Patent Application
20220251617
The great majority of Next Generation Sequencing (NGS) performed today is based on “sequencing by synthesis” (SBS), in which the sequence of a primed template molecule is determined by a signal resulting from stepwise incorporation of complementary nucleotides by a polymerase (Goodwin et al., Nat Rev Genet. 2016 May 17; 17(6):333-51). Currently, the most popular method for SBS employs fluorescent “reversible terminator” nucleotides (RTdNTPs)—nucleotides that are chemically modified to block elongation by a polymerase once they are incorporated into a primer. Post-incorporation, the free RTdNTPs are removed, and the identity of the added base is determined by a fluorescent signal from the incorporated nucleotide. Next, the fluorescent reporter and terminating group are removed from the incorporated nucleotide, rendering the primer non-fluorescent and ready for subsequent extension by a polymerase. By repeating this cycle of template-dependent extension, detection, and deprotection, the sequence of the template molecule is inferred from the sequence of fluorescence signals.
Contemporary DNA synthesis begins with chemical synthesis of tens to hundreds of 50-200 nt oligonucleotides (oligos) using the phosphoramidite method (Beaucage and Caruthers, Tetrahedron Letters 22.20 (1981): 1859-1862). These oligos are assembled into kilobase-sized products that are then isolated, sequence-verified, and amplified for subsequent recombination into the full-length target sequence if necessary (Kosuri and Church, Nature methods 11.5 (2014): 499-507.). Despite decades of incremental improvement, each chemical step of oligonucleotide synthesis results in 0.5-1.0% unreacted (or side-reacted) products, and these small losses compound exponentially to decimate the yield of the full-length oligo. Since many oligos are assembled into each kilobase-sized product, the presence of even a small fraction of erroneous oligos in the assembly reaction will result in most products containing at least one error. State of the art gene synthesis techniques apply various “error correction” strategies to enrich for error-free oligos or assembly products, but erroneous oligos are reported to be “the most crucial factor in DNA synthesis protocols today” (Czar et al., Trends in biotechnology 27.2 (2009): 63-72). Furthermore, many biologically relevant sequences, such as those with repetitive or structure-forming regions and/or high or low G/C content are difficult if not impossible to construct via assembly of oligonucleotides, impeding their use in research and engineering. Thus far, there is no practical method for de novo DNA synthesis using a polymerase to extend a nucleic acid in a cyclic manner analogous to SBS.
Arguably the key advances that lead to the NGS revolution came from the development of reversible terminator deoxynucleoside triphosphates (RTdNTPs) that can be incorporated into DNA by a polymerase and reversibly terminate further dNTP addition. Improved systems enabling single nucleotide extensions of a growing nucleic acid could benefit SBS and enable practical enzymatic de novo DNA synthesis.
Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Provided herein is a conjugate comprising a polymerase and a nucleoside triphosphate, wherein the polymerase and the nucleoside triphosphate are linked via a linker that comprises a cleavable linkage. An example of such a conjugate is shown in
In some embodiments, once the polymerase of a conjugate has incorporated its tethered nucleotide into a nucleic acid, further elongations of that nucleic acid by other polymerase-nucleotide conjugates are hindered via an effect referred to herein as “shielding”, where the term “shielding” refers to a phenomenon in which 1) the attached polymerase molecule hinders other conjugate molecules from accessing the 3′ OH of the elongated DNA molecule and 2), the nucleoside triphosphate molecules tethered to other conjugate molecules are hindered from accessing the catalytic site of the polymerase that has become attached to the end of the elongated nucleic acid. In some embodiments, further elongation of the nucleic acid may be terminated without the need for additional blocking groups on the tethered nucleoside triphosphate. The termination of elongation caused by the shielding effect may be reversed by cleavage of the linker, which releases the tethered polymerase and thereby reveals the 3′ end of the elongated nucleic acid to enable subsequent elongation by another conjugate.
In any embodiment, conjugates may comprise additional moieties that contribute to termination of elongation of a nucleic acid once the tethered nucleotide has been incorporated. For example, 3′ O-modified or base-modified reversible terminator deoxynucleoside triphosphates (RTdNTPs) that are well known and reviewed in a variety of publications, including Chen, Fei, et al. (Genomics, proteomics & bioinformatics 11.1 (2013): 34-40.), may be tethered to the polymerase. Reversible terminator nucleotide refers to a chemically modified nucleoside triphosphate analog that can be incubated in solution with a polymerase and a nucleic acid and, once incorporated into a nucleic acid molecule, hinders further elongation in the reaction. When a conjugate comprising a polymerase and an RTdNTP is used for the extension of a nucleic acid, besides cleavage of the linker, also deprotection of the RTdNTP may be required to enable an extended nucleic acid to undergo further nucleotide addition.
In some embodiments, the conjugate may be fluorescent, which may be useful in sequencing applications. In some embodiments, the nucleoside triphosphate may be linked to a cysteine residue in the polymerase. However, other chemistries may be used to link proteins and nucleoside triphosphate and, as such, in some cases, the nucleoside triphosphate may be linked to a non-cysteine residue in the polymerase.
The cleavable linker should be capable of being selectively cleaved using a stimulus (e.g., light, a change in its environment or exposure to a chemical or enzyme) without breakage of other bonds in the nucleic acid. In some embodiments, the cleavable linkage may be a disulfide bond, which can be readily broken using a reducing agent (e.g., β-mercaptoethanol or the like). Cleavable bonds that may be suitable may include, but are not limited to, the following: base-cleavable sites such as esters, particularly succinates (cleavable by, for example, ammonia or trimethylamine), quaternary ammonium salts (cleavable by, for example, diisopropylamine) and urethanes (cleavable by aqueous sodium hydroxide); acid-cleavable sites such as benzyl alcohol derivatives (cleavable using trifluoroacetic acid), teicoplanin aglycone (cleavable by trifluoroacetic acid followed by base), acetals and thioacetals (also cleavable by trifluoroacetic acid), thioethers (cleavable, for example, by HF or cresol) and sulfonyls (cleavable by trifluoromethane sulfonic acid, trifluoroacetic acid, thioanisole, or the like); nucleophile-cleavable sites such as phthalamide (cleavable by substituted hydrazines), esters (cleavable by, for example, aluminum trichloride); and Weinreb amide (cleavable by lithium aluminum hydride); and other types of chemically cleavable sites, including phosphorothioate (cleavable by silver or mercuric ions), diisopropyldialkoxysilyl (cleavable by fluoride ions), diols (cleavable by sodium periodate), and azobenzenes (cleavable by sodium dithionite). Other cleavable bonds will be apparent to those skilled in the art or are described in the pertinent literature and texts (e.g., Brown (1997) Contemporary Organic Synthesis 4(3); 216-237).
In particular embodiments, a photocleavable (“PC”) linker (e.g., a uv-cleavable linker) may be employed. Suitable photocleavable linkers for use may include ortho-nitrobenzyl-based linkers, phenacyl linkers, alkoxybenzoin linkers, chromium arene complex linkers, NpSSMpact linkers and pivaloylglycol linkers, as described in Guillier et al (Chem Rev. 2000 Jun. 14; 100(6):2091-158). Exemplary linking groups that may be employed in the subject methods may be described in Guillier et al, supra and Olejnik et al (Methods in Enzymology 1998 291:135-154), and further described in U.S. Pat. No. 6,027,890; Olejnik et al (Proc. Natl. Acad Sci, 92:7590-94); Ogata et al. (Anal. Chem. 2002 74:4702-4708); Bai et al (Nucl. Acids Res. 2004 32:535-541); Zhao et al (Anal. Chem. 2002 74:4259-4268); and Sanford et al (Chem Mater. 1998 10:1510-20), and are purchasable from Ambergen (Boston, Mass.; NHS-PC-LC-Biotin), Link Technologies (Bellshill, Scotland), Fisher Scientific (Pittsburgh, Pa.) and Calbiochem-Novabiochem Corp. (La Jolla, Calif.).
In other embodiments, the linkage may be cleaved by an enzyme. For example, an amide linkage may be cleaved by a protease, an ester linkage may be cleaved by an esterase, and a glycosidic linkage may be cleaved by a glycosylase. In some embodiments, the cleavage reagent may also break bonds in the attached polymerase, e.g. a protease may also digest the polymerase.
In a conjugate, the linker is considered to be at least the atoms that connect the base, the sugar, or the α-phosphate of a nucleotide to a Cα atom in the backbone of the polymerase. In some embodiments, the polymerase and the nucleotide are covalently linked and the distance between the linked atom of the nucleotide and the Cα atom in the backbone of the polymerase to which it is attached may be in the range of 4-100 Å, e.g., 15-40 or 20-30 Å, although this distance may vary depending on where the nucleoside triphosphate is tethered. In some embodiments, the linker may be a PEG or polypeptide linker, although, again, there is considerable flexibility on the type of linker used. In some embodiments, the linker should be joined to the base of the nucleotide at an atom that is not involved in base pairing. In such embodiments, the linker is considered to be at least the atoms that connect a Cα atom in the backbone of the polymerase to any atom in the monocyclic or polycyclic ring system bonded to the 1′ position of the sugar (e.g. pyrimidine or purine or 7-deazapurine or 8-aza-7-deazapurine). For example, in the conjugate depicted in
In all embodiments, the linker used should be sufficiently long to allow the nucleoside triphosphate to access the active site of the polymerase to which it is tethered. As will be described in greater detail below, the polymerase of a conjugate is capable of catalyzing the addition of the nucleotide to which it is linked onto the 3′ end of a nucleic acid.
A nucleic acid may be at least 3 nucleotides in length, at least 10 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 500 nucleotides, at least 1,000 nucleotides or at least 5,000 nucleotides in length and can be fully single-stranded or at least partially double-stranded, e.g., hybridized to another molecule (i.e. part of a duplex) or to itself (e.g., in the form of a hairpin). In any embodiment, a nucleic acid can be an oligonucleotide, which may be at least 3 nucleotides in length, e.g., at least 10 nucleotides, at least 50 nucleotides, at least 100 nucleotides in length, at least 500 nucleotides up to 1,000 nucleotides or more in length and can be fully single-stranded or at least partially double-stranded, e.g., hybridized to another molecule (i.e. part of a duplex) or to itself (e.g., in the form of a hairpin). In some embodiments, an oligonucleotide may be hybridized to a template nucleic acid. In these embodiments, the template nucleic acid may be at least 20 nucleotides in length, e.g., at least 80 nucleotides in length, at least 150 nucleotides in length, at least 300 nucleotides in length, at least 500 nucleotides in length, at least 2000 nucleotides in length, at least 4000 nucleotides in length or at least 10,000 nucleotides. In some cases, a nucleic acid can be part of a natural DNA substrate, e.g. it may be a strand of a plasmid. If a nucleic acid is double stranded, it can have a 3′ overhang.
