Modified nucleotides and nucleotide conjugates for polynucleotide synthesis

Abstract

Disclosed herein are improved methods and compositions of de novo synthesis of nucleic acids by cyclic extension using nucleotides that have an N-linked or O-linked scar or protecting group, and subsequent removal of the scar or protecting group. During polymerase-nucleotide conjugate-based polynucleotide synthesis or after the synthesis is completed, nucleobases having a scar from linker cleavage can be converted back into native form by removal of the scars from the nucleobases. Secondary structure formation in the nascent chain, which may inhibit extension reactions, is suppressed by the use of monomers with protected nucleobases that prevent Watson-Crick base pairing and / or other structures. After the synthesis is completed, the nucleobases can be converted back into native form by palladium-based removal of the protecting groups.

Description

Docket No.: ABB-018WOMODIFIED NUCLEOTIDES AND NUCLEOTIDE CONJUGATES FOR POLYNUCLEOTIDE SYNTHESISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application No. 63 / 680,574, filed August 7, 2024, the contents of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Polynucleotide synthesis is the process of creating chains of nucleotides, which are the building blocks of DNA and RNA. Standard de novo DNA synthesis performed today is based on the nucleoside phosphoramidite method (generally referred to as “chemical synthesis”) in which a desired sequence is synthesized by stepwise coupling of blocked monomers. The reactions are performed in organic solvents using highly reactive activated monomers, and the conditions cause side reactions that damage the growing chain, limiting the yield of full-length product. The impurities produced can be difficult or impractical to separate from the desired oligonucleotide product, limiting the usefulness of the method for producing sequences longer than approximately 200 bases.

[0003] As an alternative, different enzymatic de novo polynucleotide synthesis strategies using template-independent polymerases have more recently been developed, allowing an environmentally friendly synthesis of longer polynucleotide molecules than with chemical synthesis. These include enzymatic synthesis using conjugates comprising polymerase tethered to the nucleotide added as described in PCT Publication No. WO 2017 / 223517, incorporated herein by reference in its entirety. For conjugate-based synthesis, the nucleotide attached to the polymerase is added to an oligonucleotide at the 3' end by the attached polymerase, which then acts to shield the added nucleotide from incorporation of another nucleotide, allowing for cyclic controlled oligonucleotide synthesis. Cleavage of the polymerase from the nucleotide allows for subsequent addition of the next incoming nucleotide.

[0004] However, once a polymerase is cleaved from a nucleotide during conjugate synthesis, a portion of the linker may remain attached to the nucleotide, leaving a scar as compared to a naturally occurring nucleotide. This can negatively impact downstream use the synthesized polynucleotide, including amplification of the synthesized polynucleotide, or direct use for its intended application. Therefore, what are needed are improved conjugatesPage 1 of 26IPTS / 200089493.1Docket No.: ABB-018WO and methods of conjugate-based polynucleotide synthesis that include scars that can be removed during or after synthesis.

[0005] Furthermore, another problem for de novo polynucleotide synthesis is that, as the nucleic acid is being synthesized, it can base-pair with itself, inhibiting the extension reaction for a template-independent polymerase that has low activity on a duplex structure. Elevation of temperature has been explored to reduce secondary structure (Barthel et al., Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis, Genes 2020, 1 (1), 102). However, elevated temperature induces damage to DNA (and quickly damages RNA), and secondary structure remains even in elevated temperatures suitable for synthesis. Synthesis reactions at elevated temperatures also require thermostable polymerases. Wild-type template independent polymerases such as Terminal deoxynucleotidyl Transferase (TdT) are not thermostable. Use of bases with exocyclic amines masked as azido groups has also been explored (Nuclera Nucleics PCT Publication WO2020229831A1). However, the unmasking reagent (TCEP) causes DNA damage, and there are doubts about the stability of the azido modification.

[0006] What is needed, therefore, are improved base pair protecting groups on the nucleobase to inhibit secondary structure during polynucleotide synthesis, which can be efficiently removed after synthesis to leave a polynucleotide without modified nucleotides that might interfere with downstream applications. This can be useful for both enzymatic polynucleotide synthesis with free nucleotides and for conjugate-based polynucleotide synthesis.SUMMARY

[0007] Provided herein is a method of synthesizing a polynucleotide, comprising: providing a synthesized polynucleotide comprising one or more allylic moieties removable under palladium-catalyzed conditions, wherein said one or more allylic moieties are each bound directly or indirectly to a hydrogen base-pairing heteroatom on a nucleobase of the polynucleotide; and treating said synthesized polynucleotide with a palladium reagent to remove the one or more allylic moieties from said one or more nucleobases.

[0008] In some embodiments, providing said synthesized polynucleotide comprises: contacting a precursor polynucleotide with a polymerase and a nucleotide comprising said allylic moiety; and adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase.Page 2 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0009] In some embodiments, providing said synthesized polynucleotide comprises: contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a linker comprising said allylic moiety; adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase; and cleaving the linker, wherein said allylic moiety remains bound to the added nucleotide.

[0010] In some embodiments, the method further comprises repeating said contacting, adding, and optionally said cleaving steps one or more times to generate said polynucleotide.

[0011] In some embodiments, the polymerase is a template-independent polymerase. In some embodiments, the polymerase is TdT

[0012] In some embodiments, the nucleotide comprises a 2' or 3' modification.

