective electrodes. In some examples, the end groups covalently bond the polymer chains to the electrodes. In some examples, the end groups non-covalently bond the polymer chains to the electrodes, e.g., the polymer chains are weakly physisorbed to the electrodes via the end groups. In examples such as described with reference to
It will be appreciated that any suitable labels 131, 132, 133, 134 may be selected so as to modulate the conduction of electron flow by a single-stranded conjugated polymer bridge such as described with reference to
in which L indicates a linker and the wavy line represents coupling of linker L to the nucleotide.
The linkers L of the labeled nucleotides may be selected so as to provide suitable solubility of the labeled nucleotides in an aqueous medium and so as to enhance ionic interactions with the polymer chain. For example, to overcome the inhibitory effect of bulky acceptor labels on incorporation of nucleotides using a DNA polymerase, the acceptor may be distanced from the nucleotide using a relatively long linker, e.g., a linker including a relatively long PEG group. Additionally, or alternatively, the linker may be charged. Such a linker may include one or more charged groups such as or one or more pH-dependent polyelectrolytes, e.g., polylysines. Such a linker may also include a combination of PEG groups and pH-dependent polyelectrolytes. Alternatively, such a linker may include a relatively long polypeptide chain containing a combination of neutral and charged amino acids. Electrostatic interactions between the electrolytes coupled to the conjugated polymer chain and electrolytes on the linker of the labeled nucleotide may help to align the molecular orbitals of the conjugated polymer chain with those of the label, thereby facilitating charge-transfer complex formation. Purely by way of example, the linkers L that may be used to couple acceptors (such as those listed above) to nucleotides may be selected from the group consisting of: Polyethylene glycol, polyalanine, polyglycine, polylysine, and polyglutamic acid, example structures for which are shown below:
in which the wavy lines indicate respective couplings to the acceptor and to the nucleotide. Illustratively, the value of p for linker L may be in the range of about 2 to about 4, or about 4 to about 8, or about 8 to about 12, or about 12 to about 14, or about 14 to about 16.
It will be appreciated that in examples such as described with reference to
The present polymers may be prepared using any suitable combination of techniques, including synthetic techniques such as described with reference to examples 2 and 4. The length of a conjugated polymer may relate, at least in part, to the molecular weight of the polymer and to the conformation of that polymer in solution (e.g., in an aqueous medium). The molecular weight may be controlled using polymerization parameters such as catalyst loading, monomer concentration, solvent, reaction time, and temperature. The conformation of the polymer may relate to the polarity of the solvent and the nature of the interaction between the polymer and the solvent. Electrostatic repulsion between charges in polyelectrolytes may reduce or inhibit hydrophobic interactions between monomer units and provided a relatively extended polymer conformation which may facilitate bridge formation. As such, in some examples, charged polymers such as DNA may be used as electrostatic templates to modify the conformation of the present conjugated polymer chain in solution, e.g., to cause the present conjugated polymer to obtain an extended conformation in which the end groups of the polymer may contact, and react with, respective electrodes. For example, the present conjugated polymers may be aligned using negatively charged DNA templates. Illustratively, a mixture of DNA and polymer may be stretched on a hydrophobic surface using “molecular combing” to obtain an ordered DNA-polymer complex. Additionally, or alternatively, the present conjugated polymer chains may be electrostatically trapped between the electrodes by applying a voltage between the electrodes to polarize the polymer chains around the electrodes. The polarized polymer chains may be attracted to the area of maximum field between the electrodes.
Compositions such as described with reference to
Method 600 illustrated in
Referring again to
Additional examples are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims. It will be appreciated that the illustrative reaction schemes set forth in Examples 2 and 4 may be adapted for use in preparing any suitable conjugated polymer chain provided herein. Similarly, it will be appreciated that the illustrative reaction schemes set forth in Example 8 may be adapted for use in preparing any suitable labeled nucleotide provided herein.
In one nonlimiting example, a single-stranded conjugated polymer, which may act as a donor, had the structure of formula 1:
The polymer of formula 1 included a copolymer of monomer units M1 and M2, and a solubilizing group R was coupled to each of the monomer units M1. The starting material for the monomer units M1 of the polymer of formula 1 had the structure:
The starting material for the monomer units M2 of the polymer of formula 1 had the structure:
Using the method of Example 2 below, the bromine groups of the starting material for the M1 monomer units were reacted with the boronic acid pinacol ester groups of the starting material for the M2 monomer units, to form an M1-M2 alternating copolymer.