Also provided herein is a set of the conjugates summarized above, wherein the conjugates correspond to (i.e., have a base-pairing capability that is the same as) G, A, T (or U) and C (i.e., deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP)).
In some embodiments, these conjugates are in separate containers. In other embodiments, the conjugates may be in the same container, particularly if they are to be used for sequencing. The nucleotides used herein may contain adenine, cytosine, guanine, and thymine bases, and/or bases that base pair with a complementary nucleotide and are capable of being used as a template by a DNA or RNA polymerase, e.g., 7-deaza-7-propargylamino-adenine, 5-propargylamino-cytosine, 7-deaza-7-propargylamino-guanosine, 5-propargylamino-uridine, 7-deaza-7-hydroxymethyl-adenine, 5-hydroxymethyl-cytosine, 7-deaza-7-hydroxymethyl-guanosine, 5-hydroxymethyl-uridine, 7-deaza-adenine, 7-deaza-guanine, adenine, guanine, cytosine, thymine, uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza-guanosine, 2-thio-adenine, 2-thio-7-deaza-adenine, isoguanine, 7-deaza-guanine, 5,6-dihydrouridine, 5,6-dihydrothymine, xanthine, 7-deaza-xanthine, hypoxanthine, 7-deaza-xanthine, 2,6 diamino-7-deaza purine, 5-methyl-cytosine, 5-propynyl-uridine, 5-propynyl-cytidine, 2-thio-thymine or 2-thio-uridine are examples of such bases, although others are known. An exemplary set of conjugates for synthesizing and/or sequencing a DNA molecule may include a DNA polymerase linked to a deoxyribonucleotide triphosphate selected from deoxyriboadenosine triphosphate (dATP), deoxyriboguanosine triphosphate (dGTP), deoxyribocytidine triphosphate (dCTP), deoxyribothymidine triphosphate (dTTP), and/or other deoxyribonucleotides that base pair in the same way as those deoxyribonucleotides. An exemplary set of conjugates for synthesizing an RNA molecule may include an RNA polymerase linked to a ribonucleotide triphosphate selected from adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (dCTP), and uridine triphosphate (UTP), and/or other ribonucleotides that base pair in the same way as those ribonucleotide triphosphates.
The above described conjugates can be used in a method of nucleic acid synthesis. In some embodiments, this method may comprise: incubating a nucleic acid with a first conjugate under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the first conjugate onto the 3′ hydroxyl of the nucleic acid, to make an extension product. This reaction can be performed using a nucleic acid that is attached to a solid support or that is in solution, i.e., not tethered to a solid support. After elongation of the nucleic acid by the first desired nucleotide, the method may comprise a deprotection step wherein the cleavable linkage of the linker is cleaved, thereby releasing the polymerase from the extension product. This may be done by exposing the reaction products to reducing conditions if the cleavable linkage is a disulfide bond. However, other chemistries and reagents are available for this step. In some embodiments, the nucleoside triphosphate may be a RTdNTP and the deprotection step of the method further comprises removing the blocking group (i.e., removing the terminator group) from the added nucleotide to produce the deprotected extension product. Deprotection enables subsequent extension of the nucleic acid, and thus allows these steps to be repeated cyclically to produce an extension product of defined sequence. Specifically, in some embodiments, the method may further comprise, after deprotection: incubating the deprotected extension product with a second conjugate under conditions in which polymerase catalyzes the covalent addition of the nucleotide of the second conjugate onto the 3′ end of the extension product.
In some embodiments, the method may involve (a) incubating a nucleic acid with a first conjugate under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the first conjugate (i.e., a single nucleotide) onto the 3′ hydroxyl of the nucleic acid, to make an extension product; (b) cleaving the cleavable linkage of the linker, thereby releasing the polymerase from the extension product and deprotecting the extension product; (c) incubating the deprotected extension product with a second conjugate of claim 1 under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the second conjugate onto the 3′ end of the extension product, to make a second extension product; (d) repeating steps (b)-(c) on the second extension product multiple times (e.g., 2 to 100 or more times) to produce an extended oligonucleotide of a defined sequence. Steps (b)-(c) may be repeated as many times as necessary until an extension product of a defined sequence and length is synthesized. The end product may be 2-100 bases in length, although, in theory, the method can be used to produce products of any length, including greater than 200 bases or greater than 500 bases.
In certain embodiments, cleavage of the linker may leave a “scar” (i.e., part of the linker) on each or some of the added nucleotides. In other embodiments, cleavage of the linker does not produce a scar.
In some embodiments, scars may be further derivatized (e.g. by alkylation of thiol-containing scars using iodoacetamide) following each deprotection step. In other embodiments, all scars in the end product may be simultaneously derivatized (e.g. by acetylation of propargylamino scars using NHS acetate.)
In some embodiments, the product may be amplified, e.g., by PCR or some other method, to yield a product without scars (as demonstrated in Example 4).
A method of sequencing is also provided. These methods may comprise incubating a duplex comprising a primer and a template with a composition comprising a set of conjugates, wherein the conjugates correspond to G, A, T and C and are distinguishably labeled, e.g., fluorescently labeled; detecting which nucleotide has been added to the primer by detecting a label that is tethered to the polymerase that has added the nucleotide to the primer; deprotecting the extension product by cleaving the linker; and repeating the incubation, detection and deprotection steps to obtain the sequence of at least part of the template.
Also provided is a reagent set that can be used to make a conjugate described above. In some embodiments, this reagent set may comprise a polymerase that has been modified to contain a single cysteine on its surface; and a set of nucleoside triphosphates, wherein each of the nucleoside triphosphates is linked to a sulfhydryl-reactive group. In some embodiments, the nucleoside triphosphates correspond to G, A, T and C. As noted above, the nucleoside triphosphates may be reversible terminators. In this reagent set, the nucleoside triphosphates may comprise a linker that has a length in the range of 4-100 Å, e.g., 15-40 Å or 20-30 Å.
In any embodiment, the polymerase can be a template-independent polymerase, i.e., a terminal deoxynucleotidyl transferase or DNA nucleotidylexotransferase, which terms are used interchangeably to refer to an enzyme having activity 2.7.7.31 using the IUBMB nomenclature. A description of such enzymes can be found in Bollum, F. J. Deoxynucleotide-polymerizing enzymes of calf thymus gland. V. Homogeneous terminal deoxynucleotidyl transferase. J. Biol. Chem. 246 (1971) 909-916; Gottesman, M. E. and Canellakis, E. S. The terminal nucleotidyltransferases of calf thymus nuclei. J. Biol. Chem. 241 (1966) 4339-4352; and Krakow, J. S., Coutsogeorgopoulos, C. and Canellakis, E. S. Studies on the incorporation of deoxyribonucleic acid. Biochim Biophys. Acta 55 (1962) 639-650, among others.
Terminal transferase embodiments may be useful for DNA synthesis.
In any embodiment, the polymerase can be a template-dependent polymerase, i.e., a DNA-directed DNA polymerase (which terms are used interchangeably to refer to an enzyme having activity 2.7.7.7 using the IUBMB nomenclature), or an DNA-directed RNA polymerase. A description of such enzymes can be found in Richardson, A. Enzymatic synthesis of deoxyribonucleic acid. XIV. Further purification and properties of deoxyribonucleic acid polymerase of Escherichia coli. J. Biol. Chem. 239 (1964) 222-232; Schachman, A. Enzymatic synthesis of deoxyribonucleic acid. VII. Synthesis of a polymer of deoxyadenylate and deoxythymidylate. J. Biol. Chem. 235 (1960) 3242-3249; and Zimmerman, B. K. Purification and properties of deoxyribonucleic acid polymerase from Micrococcus lysodeikticus. J. Biol. Chem. 241 (1966) 2035-2041.
In any of the above-summarized embodiments, the nucleoside triphosphate may be a deoxyribonucleoside triphosphate or a ribonucleoside triphosphate. In some embodiments, a conjugate may comprise an RNA polymerase linked to a ribonucleoside triphosphate. In these embodiments, the nucleotide added to the nucleic acid may be a ribonucleotide. In other embodiments, a conjugate comprises an DNA polymerase linked to a deoxyribonucleoside triphosphate. In these embodiments, the nucleotide added to the nucleic acid may be a deoxyribonucleotide.
In any embodiment, the polymerase used may have an amino acid sequence that is at least 80% identical to, e.g., at least 90% or at least 95% identical to a wild type polymerase.
In some embodiments, the yield per nucleotide addition step may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, for example 91% or 99.5%. The yield per step of any implementation of the method may be increased by optimizing the conditions. As would be recognized, a nucleic acid manufactured by the presented method may be purified, e.g., by liquid chromatography, prior to use.
In any embodiment, the conjugate may additionally comprise additional polypeptide domains fused to the polymerase. For example, maltose binding protein may be fused to the N-terminus of Terminal deoxynucleotidyl transferase to enhance its solubility and/or to enable amylose affinity purification. In any embodiment, the nucleoside triphosphate may be a reversible terminator.
All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.
Further details of the reagents and methods described above may be found below. Some of this description relates to the TdT. The principle of this description can be applied to other template-independent polymerases and template-dependent polymerases, too.
Tethered Nucleotides can have a High Effective Concentration, Enabling Fast Incorporation Kinetics.
A tethered nucleotide will have a certain occupancy rate with the active site of the polymerase depending on the length and geometry of the linker and its attachment site on the protein. This rate can be expressed as an effective concentration (the concentration of free nucleotide that would give an equivalent occupancy rate). By varying the linker properties and attachment site, it is possible to control the effective concentration of the nucleotide, enabling high effective concentrations and therefore fast incorporations. For example, a very rough calculation suggests that the effective concentration of a dNTP tethered by a 20 Å linker will be ˜50 mM, (one molecule in the volume of a sphere with 20 Å radius). In this example, one could increase the local concentration of the dNTP by shortening the linker, or decrease it by lengthening the linker.
In some embodiments, the linker is specifically attached to an amino acid of the polymerase (see
In any embodiment, the length of the linker may be longer than the distance between the attachment position of the linker on the polymerase and the attachment position on the nucleoside triphosphate when it is bound to the catalytic site. In some cases, steric restrictions, e.g. due to the polymerase or due to the linker can restrict mobility of the tethered nucleoside triphosphate requiring an increased linker length to enable the tethered nucleoside triphosphate to access the catalytic site of the polymerase in a productive conformation. For example, the linker length may exceed the distance between its two attachment points by 2-3 Å or 5-10 Å, or 10-25 Å, or longer.