[0013] In some embodiments, the synthesized polynucleotide comprises at least 10, at least 20, at least 50, or at least 100 of said allylic moieties bound to said nucleobases. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the allylic moieties are removed by said treatment with said palladium reagent.

[0014] Also provided herein is a polynucleotide comprising one or more allylic moieties removable under palladium-catalyzed conditions, wherein said one or more allylic moieties are each bound directly or indirectly to a hydrogen base-pairing heteroatom on a nucleobase of the polynucleotide. In some embodiments, the polynucleotide comprises at least 10, at least 20, at least 50, or at least 100 of said allylic moieties bound to said nucleobases.

[0015] Also provided herein is a nucleotide comprising an allylic moiety removable under palladium-catalyzed conditions, wherein the allylic moiety is bound directly or indirectly to a hydrogen base-pairing heteroatom on the nucleobase of the polynucleotide.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the present disclosure, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead placed upon illustrating the principles of various embodiments of the present disclosure.

[0017] FIG. 1 depicts i) typical enzymatic DNA synthesis performed with an enzyme and free nucleotides with 3’ blocking groups, which can be inhibited by the formation of secondary structure during synthesis, and ii) a diagram of improved conjugate-basedPage 3 of 26IPTS / 200089493.1Docket No.: ABB-018WO synthesis provided herein, including use of polymerase-nucleotide conjugates comprising the polymerase linked to a base pairing N or O atom of the nucleobase. Upon cleavage of the polymerase from the nucleotide at each cycle, a scarred nucleotide comprising a portion of the linker (scar) at the N or O atom is retained in the polynucleotide, which inhibits secondary structure formation. The scar(s) can then be removed to generate a scarless polynucleotide.

[0018] FIG. 2A, 2B, and 2C show the capillary electrophoresis (CE) peaks resulting from a nucleotide comprising an allyl scar on the nucleobase before and after treatment with palladium to remove the scar.

[0019] FIG. 3 shows the capillary electrophoresis peaks resulting from a polynucleotide comprising an allyl scar and treated for 15, 30, 45, or 60 minutes with palladium (bottom row).

[0020] FIG. 4 shows CE data from a proof of concept Pd deprotection of a FAM-labeled ssDNA substrate with a single allyl scarred G or T nucleotide at the 3’ end.

[0021] FIG. 5 shows the structures of possible alternative scar designs for Pd deprotection.

[0022] FIG. 6 shows the impact of the Pd deprotectable scar design on oligonucleotide aggregation at longer oligo synthesis lengths. This is CE data of a FAM-labeled homopolymer that has been synthesized at varying lengths from 25nt up to 400nt using conjugates prepared with 4 different linker nucleotide designs bearing scars that can be removed with the Pd reagent.

[0023] FIG. 7 shows CE data from a Pd deprotection of the A112-T scar design.

[0024] FIG. 8 shows the structure of various scarred T nucleotides.

[0025] FIG. 9 is generated from next generation deep sequencing data of a lOOOmer oligonucleotide synthesis utilizing either OMe-U, Allyl-T, or A112-T as the T conjugate.DETAILED DESCRIPTION

[0026] The details of various embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the present disclosure will be apparent from the description and the drawings, and from the claims.Definitions

[0027] The term “allylic moiety” as used herein refers to an allyl group and substituted allyl variants thereof that are removeable under palladium(0)-catalyzed conditions. In some embodiments, provided herein are allylic moieties having the structure -CH2-CH=CH2,Page 4 of 26IPTS / 200089493.1Docket No.: ABB-018WO optionally substituted on the double bond or methylene carbon, and covalently bound, either directly or indirectly via a spacer, with a heteroatom (e.g., nitrogen or oxygen) of a nucleobase. The allylic moiety is used to reversibly protect a hydrogen-bond donor or acceptor atom involved in Watson-Crick base pairing, thereby inhibiting base-pair formation during synthesis. The allylic moiety may be removed under mild conditions, such as by palladium-catalyzed deprotection (e.g., Tsuji-Trost reaction), to restore natural hydrogen bonding functionality.

[0028] As used herein, a “base-pairing interference group refers” to a protecting moiety covalently attached to a hydrogen-bond donor or acceptor atom (e.g., N3 of uracil, N1 of guanine) of a nucleobase, wherein said moiety sterically and / or electronically inhibits Watson-Crick base pairing.” These are also referred to as “scars” or “protecting groups” herein. Specific examples of base-pairing interference group as described herein include allylic moieties removeable by palladium-catalyzed deprotection. The base-pairing interference groups or allylic moieties can be present on a nucleotide with a reversible terminator, or as a residual scar leftover from cleavage of a TdT conjugate during synthesis.Synthesis of polynucleotides comprising scarred or protected nucleobases

[0029] One of the major challenges in the field of polynucleotide synthesis is the synthesis of long, complex nucleotide sequences. Because nucleotides are added one at a time during the synthesis process, it can be difficult to accurately and efficiently synthesize very long sequences without introducing errors. Even small error rates during synthesis can accumulate to prevent synthesis of long sequences.