The solubilizing groups R of the polymer of formula 1 had the structure:
The first and second end groups of the polymer of formula 1 had the structure:
where the dotted line indicates covalent coupling between the thiol and a metal electrode.
The single-stranded conjugated polymer of Example 1, having the structure of formula 1, was synthesized as follows:
Step 1. Preparation of Starting Material for Monomer Unit M1
The starting material for M1 was synthesized from commercially available precursor A, 2,7-Dibromo-9,9-bis(6-bromohexyl)fluorene (Tokyo Chemical Industries Co. Ltd.). Briefly, A (500 mg, 0.77 mmol), Sodium sulfite (7.7 mmol) and Tetrabutylammonium bromide (0.23 mmol) were added to 20 mL water and refluxed for 1 day. After removing the solvent by evaporation, the residue was washed with methanol several times. The filtrate was dried to give the starting material for monomer unit M1 in 70% yield.
Step 2. Preparation of Starting Material for Monomer Unit M2
The starting material for M2 was obtained commercially from Sigma-Aldrich.
Step 3. Preparation of Polymer Backbone
The polymer backbone was synthesized using a traditional transition-metal catalyzed polycondensation reaction wherein carbon-carbon bonds were formed via coupling of aryl halides and aryl boronate esters. The starting material for monomer M1 was functionalized with terminal bromine groups while the starting material for monomer M2 was functionalized with terminal boronic acid pinacol esters. In the presence of a palladium catalyst and base, the dibrominated monomer of M1 reacts selectively with its diboronate ester coupling partner of M2, to form M1-M2 units with orthogonal terminal groups. The polymer chain propagated by reaction of two or more M1-M2 units with each other or reaction of individual monomers on either end of the M1-M2 unit. This led to the formation of polymers with a distribution of chain lengths.
In order to facilitate the conjugation of end groups to the polymer backbone, as illustrated in Example 1, pentafluorobenzene was added to the terminals of the polymer as a capping group. This was achieved by adding a bromine functionalized reagent G1, along with the starting materials for monomers M1 and M2 at the beginning of the reaction. The stoichiometric ratio of the starting material for M1, the starting material for M2, and G1 was maintained such that total number of orthogonal reactive groups (aryl bromides and aryl boronate esters) are the same. A slight excess of the starting material for monomer M2 compared to the starting material for M1 favors the formation of polymers with pentafluorobenzene-M2 end groups. After sufficient extent of polymerization, a boronic acid pinacol ester functionalized reagent G2 is added to the same pot to cap any polymer chains with the M1 terminal groups. This promotes the formation of a high fraction of polymer chains capped with pentafluorobenzene at both ends. The synthesis procedure of the polymer backbone is described below:
Briefly, the starting material for M1 (0.25 mmol), the starting material for M2 (85.73, 0.255 mmol), G1 (2.5 mg, 0.01 mmol), tetrakis(triphenylphosphine) palladium(0) (14.4 mg, 0.012 mmol), and sodium carbonate (265 mg, 2.5 mmol) were dissolved in a mixture of dry THF (0.5 mL), methanol (1.5 mL), and DI water (0.5 mL) and degassed by applying three freeze-pump-thaw cycles. After stirring the mixture for 36 hours at 85° C. under argon atmosphere, G2 (7.5 mg, 0.0255 mmol) was added and the reaction was continued for 2 hours followed by adding G1 (18.89 mg, 0.0765 mmol) and stirring for another 2 hours. The reaction was cooled down and precipitated in cold THF. The precipitate was filtered through a Soxhlet thimble and washed successively with chloroform and acetone, and finally extracted with methanol. After removing methanol in vacuo the polymer was dried under vacuum to afford an orange powder.
Step 4. Preparation of End Groups
The pentafluorobenzene capping groups of the polymer backbone then were reacted with a compound of formula:
to complete preparation of the polymer with protected end groups. 1,4-Benzenedithiol was reacted with pentafluorobenzene to introduce the benzenethiol end-group as illustrated in Example 1. However, in order to avoid cross-linking of polymer chains, one thiol group was selectively shielded by forming disulfide bonds with a protecting group.
Briefly, polymer backbone (50 mg), 6-((4-mercaptophenyl) dithio)hexan-1-ol (50 umol) and potassium carbonate (75 umol) were added to methanol (1 mL) and stirred at 70° C. for 12 h. The solution was filtered, and the filtrate was concentrated in vacuo. The solids were washed with chloroform and dried under vacuum to give the polymer with protected end groups.