In some embodiments, the tethered nucleoside triphosphate may be specifically attached to a cysteine residue of the polymerase using a sulfhydryl-specific attachment chemistry. Possible sulfhydryl specific attachment chemistries include, but are not limited to ortho-pyridyl disulfide (OPSS) (as exemplified in
In other embodiments, the linker could be attached to a lysine residue via an amine-reactive functionality (e.g. NHS esters, Sulfo-NHS esters, tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.).
In other embodiments, the linker may be attached to the polymerase via attachment to a genetically inserted unnatural amino acid, e.g. p-propargyloxyphenylalanine or p-azidophenylalanine that could undergo azide-alkyne Huisgen cycloaddition, though many suitable unnatural amino acids suitable for site-specific labeling exist and can be found in the literature (e.g. as described in Lang and Chin., Chemical reviews 114.9 (2014): 4764-4806.).
In other embodiments, the linker may be specifically attached to the polymerase N-terminus. In some embodiments, the polymerase is mutated to have an N-terminal serine or threonine residue, which may be specifically oxidized to generate an N-terminal aldehyde for subsequent coupling to e.g. a hydrazide. In other embodiments, the polymerase is mutated to have an N-terminal cysteine residue that can be specifically labeled with an aldehyde to form a thiazolidine. In other embodiments, an N-terminal cysteine residue can be labeled with a peptide linker via Native Chemical Ligation.
In other embodiments, a peptide tag sequence may be inserted into the polymerase that can be specifically labeled with a synthetic group by an enzyme, e.g. as demonstrated in the literature using biotin ligase, transglutaminase, lipoic acid ligase, bacterial sortase and phosphopantetheinyl transferase (e.g. as described in refs. 74-78 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
In other embodiments, the linker is attached to a labeling domain fused to the polymerase. For example, a linker with a corresponding reactive moiety may be used to covalently label SNAP tags, CLIP tags, HaloTags and acyl carrier protein domains (e.g. as described in refs. 79-82 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
In other embodiments, the linker is attached to an aldehyde specifically generated within the polymerase, as described in Carrico et al. (Nat. Chem. Biol. 3, (2007) 321-322). For example, after insertion of an amino acid sequence that is recognized by the enzyme formylglycine-generating enzyme (FGE) into the polymerase, it may be exposed to FGE, which will specifically convert a cysteine residue in the recognition sequence to formylglycine (i.e. producing an aldehyde). This aldehyde may then be specifically labeled with e.g. a hydrazide or aminooxy moiety of a linker.
In some embodiments, a linker may be attached to the polymerase via non-covalent binding of a moiety of the linker to a moiety fused to the polymerase. Examples of such attachment strategies include fusing a polymerase to streptavidin that can bind a biotin moiety of a linker, or fusing a polymerase to anti-digoxigenin that can bind a digoxigenin moiety of a linker.
In some embodiments, site-specific labeling may lead to an attachment of the linker to the polymerase that may readily be reversed (e.g. an ortho-pyridyl disulfide (OPSS) group that forms a disulfide bond with a cysteine that can be cleaved using reducing agents, e.g. using TCEP), other attachment chemistries will produce permanent attachments.
In any embodiment, the polymerase may be mutated to ensure specific attachment of the tethered nucleotide to a particular location of the polymerase, as will be apparent to those skilled in the art. For example, with sulfhydryl-specific attachment chemistries such as maleimides or ortho-pyridyl disulfides, accessible cysteine residues in the wild-type polymerase may be mutated to a non-cysteine residue to prevent labeling at those positions. On this “reactive cysteine-free” background, a cysteine residue may be introduced by mutation at the desired attachment position. These mutations preferentially do not interfere with the activity of the polymerase.
Other strategies for site-specific attachment of synthetic groups to proteins will be apparent to those skilled in the art and are reviewed in literature, (e.g. Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).
In some embodiments, the linker is attached the 5 position of pyrimidines or the 7 position of 7-deazapurines. In other embodiments, the linker may be attached to an exocyclic amine of a nucleobase, e.g. by N-alkylating the exocyclic amine of cytosine with a nitrobenzyl moiety as discussed below. In other embodiments, the linker may be attached to any other atom in the nucleobase, sugar, or α-phosphate, as will be apparent to those skilled in the art.
Certain polymerases have a high tolerance for modification of certain parts of a nucleotide, e.g. modifications of the 5 position of pyrimidines and the 7 position of purines are well-tolerated by some polymerases (He and Seela., Nucleic Acids Research 30.24 (2002): 5485-5496.; or Hottin et al., Chemistry. 2017 Feb. 10; 23(9):2109-2118). In some embodiments, the linker is attached to these positions.
In some examples, a polymerase-nucleotide conjugate is prepared by first synthesizing an intermediate compound comprising a linker and a nucleoside triphosphate (referred to herein as a “linker-nucleotide”), and then this intermediate compound is attached to the polymerase. In some examples, nucleosides with substitutions compared to natural nucleosides, e.g. pyrimidines with 5-hydroxymethyl or 5-propargylamino substituents, or 7-deazapurines with 7-hydroxymethyl or 7-propargylamino substituents may be useful starting materials for preparing linker-nucleotides. An exemplary set of nucleosides with 5- and 7-hydroxymethyl substituents that may be useful for preparing linker-nucleotides is shown below:
An exemplary set of nucleosides with 5- and 7-deaza-7-propargylamino substituents that may be useful for preparing linker-nucleotides is shown below:
These nucleosides are also commercially available as deoxyribonucleoside triphosphates.
In Example 2, linker-nucleotides comprising a 3-(((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group attached to the 5 position of pyrimidines and the 7 position of 7-deazapurines were prepared by reacting nucleoside triphosphates containing 5- and 7-propargylamino substituents with a precursor molecule comprising a nitrobenzyl NHS carbonate ester (as shown in
As described above, the linker may be attached to various positions on the nucleotide, and a variety of cleavage strategies may be used. Those strategies may include, but are not limited to, the following examples:
In some embodiments, the linker may be cleaved by exposure to a reducing agent such as dithiothreitol (DTT). For example, a linker comprising a 4-(disulfaneyl)butanoyloxy-methyl group attached to the 5 position of a pyrimidine or the 7 position of a 7-deazapurine may be cleaved by reducing agents (e.g. DTT) to produce a 4-mercaptobutanoyloxymethyl scar on the nucleobase. This scar may undergo intramolecular thiolactonization to eliminate a 2-oxothiolane, leaving a smaller hydroxymethyl scar on the nucleobase. An example of such a linker attached to the 5 position of cytosine is depicted below, but the strategy is applicable to any suitable nucleobase:
In other embodiments, the linker may be cleaved by exposure to light. For example a linker comprising (2-nitrobenzyl)oxymethyl group may be cleaved with 365 nm light, leaving a hydroxymethyl scar, e.g. as depicted for cytosine below, but as is applicable to any suitable nucleobase:
(where, e.g., R″═H or R″═CH3 or R″=t-Bu.)
In other embodiments, the linker may comprise a 3-(((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group that may be cleaved with 365 nm light to release a nucleobase with a propargylamino scar. This strategy was used in Example 2 and is depicted for cytosine below, but is applicable to any suitable nucleobase:
In other embodiments, the linker may comprise an acyloxymethyl group that may be cleaved with a suitable esterase to release a nucleobase with a hydroxymethyl scar, e.g. as depicted for cytosine below, but as is applicable to any suitable nucleobase:
In such embodiments, the linker may comprise additional atoms (included in R′ above) adjacent to the ester that increase the activity of the esterase towards the ester bond.
In other embodiments, the linker may comprise an N-acyl-aminopropynyl group that may be cleaved with a peptidase to release a nucleobase with propargylamino scar, e.g. as depicted for 5-propargylamino cytosine below, but as is applicable to any suitable nucleobase:
In such embodiments, the linker may comprise additional atoms (included in R′ above) adjacent to the amide that increase the activity of the peptidase towards the amide bond. In some embodiments, R′ is a peptide or polypeptide.
In some embodiments, one or more amino acids are inserted into the polymerase that can serve as part of the cleavable linker for specific attachment of the nucleotide. In this case, the linker comprises the inserted amino acids, and the cleavable linkage is considered to be one or more bonds of the inserted amino acid(s). For example, peptide bonds may be cleaved using a peptidase (which terms are used interchangeably to refer to an enzyme having activity 3.4. using the IUBMB nomenclature) such as Proteinase K (EC 3.4.21.64 using the IUBMB nomenclature).
In some embodiments, the protein itself can be cleaved to detach the tethered nucleoside triphosphate from the polymerase. For example, peptide bonds before and/or after the attachment position of the linker may be cleaved using a peptidase.
In some embodiments, amino acid positions close to the attachment point of the linker may be mutated to ensure that peptide sequences near the attachment point are good substrates for the protease, as will be apparent to those skilled in the art. E.g., mutations into aliphatic amino acids as leucine or phenylalanine may be introduced to achieve fast cleavage with proteinase K.
In certain embodiments, nucleotide analogs are tethered that, when freely available in solution, do not terminate DNA synthesis upon incorporation. However, in other embodiments nucleotide analogs containing a reversible terminator group, such as an O-azidomethyl or O—NH2 group on the 3′ position of the sugar or an (alpha-tertbutyl-2-nitrobenzyl)oxymethl group on the 5 position of pyrimidines or the 7 position of 7-deazapurines (for an overview see, e.g. Chen et al., Genomics, Proteomics & Bioinformatics 2013 11: 34-40.) are tethered. In these embodiments, the nucleotide analog prevents or hinders further elongation once incorporated into a nucleic acid and thus contributes to the conjugate's ability to achieve termination, possibly in addition to other properties of the conjugate that contribute to termination (e.g. shielding). In the case that RTdNTP-polymerase conjugates do not rely on the shielding effect to achieve termination, e.g. when a 3′ modified RTdNTP is tethered to the polymerase, the linker used may exceed 100 Å or 200 Å in length.
When a conjugate comprising a polymerase and a nucleoside triphosphate is incubated with a nucleic acid, it preferentially elongates the nucleic acid using its tethered nucleotide (as opposed to using the nucleotide of another conjugate molecule). As described above, the polymerase then remains attached to the nucleic acid via its tether to the added nucleotide (e.g.
Methods for nucleic acid synthesis and sequencing provided herein that employ the shielding effect to achieve termination comprise an extension step wherein a nucleic acid is exposed to conjugates preferentially in the absence of free (i.e. untethered) nucleoside triphosphates, because the termination mechanism of shielding may not prevent their incorporation into the nucleic acid. As shown in Example 3, exposing a primer that has been extended by a TdT-dCTP conjugate to free dCTP results in several additional elongations.