[0030] Enzymatic de novo polynucleotide synthesis strategies using template-independent polymerases has continued to develop as an important alternative and improved method of stepwise, directed-polynucleotide synthesis, allowing an environmentally friendly synthesis of longer polynucleotide molecules than with chemical synthesis. These include enzymatic synthesis using conjugates comprising polymerase tethered to the nucleotide added as described in PCT Publication No. WO 2017 / 223517, incorporated herein by reference in its entirety. For conjugate-based synthesis, the nucleotide attached to the polymerase is added to an oligonucleotide at the 3' end by the attached polymerase, which then acts to shield the added nucleotide from incorporation of another nucleotide, allowing for cyclic controlled oligonucleotide synthesis. Cleavage of the polymerase from the nucleotide allows for subsequent addition of the next incoming nucleotide.Page 5 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0031] The TdT polymerase strongly prefers single-stranded DNA (ssDNA) as its substrate of double- stranded DNA (dsDNA). As a result, the nascent formation of secondary structure at the 3’ end of a growing strand of DNA results in a substantial decrease in TdT activity. This phenomenon makes the TdT-mediated synthesis of complex DNA bearing significant secondary structure especially challenging, thereby limiting the utility of such synthesis methods.

[0032] A key characteristic of conjugate-based polynucleotide synthesis is the direct tethering of a nucleoside triphosphate within the active site of TdT via a cleavable linker, forming a polymerase-nucleotide conjugate, or simply a “conjugate” for short. Upon cleavage, some residual portion of the linker may be left covalently attached to the synthesized DNA, referred to as a “scar”. Such a scar may have both determinantal and beneficial effects on the overall performance and applicability of conjugate-based DNA synthesis depending on the scar design. In some instances, for example, residual scars left behind can increase the Tm of the DNA, which can enhance secondary structure formation and further exacerbate the deleterious effect of double stranded 3’ termini on TdT activity. In other instances, scars may be attached to the nucleobase in a manner that changes H-bonding patterns, effectively disrupting the potential for base pairing and preventing secondary structure formation - referred to as “anti-base pairing scars”. This helps maintain the single stranded structure of the oligo during synthesis regardless of sequence, enabling the synthesis of highly complex, structured DNA. However, the disruption of base pairing can also inhibit faithful PCR amplification. Therefore, in some embodiments, anti-base pairing scars should be removed from the synthesized oligonucleotide to be useful for downstream applications using the synthesized polynucleotide.

[0033] Thus, provided herein are compositions and methods of oligonucleotide synthesis that inhibit secondary structure formation and improve oligonucleotide synthesis length and accuracy by providing protecting groups attached to the nucleobase of the synthesized polynucleotide that inhibit secondary structure formation. In some embodiments, these are provided as modified nucleotides incorporated into an oligonucleotide during enzymatic synthesis. In some embodiments, these are provided as part of a linker-nucleotide conjugate used during enzymatic oligonucleotide synthesis, such that the linker is attached to a base pairing nitrogen or oxygen atom on the nucleobase, and cleavage of the linker to separate the polymerase from the nucleotide during synthesis leaves a portion of the linker attached to a base pairing nitrogen or oxygen atom, which can act as a protecting group (also referred to herein as a “scar”) to inhibit secondary structure formation, as shown in FIG.l.Page 6 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0034] Another advantage of the methods and compositions provided herein is that they allow scarless synthesis of a polynucleotide when using polymerase-nucleotide conjugates for synthesis. Preferred linker structures and methods of removing residual scars / protecting groups after oligonucleotide synthesis to leave a naturally occurring polynucleotide without scars are also provided herein.

[0035] The present disclosure includes a method of synthesizing a polynucleotide comprising one or more scarred or protected nucleobases, and removing one or more of the scars or protecting groups from the synthesized oligonucleotides. In some embodiments, provided herein are improved methods for synthesis of nucleic acids by controlled cyclic nucleotide extension using a polymerase, such as a template-independent polymerase for de novo polynucleotide synthesis.

[0036] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide, comprising: providing a polynucleotide comprising one or more scarred nucleobases and removing one or more scars from said scarred nucleobases. In some embodiments, providing a polynucleotide comprises: contacting a precursor polynucleotide with a nucleotide comprising a nucleobase linked to a template-independent polymerase; and adding said nucleotide to the 3' end of said precursor polynucleotide via said templateindependent polymerase. In some embodiments, a method of synthesizing a polynucleotide further comprises repeating contacting and adding step one or more times.

[0037] After completion of synthesis of a polynucleotide using one or more conjugates, wherein cleavage of the polymerase from the nucleotide leaves scarred nucleotides in the polynucleotide, the resulting polynucleotide can then be treated suitable conditions as described herein to remove the scar from the modified nucleotides, resulting in a polynucleotide with unmodified nucleobases. In some embodiments, removal of one or more scars from the synthesized polynucleotide comprises contacting the polynucleotide with suitable conditions capable of removing said one or more scars from a scarred nucleobase.

[0038] In some embodiments, also provided herein is a method of synthesizing a polynucleotide comprising one or more protected nucleobases, and removing one or more protecting groups from said protected nucleobases. In some embodiments, inclusion of one or more protected nucleobases prevents the formation of undesirable secondary structure during synthesis, such as by preventing Watson-Crick base pairing or other hydrogen bonding between nucleic acids.