Step 5. Reaction of End Groups with Respective Electrodes
To facilitate reaction of polymer end groups with electrodes, the protected end groups were reduced to obtain free thiol moieties. Briefly, polymer with end groups was incubated in a solution containing 100 molar equivalents of Tris(2-carboxyethyl)phosphine hydrochloride for 4 hours at room temperature to give the polymer of formula 1.
In one nonlimiting example, a single-stranded conjugated polymer, which may act as a donor, had the structure of formula 2:
The polymer of formula 2 included a copolymer of monomer units M1 and M2, and a solubilizing group R was coupled to each of the monomer units M1. The starting material for the monomer units M1 of the polymer of formula 2 had the structure:
The starting material for the monomer units M2 of the polymer of formula 2 had the structure:
The solubilizing groups R of the polymer of formula 2 had the structure:
The first and second end groups of the polymer of formula 2 had the structure:
where the dotted line indicates covalent coupling between the thiol and a metal electrode.
The single-stranded conjugated polymer of Example 3, having the structure of formula 2, was synthesized as follows:
Step 1. Preparation of Starting Material for Monomer Unit M1
The starting material for M1 was prepared in a two-step process from commercially available precursors A, 2-Bromo-3-(bromomethyl) thiophene. Briefly, A (500 mg, 1.95 mmol) and N-Bromosuccinimide (NBS, 2.9 mmol) were added to 10 mL DMF at 0° C. and stirred for 16 h. Solvent was removed by evaporation and crude product was purified by column chromatography to give intermediate B in 80% yield. In the next step, B (500 mg, 1.49 mmol), Sodium sulfite (15 mmol) and Tetrabutylammonium bromide (0.3 mmol) were added to 20 mL of 1:1 water/acetone mixture and refluxed for 1 day. After removing the solvent by evaporation, the residue was washed with methanol several times. The filtrate was evaporated to give M1 starting material in 60% yield.
Step 2. Preparation of Starting Material for Monomer Unit M2
The starting material for M2 was obtained commercially from Sigma-Aldrich.
Step 3. Preparation of Polymer Backbone
The polymer backbone was synthesized using a traditional transition-metal catalyzed polycondensation reaction wherein carbon-carbon bonds were formed via coupling of bromine functionalized monomer, the starting material for M1 and boronic acid pinacol ester functionalized monomer, the starting material for M2. Briefly, the starting material for M1 (0.25 mmol), the starting material for M2 (85.73, 0.255 mmol), G1 (2.5 mg, 0.01 mmol), tetrakis(triphenylphosphine) palladium(0) (14.4 mg, 0.012 mmol), and sodium carbonate (265 mg, 2.5 mmol) were dissolved in a mixture of dry THF (0.5 mL), methanol (1.5 mL), and DI water (0.5 mL) and degassed by applying three freeze-pump-thaw cycles. After stirring the mixture for 36 hours at 85° C. under argon atmosphere, G2 (7.5 mg, 0.0255 mmol) was added and the reaction was continued for 2 hours followed by adding G1 (18.89 mg, 0.0765 mmol) and stirring for another 2 hours. The reaction was cooled down and precipitated in cold THF. The precipitate was filtered through a Soxhlet thimble and washed successively with chloroform and acetone, and finally extracted with methanol. After removing methanol in vacuo, the polymer was dried under vacuum to afford a brown powder.
Step 4. Preparation of End Groups
The pentafluorobenzene capping groups then were reacted with a compound of formula:
to complete preparation of the polymer of formula 2. Briefly, polymer backbone (50 mg), 6-((4-mercaptophenyl) dithio)hexan-1-ol (50 umol) and potassium carbonate (75 umol) were added to methanol (1 mL) and stirred at 70° C. for 12 h. The solution was filtered, and the filtrate was concentrated in vacuo. The solids were washed with chloroform and dried under vacuum to give the polymer with protected end groups.
Step 5. Reaction of End Groups with Respective Electrodes
To facilitate reaction of the polymer end groups with electrodes, the protected end groups were reduced to obtain free thiol moieties. Briefly, polymer with —OH end groups was incubated in a solution containing 100 molar equivalents of Tris(2-carboxyethyl)phosphine hydrochloride for 4 hours at room temperature to give the polymer of formula 2.