In some embodiments, termination of further elongation may be “complete”, meaning that after a nucleic acid molecule has been elongated by a conjugate, further elongations cannot occur during the reaction. In other embodiments, termination of further elongation may be “incomplete”, meaning that further elongations can occur during the reaction but at a substantially decreased rate compared to the initial elongation, e.g. 100 times slower, or 1000 times slower, or 10,000 times slower, or more. Conjugates that achieve incomplete termination may still be used to extend a nucleic acid by predominantly a single nucleotide (e.g. in methods for nucleic acid synthesis and sequencing) when the reaction is stopped after an appropriate amount of time.
In some embodiments, the reagent containing the conjugate may additionally contain polymerases without tethered nucleoside triphosphates, but those polymerases should not significantly affect the reaction because there are no free dNTPs in the mix.
Reagents based on conjugates employing the shielding effect to achieve termination preferentially only contain polymerase-nucleotide conjugates in which all polymerases remain folded in the active conformation. In some cases, if the polymerase moiety of a conjugate is unfolded, its tethered nucleoside triphosphate may become more accessible to the polymerase moieties of other conjugate molecules. In these cases, the unshielded nucleotides may be more readily incorporated by other conjugate molecules, circumventing the termination mechanism.
Polymerase-nucleotide conjugates employing the shielding effect to achieve termination are preferentially only labeled with a single nucleoside triphosphate moiety. Polymerase-nucleotide conjugates labeled with multiple nucleoside triphosphates that can access the catalytic site can, in some cases, incorporate multiple nucleoside triphosphates into the same nucleic acid (e.g. as demonstrated with the conjugate of wt TdT labeled with up to 5 nucleoside triphosphates in Example 1). Additional tethered nucleotides may therefore lead to additional, undesired nucleotide incorporations into a nucleic acid during a reaction. Furthermore, only one tethered nucleoside triphosphates can occupy the (buried) catalytic site of its polymerase at a time so the other tethered nucleoside triphosphate(s) may have an increasing accessibility to the polymerase moieties of other conjugate molecules, as discussed below. Strategies for site-specifically tethering at most one nucleoside triphosphate to a polymerase are described above.
Polymerase-nucleotide conjugates employing the shielding effect to achieve termination preferentially comprise as short of a linker as possible that still enables the nucleoside triphosphate to frequently access the catalytic site of its tethered polymerase molecule in a productive conformation, in order to enable fast incorporation of the nucleotide into a nucleic acid. Such conjugates may also preferentially employ an attachment position of the linker to the polymerase as close to the catalytic site as possible, enabling use of a shorter linker. The length of the linker will determine the maximum distance from the attachment point a tethered nucleoside triphosphate or a tethered nucleic acid can reach. A smaller distance may lead to a reduced accessibility of the tethered moiety to other polymerase-nucleotide molecules, as discussed below. Linkers used in Examples 1 and 2 are approximately 24 and 28 Å long. Shorter linkers, e.g. with lengths of 8-15 Å may increase shielding; longer linkers, e.g. linkers longer than 50 Å, 70 Å or 100 Å, may reduce shielding.
The shielding effect may be influenced by a combination of factors including, but not limited to, to the structure of the polymerase, the length of the linker, the structure of the linker, the attachment position of the linker to the polymerase, the binding affinity of the nucleoside triphosphate to the catalytic site of the polymerase, the binding affinity of the nucleic acid to the polymerase, the preferred conformation of the polymerase, and the preferred conformation of the linker.
One contribution to shielding can be steric effects that block the 3′ OH of a nucleic acid that has been elongated by a conjugate from reaching into the catalytic site of another conjugate's polymerase moiety. Steric effects may also hinder a tethered nucleoside triphosphate from reaching into the catalytic site of another polymerase-nucleotide conjugate molecule due to clashes between the conjugates that would occur during such approaches. These steric effects may result in complete termination if they completely block productive interactions between the tethered nucleoside triphosphate (or elongated nucleic acid) of one conjugate molecule with another conjugate molecule, or may result in incomplete termination if they only hinder such intermolecular interactions.
Another contribution to shielding arises from the binding affinity of the tethered nucleoside triphosphate to the catalytic site of the polymerase. The tethered nucleoside triphosphate of a conjugate will have a high effective concentration with respect to the catalytic site of its tethered polymerase so it may remain bound to that site much of the time. When the nucleoside triphosphate is bound to the catalytic site of its tethered polymerase molecule it is unavailable for incorporation by other polymerase molecules. Thus, tethering reduces the effective concentration of nucleoside triphosphates available for intermolecular incorporation (i.e. incorporation catalyzed by a polymerase molecule to which the nucleotide is not tethered). This shielding effect can enhance termination by reducing the rate by which a nucleic acid is elongated using the nucleoside triphosphate moiety of one conjugate molecule by the polymerase moiety of another conjugate molecule.
Another contribution to shielding arises from the binding affinity of the 3′ region of a nucleic acid molecule to the catalytic site of a polymerase molecule. After elongation by a conjugate, the nucleic acid is tethered to the conjugate via it's 3′ terminal nucleotide and will have a high effective concentration with respect to the catalytic site of its tethered polymerase so it may remain bound to that site much of the time. When the nucleic acid is bound to the catalytic site of its tethered polymerase molecule it is unavailable for elongation by other conjugate molecules. This effect can enhance termination by reducing the rate by which a nucleic acid that has been elongated by a first conjugate is further elongated by other conjugate molecules.
Addition of Elements with Steric Restrictions to Increase the Shielding Effect
In some embodiments, the polymerase-nucleotide conjugates comprise additional moieties that sterically hinder the tethered nucleoside triphosphate (or a tethered nucleic acid post-elongation) from approaching the catalytic sites of another conjugate molecule. Such moieties include polypeptides or protein domains that can be inserted into a loop of the polymerase, and those and other bulky molecules such as polymers that can be site-specifically ligated e.g. to an inserted unnatural amino acid or specific polypeptide tag.
RTdNTP Termination Mechanisms in Combination with the Shielding Effect.
As described above, in some embodiments, a conjugate may comprise a polymerase and a tethered reversible terminator nucleoside triphosphate. Some RTdNTPs (particularly 3′ O-unblocked RTdNTPs) achieve incomplete termination when used freely in solution. A polymerase-nucleotide conjugate of such an RTdNTP that also employs a shielding effect may achieve more complete termination than the RTdNTP used by itself. In some embodiments, an RTdNTP with an (alpha-tertbutyl-2-nitrobenzyl)oxymethyl group attached to the 5 position of a pyrimidine or the 7 position of a 7-deazapurine in a nucleoside triphosphate e.g. described in Gardner et al. (Nucleic Acids Res. 2012 August; 40(15):7404-1); or Stupi et al. (Angewandte Chemie International Edition 51.7 (2012): 1724-1727) may be employed. In some embodiments, the linker is attached to an atom in the terminating moiety of the RTdNTP. In other embodiments the linker is attached to an atom of the RTdNTP not in the terminating moiety.
As described earlier, a tethered nucleoside triphosphate has a high effective concentration with respect to the catalytic site of its attached polymerase moiety, enabling fast incorporation. The same nucleoside triphosphate has a much lower concentration with respect to the catalytic site of other polymerase-moieties, leading to a slower intermolecular nucleotide incorporation rate if intermolecular incorporations are possible. The effective concentration of nucleoside triphosphates tethered to other conjugates is at most the absolute concentration of conjugates since each conjugate molecule comprises a single nucleotide. Due to shielding effects that hinder accessibility of these nucleoside triphosphates, the effective concentration is further reduced.
Preventing an Elongated Nucleic Acid from Shifting in the Polymerase Catalytic Site
An additional termination effect of polymerase-nucleotide conjugates can be achieved by choosing a linker and attachment position that prevent an extended (and thus tethered) nucleic acid from shifting its tethered 3′ end to the position where its 3′ OH can be activated to attack an incoming nucleoside triphosphate. This effect may be achieved if the tethered nucleoside triphosphate can access the nucleoside triphosphate binding site of the polymerase but cannot reach a position where its 3′ OH would correspond to the 3′ OH of an incoming nucleic acid.
Described herein is a method for the de novo synthesis of nucleic acids using conjugates comprising a polymerase and a nucleoside triphosphate. In some embodiments of the method, conjugates comprise the polymerase Terminal deoxynucleotidyl Transferase (TdT). In other embodiments, the method may employ conjugates comprising another template-independent polymerase or a template-dependent polymerase.
For DNA synthesis applications, in particular template-independent polymerases, i.e., a terminal deoxynucleotidyl transferase or DNA nucleotidylexotransferase, which terms are used interchangeably to refer to an enzyme having activity 2.7.7.31 may be used. Polymerases with the ability to extend single stranded nucleic acids include, but are not limited to, Polymerase Theta (Kent et al., Elife 5 (2016): e13740.), polymerase mu (Juarez et al., Nucleic acids research 34.16 (2006): 4572-4582.; or McElhinny et all., Molecular cell 19.3 (2005): 357-366.) or polymerases where template independent activity is induced, e.g. by the insertion of elements of a template independent polymerase (Juarez et al., Nucleic acids research 34.16 (2006): 4572-4582). In other DNA synthesis applications, the polymerase can be a template-dependent polymerase i.e., a DNA-directed DNA polymerase (which terms are used interchangeably to refer to an enzyme having activity 2.7.7.7 using the IUBMB nomenclature).
For RNA synthesis applications, tethered ribonucleoside triphosphates may be used. In these embodiments, a RNA specific nucleotidyl transferase, such as e. coli Poly(A) Polymerase (IUBMB EC 2.7.7.19) or Poly(U) Polymerase, among others, may be employed. The RNA nucleotidyl transferases can contain modifications, e.g. single point mutations, that influence the substrate specificity towards a specific rNTP (Lunde et al., Nucleic acids research 40.19 (2012): 9815-9824.). In some embodiments, a very short tether between an RNA nucleotidyl transferase and a ribonucleoside triphosphate may be used to induce a high effective concentration of the nucleoside triphosphate, thereby forcing incorporation of an rNTP that might not be the natural substrate of the nucleotidyl transferase.
Nucleic acid synthesis schemes using polymerase tethered nucleoside triphosphates may require a nucleic acid substrate of at least 3-5 bases (or a molecule with similar properties) to initiate the synthesis. This initial substrate can then be extended nucleotide-by-nucleotide into the desired product. In some embodiments, the initial substrate may be an oligonucleotide primer synthesized using the phosphoramidite method (as demonstrated in Example 1). In some cases, the particular sequence of the initiating primer may be used in downstream applications of the synthesized nucleic acid. In some embodiments, the sequence of the initial substrate may be removed from the synthesized nucleic acid after synthesis is complete, particularly if the initial substrate comprises a cleavable linkage near it's 3′ terminus. For example, if the initial substrate is a primer that has a 3′ terminal deoxyuridine base, exposure of the elongated primer to USER Enzyme (i.e. a mixture of Uracil DNA glycosylase and Endonuclease VIII) will cleave the synthesized sequence from the initial substrate. However, other cleavable linkages may be used, e.g. a bridging phosphorothioate in the primer that could be cleaved with silver or mercuric ions.