[0039] These methods include improved methods for synthesis of nucleic acids by cyclic extension using a template-independent polymerase. As shown herein, secondary structurePage 7 of 26IPTS / 200089493.1Docket No.: ABB-018WO formation in the nascent chain, which may inhibit extension reactions, is suppressed by the use of modified nucleotides with protecting groups on a one or more oxygens or nitrogens of nucleobases that prevent Watson-Crick base pairing and / or other structures. After the synthesis is completed, the nucleobases can be converted back into their native form by removal of the protecting group to facilitate further use and / or downstream processing of the synthesized polynucleotides.

[0040] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide, comprising: providing a polynucleotide comprising one or more protected nucleobases and removing one or more protecting groups from said protected nucleobases. In some embodiments, providing a polynucleotide comprises: contacting a precursor polynucleotide with a nucleotide comprising an protected nucleobase and a templateindependent polymerase; and adding said nucleotide to the 3' end of said precursor polynucleotide via said template-independent polymerase. In some embodiments, a method of synthesizing a polynucleotide further comprises repeating contacting and adding step one or more times.

[0041] After completion of synthesis of a polynucleotide using one or more protected nucleotides, the resulting polynucleotide can then be treated with suitable condiditions to remove the protecting group from the modified nucleotides, resulting in a polynucleotide with unmodified nucleobases. In some embodiments, removal of one or more protecting groups from the synthesized polynucleotide comprises contacting the polynucleotide with suitable conditions capable of removing said one or more protecting groups from an protected nucleobase.

[0042] Although synthesis can be completed entirely with protected nucleotides, synthesis with a combination of unmodified and protected nucleotides can also be used effectivley to improve polynucleotide synthesis. In some embodiments, only one of the four nucleotides added (e.g., from G or T) is protected during synthesis. In some embodiments, protected nucleotides are only added at targeted positions where secondary structure or ternary structure is predicted, which could interfere with synthesis. Such structures can be predicted based on the presence of complementary DNA regions in various ways and respective tools exist, such as the NUPACK algorithms (http: / / www.nupack.org / home / model). Thus, in some embodiments, synthesis of a completed polynucleotide where synthesis is improved can include the use of only 1 or 2 protected nucleotides. In some embodiments, about 5%, about 10%, about 20%, about 30%, about 50%, substantially all, or 100% of a specific nucleotide is incorporated into the polynucleotide in their protected version. In some embodiments, lessPage 8 of 26IPTS / 200089493.1Docket No.: ABB-018WO than 5%, less than 10%, less than 20%, less than 30%, or less than 50% of a specific nucleotide is incorporated into the polynucleotide in its protected version. In some embodiments, more than 5%, more than 10%, more than 20%, more than 30%, or more than 50% of a specific nucleotide is incorporated into the polynucleotide in its protected version. In some embodiments, only protected guanine nucleotides are used in the nucleotide synthesis reaction. The removal of protecting groups in the terminal positions of a nucleic acid may be more challenging than the removal from internal DNA positions. Therefore, in some embodiments, nucleotide synthesis is performed such that the last and first 1, 2, or 3 positions of the synthesized nucleic acid does not comprise protected nucleotides.

[0043] As used herein, a “scarred” nucleotide refers to a nucleotide that has a portion of a linker still attached to the nucleotide after cleavage of the linker to release an attached biomolecule, such as a polymerase of a polymerase-nucleotide conjugate.

[0044] As used herein, a “protected” nucleotide, as used herein, refers to a nucleotide that has biomolecule attached to a base pairing oxygen or nitrogen on the nucleobase. In some embodiments, the biomolecule inhibits hydrogen bonding of the oxygen or nitrogen to other nucleotides, such as in Watson-Crick base pairing, G-quadruplex formation, and other types of hydrogen bonding that can generate secondary structure. Thus, in some embodiments, the biomolecule inhibits formation of secondary structure during oligonucleotide synthesis. A “scarred” nucleotide and a “protected” nucleotide can both refer to the same structure when a linker is bound to an oxygen or nitrogen on the nucleobase. Such that cleavage of the linker leaves a “scarred” nucleotide that is also a “protected” nucleotide. In some embodiments, a conjugate linker is attached to a base pairing oxygen or nitrogen to take advantage of the presence of a scar to provide a protected nucleotide to inhibit secondary structure formation during synthesis. Protected nucleotides can also refer to modified nucleotides using during oligonucleotide synthesis that are not part of a conjugate, but are useful to prevent secondary structure during oligonucleotide synthesis.

[0045] In some embodiments, the present disclosure includes a method of treating a polynucleotide synthesized with conjugates, leaving scarred nucleobases, comprising: providing a polynucleotide comprising one or more scarred nucleobases; and removing one or more scars from said one or more scarred nucleobases.

[0046] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide comprising a scarred nucleobase, comprising:Page 9 of 26IPTS / 200089493.1Docket No.: ABB-018WO contacting a precursor polynucleotide with a polymerase bound to said nucleobase via a cleavible linker; adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase; and cleaving said cleavable linker, leaving a scarred nucleobase at the end of said polynucleotide.

[0047] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a cleavable linker, wherein cleavage of said nucleotide from said polymerase generates a scarred nucleobase. In some embodiments, the method of synthesizing a polynucleotide comprises cleaving a cleavable linker after addition of a nucleotide to a precursor polynucleotide. In some embodiments, the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally cleaving steps described herein one or more times. In some embodiments, removal of one or more scars described herein comprises contacting said polynucleotide with an chemical of photolytic condition capable of removing said one or more scars from said scarred nucleobases.