In one nonlimiting example, an acceptor-labeled nucleotide, which may form respective charge-transfer complexes with the polymers of formula 1 and formula 2, has the structure of formula 3:
The acceptor-labeled nucleotide of formula 3 may include a nucleotide coupled to an acceptor via a linker. The nucleotide dT6P of the acceptor-labeled nucleotide of formula 3 has the structure:
where the dotted line indicates coupling of the nucleotide to the linker.
The linker of the acceptor-labeled nucleotide of formula 3 has the structure:
where the dotted lines indicate respective couplings of the linker to the acceptor and the nucleotide.
The acceptor of the acceptor-labeled nucleotide of formula 3 have the structure:
where the dotted line indicates coupling of the acceptor to the linker.
In one nonlimiting example, an acceptor-labeled nucleotide, which may form respective charge-transfer complexes with the polymers of formula 1 and formula 2, has the structure of formula 4:
The acceptor-labeled nucleotide of formula 4 may include a nucleotide coupled to an acceptor via a linker. The nucleotide dT6P of the acceptor-labeled nucleotide of formula 4 has the same structure as for the acceptor-labeled nucleotide of formula 3. The linker of the acceptor-labeled nucleotide of formula 4 has the below structure, where the dotted lines indicate respective attachments to the acceptor and to the nucleotide:
The acceptor of the acceptor-labeled nucleotide of formula 4 has the structure:
where the dotted line indicates coupling of the acceptor to the linker.’
In one nonlimiting example, an acceptor-labeled nucleotide, which may form respective charge-transfer complexes with the polymers of formula 1 and formula 2, has the structure of formula 5:
The acceptor-labeled nucleotide of formula 5 may include a nucleotide coupled to an acceptor via a linker. The nucleotide of the acceptor-labeled nucleotide of formula 5 has the structure:
The starting material of the linker of the acceptor-labeled nucleotide of formula 5 may have the structure:
The starting material of the acceptor of the acceptor-labeled nucleotide of formula 5 may have the structure:
The acceptor-labeled nucleotide of example 7, having the structure of formula 5, may be synthesized as follows:
Step 1. Preparation of Acceptor
The acceptor is synthesized from commercially available precursors A, Perylene-3,4,9,10-tetracarboxylic dianhydride (Sigma Aldrich) and B, DBCO-PEG4-amine (BroadPharm). Briefly, A (100 mg, 0.25 mmol), B (0.75 mmol) and imidazole (3.75 mmol) are added to a flask and heated at 130° C. for 2 days under argon atmosphere. The crude products are dissolved in dichloromethane and washed with 1 M HCl and water. The organic phase is concentrated, and the residue was purified by column chromatography to give the acceptor as a red solid in expected 40% yield.
Step 2. Preparation of Nucleotide
The nucleotide used in this example is commercially obtained from MyChem LLC.
Step 3. Preparation of Acceptor-Labelled Nucleotide
Acceptor-labelled nucleotide is prepared by a two-step process wherein the first step involves addition of the linker group, Azido-PEG24-NHS (BroadPharm) to the nucleotide. The second step involves conjugation of nucleotide-linker unit to the acceptor.
Briefly, nucleotide (1.6 mg, 2 umol) and Azido-PEG24-NHS (5 umol) are mixed in 1× phosphate-buffered saline solution, pH 7.4 at room temperature for 16 hours. The crude product is purified by reverse phase chromatography to yield nucleotide-linker unit in expected 50% yield.
Nucleotide-linker unit (2 mg, 1 umol) and acceptor (1 umol) are stirred in 1× phosphate-buffered saline solution, pH 7.4 at room temperature for 16 hours. The crude product is purified by reverse phase chromatography to yield the acceptor-labeled nucleotide of formula 5 in expected 40% yield.
Accordingly, it may be understood from Examples 1, 2, 3, and 4 that various conjugated polymer bridges may be prepared, e.g., using reaction schemes such as described in Examples 2 and 4. Additionally, it may be understood from Examples 5, 6, 7, and 8 that various acceptor-labeled nucleotides may be prepared, e.g., using reaction schemes such as described in Example 8.
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.
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.
This application claims the benefit of U.S. Provisional Patent Application No. 63/046,663, filed Jun. 30, 2020 and entitled “Compositions and Methods for Sequencing Using at Least Altering Electrical Characteristics of Bridges Between Electrodes,” the entire contents of which are incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050367 | 6/24/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63046663 | Jun 2020 | US |