In some embodiments, a double-stranded DNA molecule may be employed to initiate the synthesis, particularly if it has a 3′ overhang (as demonstrated in Example 2). If the initial substrate is a linearized plasmid backbone, the DNA synthesis method could be used to elongate the DNA molecule by one or more synthetic gene sequences and the elongated DNA could then be (optionally amplified and) circularized into a plasmid. In general, the methods for nucleic acid synthesis described herein enable initiation of de novo DNA synthesis from natural nucleic acid molecules; in contrast, it is not possible to directly extend natural nucleic acid molecules by a defined sequence using the phosphoramidite method.
In some embodiments, cleavage of a linker attached to a nucleotide may result in the production of a natural nucleotide upon cleavage. For example a linker comprising a nitrobenzyl moiety alkylating an amine of the nucleobase may be cleaved with light, e.g. as depicted for the exocyclic amines of cytosine and adenine below, but as is applicable to any suitable nitrogen atom on any nucleobase:
In other embodiments, a linker attached to an amine of the nucleobase via an amide linkage may be cleaved by a suitable peptidase, e.g. as depicted for the exocyclic amines of cytosine and adenine below, but is applicable to any suitable amino group on any nucleobase:
In such embodiments, the linker may comprise additional atoms (included in R′ above) adjacent to the amide that increase the activity of the peptidase towards the amide bond. In some embodiments, R′ is a peptide or polypeptide.
In some embodiments, cleavage of the linker in the deprotection step may leave a scar that persists throughout the stepwise synthesis but that is removed or further reduced once the stepwise synthesis is completed. This attachment strategy may enable the introduction of additional distance between the cleavable moiety of the linker and the nucleotide, which may be useful with certain (e.g. bulky) cleavable groups. Scars may be useful to prevent base pairing of incorporated nucleotides and therefore prevent the formation of secondary structures during synthesis, e.g. by preventing the exocyclic amino group from engaging in base-pairing as discussed below. Once synthesis of such a molecule is complete, a single “scar-removal” step can be used to prevent interference of the scars with downstream applications and to restore the nucleic acid's base-pairing ability.
For example, acyl scars left on the exocyclic amino group of adenine, cytosine, and guanine after cleavage may hinder the formation of some types of secondary structures during the synthesis. After the synthesis is completed, such scars may be quantitatively removed using a mild ammonia treatment (Schulhof et al., Nucleic Acids Research. 1987; 15(2):397-416.) as depicted below:
An example of a linker employing these groups would be to attach a linker comprising a 2-((4-(disulfaneyl)butanoyl)oxy)acetyl group to an exocyclic amino group of a nucleobase (forming an amide), as depicted for cytosine and adenine below:
Cleavage of the disulfide (e.g. by DTT) may result in elimination of a 2-oxothiolane by intramolecular thiolactonization, leaving a glycolyl scar. Another example of this strategy would be to attach a linker comprising a photocleavable group (e.g. NPPOC) to a glycolyl of an exocyclic amino group of a nucleobase, as depicted for cytosine and adenine below:
After photo-cleavage of the linker, the bases still comprise a glycolyl (acyl) scar which may not terminate further elongation by other nucleotide-polymerase conjugates, but may ultimately be removed by treatment with ammonia, as depicted below:
Other strategies for the attachment of the nucleoside triphosphate to the polymerase following the above described principles may be used and will be apparent to those skilled in the art.
Template independent polymerases may have a high tolerance for base modifications (e.g., for TdT see Figeys et al. (Anal Chem. 1994 Dec. 1; 66(23):4382-3.) and Li et al. (Cytometry. 1995 Jun. 1; 20(2):172-80.)) so that certain scars may be well tolerated in following nucleic acid extension steps.
In some embodiments, linkers with multiple cleavable groups inserted in tandem may be used to increase the cleavage rate compared to a linker with single cleavable group.
In certain embodiments, modified nucleoside triphosphates with attached chemical moieties that prevent base pairing or the formation of undesirable secondary structure during synthesis may be used. Such modifications may include, but are not limited to, N3-methylation of cytosine, N1-methylation of adenine, 06-methylation of guanine, and acetylation of the exocyclic amine of guanine. Similar modifications were shown to significantly enhance the rate of dGTP homopolymer synthesis using TdT (Lefler and Bollum, Journal of Biological Chemistry 244.3 (1969): 594-601.). After completion of the synthesis, such base modifications may be simultaneously removed to restore base pairing for downstream applications. For example, N-acetylation of guanine may be removed by ammonia treatment as described above. N3-methylation of cytosine and N1-methylation of adenine may be removed by the enzyme AlkB, and 06-methylation of guanine may be removed by the enzyme MGMT, as depicted below:
In some embodiments, the de novo nucleic acid synthesis may be paused at an intermediate step and synthesis of complementary DNA may be performed, e.g. by hybridization of a suitable primer (e.g. random hexamers) that may be extended by a template-dependent polymerase using nucleoside triphosphates. After the complementary DNA synthesis, the de novo DNA synthesis may resume, and the double stranded part of the nucleic acid may be hindered from forming secondary structures. In some cases, the complementary DNA synthesis step may comprise leaving a 3′ overhang on the de novo synthesized nucleic acid to enable efficient subsequent extensions by polymerase-nucleotide conjugates.
In some embodiments, single-stranded binding proteins (e.g. E. coli SSB) may be included in extension reactions to hinder the formation of secondary structures in the nucleic acid being synthesized.
Incorporation of Unnatural or Modified dNTPs.
Conjugates useful for de novo nucleic acid synthesis may comprise nucleoside triphosphate analogs, including nucleotides without base-pairing ability (e.g. abasic nucleotide analogs) or nucleoside triphosphates with base-pairing ability different from the natural nucleotides, e.g. deoxyinosine or nitroindole nucleoside triphosphates.
In some embodiments, during synthesis the nucleic acid molecules are immobilized on a solid support that can be washed and exposed to the reaction cycle enzymes and buffers via automated liquid handing equipment. Examples of a solid support include, but are not limited to, a microtiter plate, into which reagents could be dispensed and removed by a liquid-handling robot, magnetic beads, which can be magnetically separated from a suspension and then resuspended in a new reagent in microtiter format, or an interior surface of a microfluidic device that can dispense the reaction cycle reagents to that location in an automated fashion.
An application of an automated system for nucleic acid synthesis employing conjugates comprising a polymerase and a nucleoside triphosphate is the synthesis of 10-100 nt oligonucleotides for molecular biology applications such as PCR. Another application is the picomole-scale or femtomole-scale synthesis of 50-500 nt or longer oligonucleotides using inkjet-based liquid handling techniques to produce DNA molecules that serve, e.g. as input to conventional DNA assembly methods (Kosuri and Church, Nature methods 11.5 (2014): 499-507.).
In some embodiments, the DNA synthesis method can be implemented in single-molecule format. In this approach, the reaction chamber of an automated microfluidic device is loaded with a single primer molecule of DNA that is iteratively extended into the desired sequence using a modified version of the reaction cycle described above (
Provided herein is a method for nucleic acid sequencing using conjugates of a template-dependent polymerase and a nucleoside triphosphate. The method is analogous to Sequencing By Synthesis (SBS).
In some embodiments, the method employs an “ACGT extension reagent” comprising four conjugates with base-pairing ability equivalent to A, C, G, and T, wherein the conjugates are labeled with distinguishable labels, e.g. distinct fluorophores. In other embodiments, the method employs “four distinct extension reagents” in separate containers, each comprising a conjugate with base-pairing ability equivalent to A, C, G, and T respectively. In some embodiments, these four extension reagents may be labeled for detection, e.g. with fluorophores.
In some embodiments, the method comprises: (a) immobilizing a duplex comprising a primer and template nucleic acid on a support (b) exposing the duplex to the “ACGT extension reagent” to extend the primer by a nucleotide complementary to the template; (c) detecting the label of the attached conjugate to infer the complementary base of the template; (d) exposing the duplex to the deprotection reagent, which cleaves the linkage between the polymerase and the added nucleotide, rendering the duplex unlabeled; and (e) repeating steps (b-d) 10 or more times to determine the sequence of at least part of the template molecule. An example of this method employing polymerase-conjugates with four distinguishable fluorophores is depicted in
In other embodiments, the method comprises: (a) immobilizing a duplex comprising a primer and template nucleic acid on a support (b) exposing the duplex to the first extension reagent to extend the primer by a nucleotide complementary to the template if the nucleotide of the conjugate is complementary; (c) detecting the label of the attached conjugate to infer if an extension has occurred; (d) exposing the nucleic acid to the deprotection reagent, which cleaves the linkage between the polymerase and the added nucleotide, rendering the duplex unlabeled; (e) repeating steps (b-d) three more times with the remaining three extension reagents, and (f) repeating steps (b-e) 10 or more times to determine the sequence of at least part of the template molecule.
In some embodiments, the detectible label may be a fluorescent protein fused to the polymerase. In other embodiments, the detectible label may be a quantum dot that is specifically attached to the polymerase.
Particularly in embodiments that employ four distinct extension reagents, the conjugates may be non-fluorescent or without a detectible label. In such embodiments, extension may be detected by other signals of the extension reaction such as release of H+ or pyrophosphate. In other embodiments, the polymerase may be fused to a reporter enzyme such as luciferase or peroxidase that may be detected when they produce light by catalyzing a reaction. In other embodiments, the polymerase may be fused to a nanoparticle detectible by scatting light. In other embodiments, an otherwise unlabeled polymerase of a conjugate that has extended a nucleic acid may be detected by a change in surface plasmon resonance.
Particularly in embodiments that employ conjugates with detectible labels, the method may be useful for determining the sequence of individual molecules, i.e. “single-molecule sequencing”.
In some embodiments, the method may additionally comprise an initial step of making 10, 100, 1000, or more copies of the template molecule and then applying a method described above to all copies simultaneously.
In any embodiment of the nucleic acid sequencing method, the length of the linker between the nucleoside triphosphate and the polymerase of the conjugates may be selected to maximize the fidelity of nucleotide incorporation by the conjugates, i.e. to minimize incorporation of mismatched bases with the template. Likewise, in any embodiment, the concentration of divalent cation(s) in the extension reaction (e.g. Mg2+) may be adjusted to maximize the fidelity of nucleotide incorporation by the conjugates.
In some embodiments, the polymerase of the conjugates may be a polymerase with “random binding order”, i.e. the primer-template duplex can bind to the catalytic site before or after nucleoside triphosphate.