[0048] In some embodiments, synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3' end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized. As described herein, the use of nucleotides comprising protected nucleobases during polynucleotide synthesis helps to improve the efficiency and accuracy of synthesis by inhibiting secondary structure formation which can interfere with the addition of the incoming nucleotide by the polymerase during synthesis.

[0049] In some embodiments, synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3' end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.Page 10 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0050] In some embodiments, the present disclosure includes a method of treating a polynucleotide synthesized with protected nucleobases, comprising: providing a polynucleotide comprising one or more protected nucleobases; and removing one or more protecting groups from said one or more protected nucleobases.

[0051] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide comprising a protected nucleobase, comprising: contacting a precursor polynucleotide with a polymerase and a nucleotide comprising said protected nucleobase; adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase.

[0052] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a nucleotide and a polymerase, wherein said nucleotide comprises comprising a protecting group bound to a base pairing oxygen or nitrogen on the nucleobase. In some embodiments, the method of synthesizing a polynucleotide comprises removing a blocking group, such as a conjugated polymerase or a reversible terminator, after addition of a nucleotide to a precursor polynucleotide. In some embodiments, the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally removing a blocking group described herein one or more times. In some embodiments, removal of one or more protecting groups described herein comprises exposing said polynucleotide to a palladium reagent capable of removing said one or more protecting groups from said protected nucleobases.Reversible Terminators

[0053] While a Tsuji-Trost like deallylation reaction can be used to deprotect scars left behind using the conjugate -based oligonucleotide synthesis described above, such an approach may be used more generally to reduce secondary structure and improve the efficiency of oligonucleotide synthesis. A common alternative to the use of conjugates for enzymatic oligonucleotide synthesis is the use of 3’ reversible terminators (RT). Allyl groups, as well as similar functional groups removable under Tsuji-Trost like conditions, may be added to an anti-base pairing position of the nucleobase of nucleoside triphosphates bearing 3’ RTs such that removal of the RT leaves the allyl group intact for subsequentPage 11 of 26IPTS / 200089493.1Docket No.: ABB-018WO nucleotide addition steps. Similarly to the conjugate based approach described above, this would disrupt secondary structure throughout a RT-based oligonucleotide synthesis. The scars could then be removed using the same or similar Pd reagent and deprotection conditions as described above.

[0054] Shown below is a generalized structure for a nucleoside triphosphate bearing both an anti-base pairing group (Rl) on the nucleobase (Nuc) and a reversible terminator group (R2) on the 3’ hydroxyl.

[0055] Several embodiments of possible nucleoside triphosphate structures that could be used with the Pd-based deprotection approach to enable the combination of reversible terminator nucleotide functionality with anti-base pairing functionality are shown below:

[0056] In some embodiments, the nucleotides analogs described herein comprise a reversible terminator group, such as such as an O- azidomethyl, O-NH2, or phosphate groupPage 12 of 26IPTS / 200089493.1Docket No.: ABB-018WO on the 3' position of the sugar (for an overview see, e.g. Chen et al., Genomics, Proteomics & Bioinformatics 2013 11: 34-40). In these embodiments, the nucleotide analog prevents or hinders further elongation once incorporated into a nucleic acid to achieve controlled termination of synthesis.Protecting Group / Scar Removal

[0057] In some embodiments, suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with conditions to remove an allyl group. In some embodiments, suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with Pd. In some embodiments, suitable conditions for removal of a protecting group comprises the step of treating a protected nucleobase with Pd(OAc), Pd2(dba)3, and Pd2(pmdba)3.

[0058] Palladium catalysts readily may be used to remove the allylic blocking group or scars from the polynucleotide after synthesis.Chemically Removeable Protecting Groups / Scars

[0059] Described herein are removeable protecting group or scar structures that can be chemically removed from a nitrogen or oxygen of a nucleobase, leaving the natural nucleotide. For a polymerase-nucleotide conjugate, a linker connecting the nucleotide to the polymerase is attached to the nucleotide at a nitrogen or oxygen on the nucleobase, such that cleavage of the linker leaves the removeable protecting group on the nitrogen or oxygen. In some embodiments, the protecting group / scar structures described herein remain attached to the nitrogen or oxygen on the nucleobase during synthesis, but are removed before downstream processing or use of the newly synthesized oligonucleotide. In some embodiments, a nucleotide comprising a removeable protecting group structure described herein bound to a nitrogen or oxygen atom of the nucleobase (with a structure corresponding to a removeable scar after cleavage of a conjugate linker) can be used for oligonucleotide synthesis.

[0060] In some embodiments, an allyl group is used in a protecting group or scar that can be removed from the nucleobase. In some embodiments, a linker comprises an allyl group. In some embodiments, a scar comprises an allyl group. In some embodiments, a scar comprises an allyl group that is removed upon exposure to an appropriate transition metal catalyst. In some embodiments, a appropriate transition metal catalyst is a palladium catalyst. In somePage 13 of 26IPTS / 200089493.1Docket No.: ABB-018WO embodiments, a appropriate transition metal catalyst is selected from the group consisting ofPd2(dba)3, Pd2pmdba)3, PdCh, Pd(TFA)2, and Na2PdCl4.