In other embodiments, the polymerase of the conjugates may be a polymerase with defined binding order, i.e. the primer-template duplex must bind to the catalytic site before the nucleoside triphosphate. In such embodiments, the length of the linker between the nucleoside triphosphate and the polymerase of the conjugates may be selected to minimize inhibition of primer-template duplex binding to the conjugate, i.e. by using a linker longer than 10 Å, or 100 Å, or 200 Å.
In any embodiment, the conjugate may comprise a reversible terminator nucleoside triphosphate.
Embodiment 1. A conjugate comprising a polymerase and a nucleoside triphosphate, wherein the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage.
Embodiment 2. The conjugate of embodiment 1, wherein the polymerase is capable of catalyzing the addition of the nucleotide that is linked to the polymerase to the 3′ end of a nucleic acid.
Embodiment 3. The conjugate of any prior embodiment, wherein the polymerase is linked to the nucleoside triphosphate via a linker that has a length in the range of 4-100 Å, and wherein the length of the linker is sufficient for the nucleoside triphosphate to access the active site of the polymerase.
Embodiment 4. The conjugate of any prior embodiment, wherein the nucleoside triphosphate is linked to a cysteine residue in the polymerase.
Embodiment 5. The conjugate of any prior embodiment, wherein the cleavable linkage is a light or enzyme-cleavable linkage.
Embodiment 6. The conjugate of any prior embodiment, wherein the polymerase is a DNA polymerase.
Embodiment 7. The conjugate of any of embodiments 1-5, wherein the polymerase is an RNA polymerase.
Embodiment 8. The conjugate of any prior embodiment, wherein the polymerase is a template-independent polymerase.
Embodiment 9. The conjugate of any of embodiments 1-7, wherein the polymerase is a template-dependent polymerase.
Embodiment 10. The conjugate of any prior embodiment, wherein the nucleoside triphosphate or the polymerase comprises a fluorescent label.
Embodiment 11. The conjugate of any prior embodiment, wherein the nucleoside triphosphate is a deoxyribonucleoside triphosphate.
Embodiment 12. The conjugate of any prior embodiment, wherein the nucleoside triphosphate is a ribonucleoside triphosphate.
Embodiment 13. A set of conjugates of any prior embodiment, wherein the conjugates correspond to G, A, T and C and are in separate containers.
Embodiment 14. A method of nucleic acid synthesis, comprising:
incubating a nucleic acid with a first conjugate, wherein the first conjugate is a conjugate of any prior embodiment and the incubating is done under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the first conjugate onto the 3′ hydroxyl of the nucleic acid, to make an extension product.
Embodiment 15. The method of embodiment 14, wherein nucleic acid is tethered to a support.
Embodiment 16. The method of embodiment 14 or 15, wherein the method comprises, after addition of the nucleotide onto the nucleic acid, cleaving the cleavable linkage of the linker, thereby releasing the polymerase from the extension product.
Embodiment 17. The method of embodiment 16, wherein the cleavable linkage is an enzyme- or light-cleavable linkage and the cleaving comprises exposing the extension product to an enzyme or to light.
Embodiment 18. The method of embodiments 16 or 17, wherein the cleavage of the cleavable linkage deprotects the added nucleotide to produce a deprotected extension product.
Embodiment 19. The method of embodiment 18, further comprising, after deprotection of the added nucleotide:
incubating the deprotected extension product with a second conjugate, wherein the second conjugate is a conjugate of any of embodiments 1-12 and the incubating is done under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the second conjugate onto the 3′ end of the deprotected extension product.
Embodiment 20. The method of any of embodiments 14-19, wherein the method comprises:
(a) incubating a nucleic acid with a first conjugate of any of embodiments 1-12 under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the first conjugate onto the 3′ hydroxyl of the nucleic acid, to make an extension product;
(b) cleaving the cleavable linkage of the linker, thereby releasing the polymerase from the extension product and deprotecting the extension product;
(c) incubating the deprotected extension product with a second conjugate of any of embodiments 1-12 under conditions in which the polymerase catalyzes the covalent addition of the nucleotide of the second conjugate onto the 3′ end of the extension product, to make a second extension product;
(d) repeating steps (b)-(c) on the second extension product multiple times to produce an extended nucleic acid of a defined sequence.
Embodiment 21. The method of embodiment 20, wherein the nucleotide is a reversible terminator, and wherein deprotection of the extension product comprises removal of the blocking group of the reversible terminator.
Embodiment 22. The method of any of embodiments 14-21, wherein the nucleic acid is an oligonucleotide.
Embodiment 23. A method of sequencing, comprising:
incubating a duplex comprising a primer and a template with a composition comprising a set of conjugates of embodiment 13, wherein the conjugates correspond to G, A, T (or U) and C and are distinguishably labeled;
detecting which nucleotide has been added to the primer by detecting a label that is tethered to the polymerase that has added the nucleotide to the primer;
deprotecting the extension product by cleaving the linker; and
repeating the incubation, detection and deprotection steps to obtain the sequence of at least part of the template.
Embodiment 24. The method of embodiment 23, wherein the method of sequencing is a method of DNA sequencing.
Embodiment 25. The method of embodiment 23, wherein the method of sequencing is a method of RNA sequencing.
Embodiment 26. The method of any of embodiments 23-25, wherein the nucleotide is a reversible terminator, and wherein deprotection of the extension product comprises removal of the blocking group.
Embodiment 27. A reagent set, comprising:
a polymerase that has been modified to contain a single cysteine on its surface; and
a set of nucleoside triphosphates, wherein each of the nucleoside triphosphates is linked to a sulfhydryl-reactive group.
Embodiment 28. The reagent set of embodiment 27, wherein the nucleoside triphosphates correspond to G, A, T (or U) and C.
Embodiment 29. The reagent set of any of embodiments 27-28, wherein the nucleoside triphosphates are reversible terminators.
Embodiment 30. The reagent set of any of embodiments 27-29, wherein the nucleoside triphosphates comprise a linker that has a length in the range of 4-100 Å.
Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
1. Generation of Polymerase (TdT) Mutants with Various Attachment Positions for the Linker.
A gBlock coding for the Mus musculus TdT amino acid sequence used by Boule et al. (Molecular biotechnology 10.3 (1998): 199-208.) was ordered from IDT (Coralville, Iowa). The sequence was cloned into a pET19b vector, fusing the N-terminal his-tag of the vector to the protein using isothermal assembly. QuickChange PCR was used to generate a TdT mutant lacking all surface cysteines that might allow incorporation of tethered dNTPs (TdT5cysX). The cysteines in positions 188, 302, and 378 were mutated into alanine, the cysteines in positions 216 and 438 were mutated into serine (positions refer to the numbering in PDB structure 4127). TdT mutants containing one surface cysteine close to the catalytic site were generated by QuickChange PCR on TdT5cysX. Cysteines were inserted in positions 188, 302, 180 or 253, respectively.
Listed below is the amino acid sequence of the “wildtype” TdT protein used in this example prior to the mutation of cysteine residues (TdTwt):
As described above, plasmids coding for 6 TdT mutants with varying cysteine residues (therefore with different attachment positions for a linker) were generated:
(a) A plasmid coding for “wildtype” TdT with 7 cysteines (TdTwt)
(b) A plasmid coding for a TdT mutant with no surface cysteines and only 2 cysteines that are both buried (TdT5cysX)
(c) Four plasmids coding for TdT mutants with 2 buried cysteines plus one exposed surface cysteine in different positions (188, 180, 253, and 302) referred to herein as TdTcys188, TdTcys 180, TdTcys253, and TdTcys302
2. Protein Expression and Purification of the Mutants.
TdT expression was performed using Rosetta-gami B(DE3)pLysS cells (Novagen) in LB media containing antibiotics for all four resistance markers of the cells (Kan, Cmp, Tet, and Carb, which is introduced by the pET19 vector). An overnight culture of 50 mL was used to inoculate a 400 mL expression culture with 1/20 vol. Cells were grown at 37° C. and 200 rpm shaking until they reached OD 0.6. IPTG was added to a final concentration of 0.5 mM and the expression was performed for 12h at 30° C. Cells were harvested by centrifugation at 8000 G for 10 min and resuspended in 20 mL buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 8.3)+5 min imidazole. Cell lysis was performed using sonication followed by centrifugation at 15000 G for 20 min. The supernatant was applied to a gravity column containing 1 mL of Ni-NTA agarose (Qiagen). The column was washed with 20 volumes of buffer A+40 mM imidazole, and bound protein was eluted using 4 mL buffer A+500 mM imidazole. The protein was concentrated to ˜0.15 mL with Vivaspin 20 columns (MWCO 10 kDa, Sartorius) and then dialyzed against 200 mL TdT storage buffer (100 mM NaCl, 200 mM K2HPO4, pH 7.5) over night using Pur-A-Lyzer™ Dialysis Kit Mini 12000 tubes (Sigma).
All 6 TdT mutants with varying cysteine residues were expressed and purified.
3. Attachment of Tethered Nucleoside Triphosphates to the Polymerase
To prepare TdT-dUTP conjugates, the linker-nucleotide OPSS-PEG4-aa-dUTP was first synthesized and then reacted with TdT. OPSS-PEG4-aa-dUTP was synthesized by reacting amino-allyl dUTP (aa-dUTP) with the heterobifunctional crosslinker PEG4-SPDP (
The scheme for the preparation of the polymerase-nucleotide conjugates is shown in
All 6 TdT mutants with varying cysteine residues were separately exposed to OPSS-PEG4-aa-dUTP tethering reactions.
(a) TdTwt contains five surface cysteine residues that result in labeling with up to five OPSS-PEG4-aa-dUTP moieties.
(b) TdT5cysX only contains two buried but no surface cysteine residues, presumably not resulting in substantial labeling by OPSS-PEG4-aa-dUTP.
(c) TdTcys188, TdTcys180, TdTcys253, and TdTcys302 have a single surface cysteine that can be labeled with a single OPSS-PEG4-aa-dUTP moiety (at residue position 188, 180, 253, and 302, respectively). These TdT mutants also contain the two buried cysteines present in TdT5cysX that presumably are not labeled.
4. Generation of the Ladder of Elongation Product Standards.
The ladder of linked incorporation product standards was generated by incorporating free OPSS-PEG4-aa-dUTP using TdT. The reaction to synthesize OPSS-PEG4-aa-dUTP was performed by mixing 6 μL 50 mM aa-dUTP, 5 μL 180 mM PEG4-SPDP, 5 μL 1M NaHCO3 and 4 μL ddH2O. The reaction was incubated at RT for 1h and another 5 μL of 180 mM PEG4-SPDP was added. After 1h, the reaction was quenched using 5 μL of 100 mM glycine in PBS. To achieve varying numbers of incorporations, 6 TdT incorporation reactions using free OPSS-PEG4-aa-dUTP as substrate were performed. The reactions contained 1.5 μL 10×NEB TdT reaction buffer, 1.5 μL NEB TdT CoCl2, 1.5 μL 10 μM 5′-FAM labeled 35-mer dT-oligonucleotide (5′-FAM-dT(35)), 1 μL 10 mM OPSS-PEG4-aa-dUTP, 4.5 μL ddH2O and varied in their TdT concentration (100, 50, 25, 12.5, 6.3, 3.13 units of NEB TdT in 5 μL 1×NEB reaction buffer). Reactions were performed for 5 mM at 37° C. and stopped by the addition of 0.3 mM EDTA. Before running the ladder on the polyacrylamide gel, reaction products were mixed with an equal volume of 2× Novex Tris-Glycine buffer+1% v/v f3-mercaptoethanol and heated to 95° C. for 5 mM.
The 5′-FAM-dT(60) oligo was extended by 0 to 5 or more OPSS-PEG4-aa-dUMP nucleotides. After reduction of the ladder in the loading dye, the HS-PEG4-aa-dUTP elongation product standards (structure of one HS-PEG4-aa-dUTP elongation product is depicted in
5. Incorporation of Tethered Nucleoside Triphosphates into a Nucleic Acid.
The OPSS-PEG4-aa-dUTP conjugates of TdTwt, TdT5cysX, TdTcys188, TdTcys180, TdTcys253, and TdTcys302 were reacted with 5′-FAM-dT(35). Reactions that are shown in
As shown in chemical detail in
To demonstrate that polymerase-nucleotide conjugates add their tethered nucleotides to an oligonucleotide, TdT conjugates of OPSS-PEG4-aa-dUTP were incubated with a 5′ FAM-labeled dT(35) primer. The reaction was performed with conjugates of TdTwt, which have multiple tethered nucleotides, conjugates of TdT5cysX, which do not have tethered nucleotides, and conjugates of TdTcys302 which has a single nucleotide tethered to the cysteine at position 302. As described above, the reactions were stopped and the products were resolved by SDS-PAGE (
These data show that the polymerase moiety of polymerase-nucleotide conjugates can incorporate one or more tethered nucleoside triphosphates into a primer. They also show that conjugates labeled with a single nucleotide triphosphate can perform a single elongation of a primer and that further elongations of the primer by other polymerase-nucleotide conjugates can be hindered, enabling elongation of a nucleic acid by a single nucleotide.
To demonstrate that functional polymerase-nucleotide conjugates can be generated using a variety of attachment positions on the polymerase, OPSS-PEG4-aa-dUTP conjugates of TdT mutants with a single surface cysteine at positions 188, 302, 180, and 253 (TdTcys188, TdTcys302, TdTcys180, and TdTcys253, respectively) were used to extend a primer by a single nucleotide. The conjugates were separately exposed to a 5′ fluorescently-labeled poly-dT primer. Separation of the products on SDS-PAGE (
These data show that the principle of tethering a single nucleotide to a polymerase for achieving single nucleotide elongation of a nucleic acid is generalizable across attachment points on the polymerase.
The sequence encoding Maltose Binding Protein (MBP) was amplified from pMAL-c5X (NEB) and N-terminally fused to the TdTcys302 construct used in Example 1 using isothermal assembly. The resulting MBP-TdT fusion protein (herein referred to as TdTcys) was used throughout Example 2.
Unless mentioned otherwise, E. coli BL21(DE3) harboring pET19-TdTcys was grown in LB (Miller) with 100 μg/mL carbenicillin while shaking at 200 RPM. An overnight culture was diluted 1/60 into 400 mL LB in a 2 L flask without baffles and grown at 37° C. until an OD600 of 0.40-0.45 was reached. The flasks were then cooled down to RT for 45 min without shaking and then shaken at 15° C. for 45 min. Protein expression was induced with IPTG (final conc. 1 mM) and cells were grown overnight at 15° C. and harvested by centrifugation. All protein purification steps were performed at 4° C. Cells were lysed in Buffer A (20 mM Tris-HCl, pH 8.3, 0.5 M NaCl)+5 mM imidazole and the lysate was subjected to nickel affinity chromatography (HisTrap FF 5 mL, GE Healthcare) with an imidazole gradient (Buffer A+5 mM imidazole to Buffer A+500 mM imidazole). Fractions with sufficient purity were pooled, diluted 1:40 into 20 mM Tris-HCl, pH 8.3 and subjected to anion-exchange chromatography (HiTrap Q HP 5 mL, GE Healthcare) in 20 mM Tris-HCl using a gradient of 0 to 1 M NaCl. The protein eluted at 200 mM NaCl. The protein was stored at −20° C. after the addition of 50% glycerol.
3. Preparation of TdT-dNTP Conjugates.
Propargylamino-dNTPs (pa-dNTPs) were coupled to the photocleavable NHS carbonate-maleimide crosslinker BP-23354 (
Analogs of the four nucleotides dATP, dCTP, dGTP, and dTTP were separately coupled to the photocleavable crosslinker and tethered to TdT. The different polymerase-nucleotide conjugates were purified using amylose affinity chromatography.
4. Capillary Electrophoresis Analysis and Ladder Generation.
Capillary electrophoresis (CE) analysis throughout Example 2 was run on an ABI 3730x1 DNA Analyzer. GeneScan Liz600 v1.0 (Thermo) was added to all samples for use as internal size standards. Ladders (size standards) for 5′-FAM labeled 60-mer dT-oligonucleotide (5′-FAM-dT(60)) extension products were generated by the incorporation of free pa-dNTPs with TdT. Reactions contained 100 nM 5′-FAM-dT(60), 100 μM of one type of pa-dNTP, 1×RBC and either 0.05 U/μL or 0.03 U/μL NEB TdT. Reactions were performed at 37° C. Aliquots were taken after 2, 5 and 10 min and quenched with EDTA to a final concentration of 33.3 mM. Quenched samples were then acetylated using NHS-acetate, purified Oligo Clean & Concentrator kit (“OCC”, Zymo Research) and analyzed by capillary electrophoresis. Samples with detectable peaks for 5′-FAM-dT(60) as well as the +1 and +2 pa-dNTP extension products were chosen as size standards (ladders).
5. Demonstration of Two Reaction Cycles on PAGE and Capillary Electrophoresis.
Throughout the experiment, primer extension reactions were performed for 2 min at 37° C. and quenched by the addition of an equal volume of 200 mM EDTA. All reactions contained 50 nM 5′-FAM-dT(60), TdTcys(-linker)/TdT-dCTP at 0.25 mg/mL and 1×RBC (1×NEB TdT reaction buffer, 0.25 mM cobalt). Light induced cleavage of the linker was performed using a Benchtop 2UV Transilluminator (UVP, LLC) on the 365 nm setting for 1 h on ice. Measured irradiance was approximately 5 mW/cm2. Two cycle experiment: a reaction containing TdT-dCTP conjugate and 5′-FAM-dT(60) was performed and the product was cleaved with 365 nm light. The oligo was then purified (Zymo OCC), and subjected to another reaction with TdT-dCTP, again followed by a light cleavage step. Aliquots were taken after both extension reactions (for PAGE) and after both light cleavage reactions (for PAGE and CE). For the control experiment (“control unlinked”), TdT-dCTP conjugate was cleaved by irradiation with 365 nm light for 1 h on ice to generate an equimolar mix of unlinked TdTcys(-linker)+pa-dCTP. The products were then reacted with 5′-FAM-dT(60). Aliquots for PAGE and CE were taken after quenching the reaction with EDTA. Sample preparation: all CE samples were acetylated using 20 mM NHS-acetate in bicarbonate buffer prior to analysis. Samples were combined with SDS loading dye and analyzed by PAGE, the gel was imaged for green fluorescence (of the 5′FAM-labeled primer) and, after staining with Lumitein UV (Biotium), imaged for red fluorescence (total protein).
Exposure of a 5′ PAM-labeled oligonucleotide primer to TdT-dCTP conjugate resulted in a covalent complex visible on SDS-PAGE containing both the DNA primer and the protein (
These data show that the process of extending a primer by one nucleotide using a polymerase-nucleotide conjugate can be repeated to elongate a primer by a defined sequence.
6. Rapid Single Nucleotide Incorporation by TdT-dCTP, TdT-dGTP, TdT-dTTP, and TdT-dATP.
Oligonucleotide extension yield by 1.5 mg/mL TdT-dNTP conjugate was measured at 8, 15, and 120 seconds. Reactions were performed at 37° C. by adding 4.5 μL of TdT-dNTP conjugate (2 mg/mL) to 1.5 μL 5′-FAM-dT(60) (100 nM, final 25 nM), both in 1×RBC. After rapid mixing, 4.5 μL of the reaction were quenched in 18 μL QS (94% HiDi formamide, 10 mM EDTA) after 8 or 15 sec. The remaining reaction volume was quenched with 6 μL QS after 2 min. All samples were irradiated at 365 nm on a Benchtop 2UV Transilluminator (UVP, LLC) for 30 min to cleave the linker. Cleavage products were diluted with wash buffer (0.67 M NaH2PO4, 0.67 M NaCl, 0.17 M EDTA, pH 8) and captured onto DynaBeads M-280 StreptAvidin (Thermo) saturated with a 5′ biotinyl dA(60) oligo, washed, acetylated using 100 mM NHS-acetate in bicarbonate buffer, and eluted with 75% deionized formamide for CE.
The CE data in
7. Cyclic Synthesis of a Defined DNA Sequence.
Four iterations of nucleic acid extension and deprotection were performed using TdT-dNTP conjugates at 0.25 mg/mL. Extension reactions were performed with 2 min incubations at 37° C. in 1×RBC and were quenched by the addition of an equal volume of quenching buffer (250 mM EDTA, 500 mM NaCl). Cleavage of the linker was performed by irradiation at 365 nm. The first extension reaction contained TdT-dCTP and 50 nM oligo C1 (/5Phos/UTGAAGAGCGAGAGTGAGTGA/iFluorT/CATTAAAGACGTGGGCCTGGAttt (SEQ ID NO: 3) where/5Phos/refers to a 5′ phosphorylation, /iFluorT/ refers to a dT nucleotide base-modified with fluorescein). After photolysis, the extension product was purified (Zymo OCC), and the recovered DNA was subjected to the next extension step with TdT-dTTP. Two more cycles were performed with TdT-dATP and subsequently with TdT-dGTP, and the ultimate product was T-tailed using TdT and free dTTP+ddTTP at a ratio of 100:1. The tailed product was then PCR-amplified using HotStart Taq (NEB) with primers C2 (GTGCCGTGAGACCTGGCTCCTGACGATATGGATaagcttTGAAGA GCGAGAGTGAGTGA; SEQ ID NO:4) and C3 (AAAAgaattcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAA; SEQ ID NO:5) (PCR program: Initial cycle of 98° C. for 2 min, 49° C. for 20 sec, 68° C. for 5 min, then 30 cycles of three step protocol: 98° C. for 30 sec, 49° C. for 20 sec, 68° C. for 30 sec). The PCR product was inserted into the pUC19 plasmid using EcoRI and HindIII sites that were introduced by the PCR primers. The plasmid was transformed into DH10B cells and the plasmids of single colonies were extracted after overnight growth in LB and sequenced.