[0061] In some embodiments, the protecting group comprises.

[0062] In some embodiments, the protecting group comprises

[0063] The present disclosure includes a method of preparing a polynucleotide comprising treating a scarred nucleobase comprising an allyl group with a transition metyl catalyst and, optionally, one or more suitable ligand. In some embodiments, a suitable ligand isP(PhSO3Na)3. Without being bound by any particular theory, treatment of a scarred nucleobase comprising an allyl group with a suitable transition metal catalyst results in catalytic deallylation:

[0064] In some embodiments, a conjugate or nucleotide comprising an allyl group can be prepared as outlined in Scheme 4:Page 14 of 26IPTS / 200089493.1Docket No.: ABB-018WOScheme 4

[0065] In some embodiments, a conjugate comprising a linker with a removable scar is bound to a nitrogen on the nucleobase of the nucleotide. In some embodiments, a scar or a protecting group is bount to a nitrogen on the nucleobase of the nucleotide. In some embodiments, the nitrogen is a base pairing nitrogen on the nucleobase.

[0066] In some embodiments, the linker, scar or protecting group is bound to the N1 or N2 nitrogen of guanine, the N4 nitrogen of cytosine, or the N6 nitrogen of adenine. Examples of chemically removeable scars or protecting groups are described below:

[0067] In some embodiments, the allylic moiety is bound to the nucleobase via a carbamate, which is attached to a nitrogen of the nucleobase.

[0068] As described above, the substrate scope of the Pd-based scar deprotection approach is quite broad and enables a diversity of scar designs to be tested for varying biochemical and biophysical properties, as was demonstrated using the A112 scar to resolve the aggregation issue observed with allyl-T. Many other scar designs would be compatible with thisPage 15 of 26IPTS / 200089493.1Docket No.: ABB-018WO deprotection approach, not only allylic structures, but also propargylic, benzylic, and any substituted variation thereof.

[0069] Some embodiments of structures comprising protecting groups compatible with the Tsuji-Trost inspired deprotection approach using palladium are shown below:

[0070] Such protecting groups may be optionally substituted while still retaining the capability of being removed by a palladium reagent. Generalized structures including optionally substituted R groups that may be compatible with the Tsuji-Trost inspired deprotection approach using palladium are shown below:Page 16 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0071] Furthermore, the removeable protecting group may be indirectly bound to the nucleobase via a linker or spacer. Generalized structures above where R1 represents allylic, propargylic, and benzylic groups and their substituted derivatives thereof are shown below:Conjugates

[0072] In some embodiments, the present disclosure includes use of TdT with free nucleotides that have a 3' modification to enable controlled cyclic oligo synthesis. In some embodiments, the present disclosure also includes use of TdT with a tethered nucleotide (we call this polymerase-nucleotide conjugate). Linkage of the dNTP can occur via a tether to the nucleobase. Further description of polymerase-nucleotide conjugates can be found, e.g., in PCT Publication WO2017 / 223517 “Nucleic Acid Synthesis and Sequencing Using Tethered Nucleoside Triphosphates,” the entirety of which is incorporated by reference.

[0073] In some embodiments, the conjugates comprise the polymerase Terminal deoxynucleotidyl Transferase (TdT). In other embodiments, the method may employ conjugates comprising another template-independent polymerase.

[0074] In some embodiments, a process for the stepwise synthesis of a defined sequence uses a template-independent polymerase. A nucleic acid that serves as an initial substrate for elongation (i.e. "starter molecule") is incubated with a first polymerase-nucleotide conjugate. Once the nucleic acid has been elongated by the tethered nucleotide of a conjugate, no further elongations occur because the conjugates implement a termination mechanism. In the second step of the process, the linker is cleaved to release the polymerase and reverse the termination mechanism, thus enabling subsequent elongations. The elongation products are then exposed to the second conjugate, and these two steps are iterated to elongate the nucleic acid by a defined sequence. In some embodiments, synthesis is performed using a conjugate comprising TdT and a photocleavable linker. As described above, other strategies are available for the attachment and cleavage of the linker.

[0075] For DNA synthesis applications, in particular template- independent polymerases, i.e., a terminal deoxynucleotidyl transferase or DNA nucleotidylexo transferase, which termsPage 17 of 26IPTS / 200089493.1Docket No.: ABB-018WO are used interchangeably to refer to an enzyme having activity as described for E.C. class 2.7.7.31 may be used.

[0076] Polymerases with the ability to extend single stranded nucleic acids include, but are not limited to, Polymerase Theta (Kent et al., eLife 5 (2016): el3740.), 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).

[0077] For RNA synthesis applications, tethered ribonucleotides may be used. In these embodiments, an 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 ribonucleotide may be used to induce a high effective concentration of the nucleotide, thereby forcing incorporation of an rNTP that might not be the natural substrate of the nucleotidyl transferase.