As described in detail above, four iterations of the reaction cycle were performed on a starter DNA molecule, the tailed products were PCR-amplified, and the amplicon was cloned for sequencing as depicted in
The experiment was performed using TdT-dCTP conjugates as prepared in Example 2. Capillary electrophoresis analysis was also performed as described in Example 2, and the same oligonucleotide ladder was used as size reference. 5′-FAM-dT60 (50 nM) was incubated with TdT-dCTP (0.25 mg/mL) for 120 sec at 37° C. in 1×RBC to yield a primer-polymerase complex and the reaction was split into aliquots. One aliquot was quenched by the addition EDTA to a final concentration of 100 mM. Another aliquot was diluted 10-fold with 1×RBC. Another aliquot was diluted 10-fold with 1×RBC containing free pa-dCTP to a final concentration of 500 μM pa-dCTP. After incubation at 37° C. for another 60 sec, both reactions were quenched by the addition of EDTA to a final concentration of 100 mM. Light induced cleavage of the linker for all samples was performed using a Benchtop 2UV Transilluminator (UVP, LLC) on the 365 nm setting for 1 h on ice. The samples were acetylated using 20 mM NHS-acetate in bicarbonate buffer, purified and analyzed by CE. During the initial incubation of the primer with TdT-dCTP, a primer-polymerase complex forms, as indicated by the extension of the primer by a single nucleotide observed by CE analysis (
These results show that a nucleic acid-polymerase complex that is formed upon tethered incorporation of a nucleoside triphosphate by a polymerase-nucleotide conjugate may still be able to incorporate free nucleoside triphosphates.
Fluorescent primers were prepared by labeling amine-containing oligos with 4.5 mM fluorophore NHS ester in sodium bicarbonate buffer followed by OCC. A 639 nt DNA product containing deoxyuridine bases was obtained by 35 cycles of PCR using Phusion U (Thermo) following the manufacturer's instructions (two step protocol: 98° C. denaturation for 10 sec, 72° C. annealing/extension for 1 min) from the plasmid template pMal-c5x (NEB) using primers PA1 (/5Phos/cattaaagacgtgggcgtgga; SEQ ID NO:6) and PA2 (t*t*t/iUniAmM/tgtgaaatccttccctcgatcc; SEQ ID NO:7). Primer PA1 is 5′ phosphorylated; primer PA2 contains an internal amino group (/iUniAmM/) labeled with Cy3 NHS (GE Healtcare) and begins with two phosphorothioate linkages (*) to render it exonuclease-resistant. The PCR product was purified from a 1% TAE-agarose gel and ˜6.7 μg of product was digested with 5 U of Lambda exonuclease (NEB) in a 100 μL reaction for 20 min at 37° C. to isolate the Cy3-labeled strand. Digestion products were purified by OCC and then hybridized at ˜1 μM with equimolar 5′FAM-labeled primer PA3 (/5AmMC6/CAACACACCACCCACCCAACcgcagatgtccgctttctgg (SEQ ID NO:14); /5AmMC6/refers to an aminohexyl modification of a 5′ phosphate) in 1× CutSmart buffer (NEB) by heating to 85° C. and cooling to 25° C. at 1° C./min. N-acetyl propargylamino dNTPs were prepared by acetylating 13 mM propargylamino dNTPs with 25 mM NHS acetate in 100 mM sodium bicarbonate buffer and quenched with glycine to 25 mM final. The primer was then elongated using 7.5 U of Klenow(exo-) (NEB) in 30 μL reactions at 37° C. with 200 μM (each) N-acetyl propargylamino dNTPs (reaction ii) or without any dNTPs (reaction i). After 1 hour, 3 μL of 2.5 mM (each) ddNTPs (Affymetrix) were added to both reactions for an additional 15 min incubation followed by inactivation of the polymerase by heating to 75° C. for 20 minutes. The products were then immediately digested in 50 μL reactions using 5 U of USER Enzyme (NEB) at 37° C. for 1 hour to remove the dU-containing ssDNA template. Digestion products were purified by OCC and propargylamino dNTP-dependent elongation of the 5′FAM-labeled primer and USER digestion of the Cy3-labeled template were confirmed by CE. Both products were then used as templates for complementary DNA synthesis (“reading”) by 5 U of Taq (Thermo) using 200 μM (each) natural dNTPs and 200 nM of the 5′ Cy3-labeled primer PA4 (/5AmMC6/CGACTCACCTCACGTCCTCAtgtgaaatccttccctcgatcc; SEQ ID NO:8) in a 20 μL reaction by heating to 95° C. for 2 min and then incubating at 45° C. After 30 minutes, 1 μL of 2.5 mM (each) ddNTPs was added to both reactions for an additional 15 min incubation, and the DNA products were purified. Equal volumes of both reading products were then analyzed by qPCR on a CFX96 instrument (Bio-Rad), using Phusion HS II (Thermo) with 1× EvaGreen (Biotium) and primers PA5 (ttttGAATTCCAACACACCACCCACCCAAC; SEQ ID NO:9) and PA6 (ttttAAGCTTCGACTCACCTCACGTCCTCA; SEQ ID NO:10) for 30 cycles of 98° C. for 5 sec, 67° C. for 15 sec, and 72° C. for 30 sec. qPCR products from reaction ii were inserted into a pUC19 plasmid using EcoRI and HindIII sites introduced by the PCR primers and 81 clones were sequenced as described above.
The dNTP analogs used in this example contain a propargylamino group extending from the nucleobase (5 position of pyrimidines, 7 position of 7-deazapurines) which is the same moiety the polymerase-nucleotide conjugates from Example 2 leave as a scar. The propargylamino moiety was further derivitized by N-acetylation. To demonstrate that DNA comprising scarred bases as produced in Example 2 can serve as template for accurate synthesis of complementary DNA by a template-dependent polymerase using natural dNTPs, in this example a DNA molecule containing 141 sequential 3-acetamidopropynyl (i.e. N-acetylated propargylamino) nucleotides was prepared. This DNA product was isolated and used as template for PCR (
This data shows that scarred nucleic acids (in this case a polynucleotide harboring a moiety derivable from the propargylamino scar from Example 2) can be PCR-amplified with high fidelity, thereby generating natural DNA that can be used in biological applications.
Ten extensions of the 3′ overhang of a double-stranded DNA molecule were performed using TdT-dNTP conjugates as prepared in Example 2. The double stranded DNA used as initial substrate was prepared from a ˜350 bp PCR product derived from the pET19b plasmid using Phusion Polymerase (Thermo) following the manufacturer's instructions (two step protocol: 98° C. for 10 sec, 72° C. for 45 sec) with the primers T1 (/5Phos/GCAGCCAACTCAGCTTCTGCAGGGGCTTTGTTAGCAGCCGGATCCTC; SEQ ID NO:11) and T2 (AAACAAGCGCTCATGAGCCAGAAATCTGGAGCCCGATCTTCCCCATCGG; SEQ ID NO:12). The PCR product was digested with PstI to generate a 3′ overhang on one side, that was then tailed with ddTTP using TdT to prevent elongation of the generated 3′ overhang. After tailing, the DNA was digested with BstXI to generate a 3′ overhang on the other end of the amplicon to enable extensions by polymerase-nucleotide conjugates. The digestion product was isolated by 2% agarose gel electrophoresis and purified to yield the initial substrate for extensions by polymerase-nucleotide conjugates.
The extension reactions were performed for 90 sec at 37° C. in 1×RBC with 1 mg/mL of the respective polymerase-nucleotide conjugate and were quenched by the addition of an equal volume of quenching buffer (250 mM EDTA, 500 mM NaCl). Cleavage of the linker was performed by irradiation at 365 nm. The first extension reaction contained ˜40 nM of the initial substrate. After each cleavage step, the DNA products were purified (Zymo OCC), and the recovered DNA was subjected to the next extension step. The following conjugates were used in the extension steps: 1) TdT-dCTP, 2) TdT-dTTP, 3) TdT-dATP, 4) TdT-dCTP, 5) TdT-dTTP, 6) TdT-dGTP, 7) TdT-dATP, 8) TdT-dCTP, 9) TdT-dTTP, 10) TdT-dGTP. The ten-cycle product was T-tailed using TdT and free dTTP+ddTTP at a ratio of 100:1 and acetylated using 20 mM NHS-acetate in bicarbonate buffer. The tailed product was then PCR-amplified using HotStart Taq (NEB) with primers C3 and C4 (GTGCCGTGAGACCTGGCTCCTGACGAGGAtaagcttCTATAGTGAGTCGT ATTAATTTCG; SEQ ID NO:13) (PCR program: Initial cycle of 98° C. for 2 min, 49° C. for 20 sec, 68° C. for 12 min, then 30 cycles of three step protocol: 98° C. for 30 sec, 49° C. for 20 sec, 68° C. for 30 sec). The PCR product was inserted into pUC19 using EcoRI and HindIII sites that were introduced by the PCR primers. The plasmids were transformed into DH10B cells and the plasmids of single colonies were extracted after overnight growth in LB and sequenced.
As described in more detail above, a double stranded DNA template was elongated by 10 cycles using polymerase-nucleotide conjugates, the synthesis product was amplified and cloned for sequencing (
This result demonstrates that the cyclic process of DNA extension can be repeated many times to write nucleic acid molecules of the desired sequence and length.
This application is a continuation of Ser. No. 17/571,529; filed: Jan. 9, 2022, which is a continuation of Ser. No. 16/230,438; filed: Dec. 21, 2018, which is a continuation of PCT/US17/39120; filed: Jun. 23, 2017, which claims the benefit of Ser. No. 62/354,635, filed on Jun. 24, 2016, which application is incorporated by reference herein.
This invention was made with government support under Grant Number DE-AC02-05SC11231 awarded by the US Department of Energy. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62354635 | Jun 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17571529 | Jan 2022 | US |
| Child | 17691029 | US | |
| Parent | 16230438 | Dec 2018 | US |
| Child | 17571529 | US | |
| Parent | PCT/US17/39120 | Jun 2017 | US |
| Child | 16230438 | US |