[0078] In some embodiments, the linker is specifically attached to an amino acid of the polymerase. In these cases, it is preferable to attach the linker to an amino acid at a position that can be mutated without loss of the polymerase activity, e.g. positions 180, 188, 253 or 302 of murine TdT (numbering as in the crystal structure PDB ID: 4127). It is preferable to not attach the linker to an amino acid involved in the catalytic activity of the polymerase to avoid interfering with catalysis. Residues known to be involved with catalysis and methods for determining if a residue is involved with catalysis (e.g. by site-specific mutagenesis) will be apparent to those skilled in the art and are reviewed in literature (e.g. Joyce et al. (Journal of Bacteriology 177.22 (1995): 6321.) and Jara and Martinez (The Journal of Physical Chemistry B 120.27 (2016): 6504-6514.))

[0079] In some embodiments, a linker of a conjugate may be attached to an oxygen or nitrogen of the nucleobase. In particular embodiments, the linker of a conjugate attached to an oxygen or nitrogen of the nucleobase is cleaved to leave a scar, which can also be a protecting group that inhibits secondary structure formation. In some embodiments, a scar isPage 18 of 26IPTS / 200089493.1Docket No.: ABB-018WO removed using a chemical or photolytic condition capable of removing said one or more protecting groups from a protected nucleobase.

[0080] In some embodiments, a nucleotide is a ribose polyphosphate. In some embodiments, a ribose polyphosphate is selected from the group consisting of ribose triphosphate, ribose tetraphosphate, ribose pentaphosphate, and ribose hexaphosphate. In some embodiments, a ribose polyphosphate is a ribose triphosphate. In some embodiments, a ribose polyphosphate is a ribose hexaphosphate. In some embodiments, ribose polyphosphate is a ribose pentaphosphate. In some embodiments, ribose polyphosphate is a ribose tetraphosphate.

[0081] In some embodiments, a nucleotide is a deoxyribose polyphosphate. In some embodiments, a deoxyribose polyphosphate is selected from the group consisting of deoxyribose triphosphate, deoxyribose tetraphosphate, deoxyribose pentaphosphate, and deoxyribose hexaphosphate. In some embodiments, a deoxyribose polyphosphate is a ribose triphosphate. In some embodiments, a deoxyribose polyphosphate is a deoxyribose hexaphosphate. In some embodiments, deoxyribose polyphosphate is a deoxyribose pentaphosphate. In some embodiments, deoxyribose polyphosphate is a deoxyribose tetraphosphate.Equivalents and Scope

[0082] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0083] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.Page 19 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0084] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.

[0085] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0086] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0087] Section and table headings are not intended to be limiting.EXAMPLES

[0088] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0089] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B( 1992).Page 20 of 26IPTS / 200089493.1Docket No.: ABB-018WOExample 1 : Allyl Scar Removal

[0090] This example describes an exemplary method for removing a scar comprising an allyl group from nucleobases.

[0091] Stock solutions of NaiPdCE (6 mM), Triphenylphosphine-3,3’,3”-trisulfonic acid trisodium salt (60 mM), and dN-allyl-OH (10 mM for allyl nucleosides or 100 nM for allyl protected DNA oligomer) were prepared. The components were mixed together to a final target concentration of 0.3 mM NaiPdCE. 30 mM Triphenylphosphine-3,3’,3”-trisulfonic acid trisodium salt, and 1 mM allyl nucleoside or 10 nM allyl oligomer. The mixture was incubated at 50 °C for 1 hour before analysis on SQ LCMS (for nucleosides) or CE (for oligos).

[0092] The following nucleotides comprising an allylic moiety bound to the nucleobase were tested• dG-allyl (FIG. 2A),• dG-allyl-OH (FIG. 2B), and• dT-allyl-OH (FIG. 2C).

[0093] Exemplary results are shown in FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 3. FIGS. 2A, 2B and 2C show a shift of the peak to the left after the reaction, indicating successful removal of the allyl scar from the nucleotide. FIG. 3 shows the capillary electrophoresis peaks resulting from a polynucleotide comprising an allyl scar and treated for 15, 30, 45, or 60 minutes with palladium (bottom row). The peak shifts to the native G nucleotide without the allyl scar over time with palladium treatment.Example 2: Allyl Scar Removal

[0094] A solution of lOOmM DEEA at pH 9.6 was prepared. To this solution was added THP (lOOmM final concentration) in Tris pH 9 (lOmM final concentration).Allylpalladium(II) chloride dimer was then added (lOmM final concentration) and the resulting solution was vigorously vortexed and sonicated multiple times until all solid has been fully dissolved. Sodium ascorbate (lOmM final concentration), NaCl (IM final concentration), and Tween 20 (0.1% v / v final concentration) were then added to the solution. This final mixture may be referred to as Pd reagent. To deprotect immobilized oligonucleotide, Pd reagent is added to the surface and incubated at 28°C for Ihr. The Pd reagent is then removed, and the surface is thoroughly washed with a suitable wash buffer before elution of the deprotected oligonucleotide off of the surface.Page 21 of 26IPTS / 200089493.1Docket No.: ABB-018WO

[0095] FIG. 4 shows CE data from a proof of concept Pd deprotection of a FAM-labeled ssDNA substrate with a single allyl scarred G or T nucleotide at the 3’ end. In this example, the scarred oligo is mixed with IX Pd reagent and incubated at 27.5° C for 3 minutes before diluting 1:10 into formamide to quench and for CE analysis. The shift in retention time between the samples that received the Pd reagent versus the samples that did not demonstrates the complete removal of the allyl scar.Example 3: Allyl scar removal

[0096] FIG. 5 shows the structures of possible alternative scar designs for Pd deprotection.

[0097] FIG. 6 shows the impact of the Pd deprotectable scar design on oligonucleotide aggregation at longer oligo synthesis lengths. This is CE data of a FAM-labeled homopolymer that has been synthesized at varying lengths from 25nt up to 400nt using conjugates prepared with 4 different linker nucleotide designs bearing scars that can be removed with the Pd reagent. It is observed that for allyl T, allyl U, and Prg U the signal on CE decreases significantly then disappears as the length of the oligo increases, but the CE signal returns at the appropriate length upon deprotection with the Pd reagent, indicating that the loss of CE signal is caused by the presence of the scar and is presumed to be due to oligonucleotide aggregation interfering with CE analysis. Homopolymers prepared with the A112 conjugate, however, do not show this effect and give a strong CE signal both before and after deprotection with the Pd reagent.Example 4: Deprotection of A112-T nucleotide

[0098] FIG. 7 shows CE data from a Pd deprotection of the A112-T scar design. A FAM- labeled ssDNA substrate with a single A112-scarred T nucleotide at the 3’ end is treated with the Pd reagent at 28°C for 1 min. It can be seen that after 1 min, the peak corresponding to the starting material has been fully consumed and has shifted to an apparently smaller peak that corresponds to the deprotected T nucleotide at the 3’ end, indicating complete deprotection of the A112-T scar.Example 5: Synthesis error rate using allyl-scarred nucleotides in a polynucleotide

[0099] FIG. 8 shows the structure of various scarred T nucleotides.

[0100] FIG. 9 is generated from next generation deep sequencing data of a lOOOmer oligonucleotide synthesis utilizing either OMe-U, Allyl-T, or A112-T as the T conjugate. The upon completion of the lOOOmer synthesis, the oligos containing Allyl-T and A112-T were deprotected using the Pd reagent. Oligos were then amplified by PCR and sequenced. ThePage 22 of 26IPTS / 200089493.1Docket No.: ABB-018WO plots here show the resulting error rates (plotted as SWY = 1 - error rate) for each error type as a function of synthesis cycle, or synth_period, where each period represents 200 cycles (i.e. synth_period 1 = cycle 1 to 200, 2 = cycles 201 to 400, and so on). Each line represents data collected from oligos synthesized with the varying T conjugates (solid = OMe-U, dashed = allyl-T, dotted = A112-T). The error rate of synthesis with OMe-U is relatively stable from 0 to lOOOnt. However, the error rate increases dramatically past 400nt when Allyl-T conjugate is used during synthesis, primarily due to an increase in deletion rate. The A112-T conjugate, on the other hand, displays a similarly stable error rate with length as with OMe-U.OTHER EMBODIMENTS

[0101] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0102] While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the present disclosure.

[0103] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.Page 23 of 26IPTS / 200089493.1

Claims

Docket No.: ABB-018WOCLAIMS1. A method of synthesizing a polynucleotide, comprising: providing a synthesized polynucleotide comprising one or more allylic moieties removable under palladium-catalyzed conditions, wherein said one or more allylic moieties are each bound directly or indirectly to a hydrogen basepairing heteroatom on a nucleobase of the polynucleotide; and treating said synthesized polynucleotide with a palladium reagent to remove the one or more allylic moieties from said one or more nucleobases.

2. The method of claim 1, wherein providing said synthesized polynucleotide comprises: contacting a precursor polynucleotide with a polymerase and a nucleotide comprising said allylic moiety; and adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase.

3. The method of claim 1, wherein providing said synthesized polynucleotide comprises: contacting a precursor polynucleotide with a conjugate comprising a nucleotide covalently linked to a polymerase via a linker comprising said allylic moiety; adding said nucleotide to the 3' end of said precursor polynucleotide via said polymerase; and cleaving the linker, wherein said allylic moiety remains bound to the added nucleotide.

4. The method of any one of claims 2-3, further comprising repeating said contacting, adding, and optionally said cleaving steps one or more times to generate said polynucleotide.

5. The method of any one of claims 2-4, wherein said polymerase is a template-independent polymerase.

6. The method of claim 5, wherein the polymerase is TdT.

7. The method of claim 2, wherein the nucleotide comprises a 2' or 3' modification.

8. The method of any one of claims 2-7, wherein the synthesized polynucleotide comprises at least 10, at least 20, at least 50, or at least 100 of said allylic moieties bound to said nucleobases.Page 24 of 26IPTS / 200089493.1Docket No.: ABB-018WO9. The method of any one of claims 1-8, wherein at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the allylic moieties are removed by said treatment with said palladium reagent.

10. A polynucleotide comprising one or more allylic moieties removable under palladium- catalyzed conditions, wherein said one or more allylic moieties are each bound directly or indirectly to a hydrogen base-pairing heteroatom on a nucleobase of the polynucleotide.

11. The composition of claim 10, wherein the polynucleotide comprises at least 10, at least 20, at least 50, or at least 100 of said allylic moieties bound to said nucleobases.

12. A nucleotide comprising an allylic moiety removable under palladium-catalyzed conditions, wherein the allylic moiety is bound directly or indirectly to a hydrogen base-pairing heteroatom on the nucleobase of the polynucleotide.Page 25 of 26IPTS / 200089493.1

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