Novel methods for the production of polynucleotides (including oligonucleotides)

Abstract

The present invention relates to novel methods for the production of polynucleotides or oligonucleotides using enzymes, which are suitable for use in producing modified polynucleotides or oligonucleotides, such as for use in therapy.

Description

[Technical field]

[0001] The present invention relates to novel methods for the production of polynucleotides (including oligonucleotides) using polymerases and ligases, which are suitable for use in producing modified polynucleotides (including modified oligonucleotides), such as for use in therapy. [Background technology]

[0002] Chemical synthesis of polynucleotides and modified polynucleotides, particularly oligonucleotides and modified oligonucleotides, for example via phosphoramidite chemistry, is well established and has been the method of choice for synthesizing these defined sequence biopolymers for decades. This synthesis process is usually carried out as a solid-supported synthesis (commonly referred to as solid-phase synthesis), whereby single nucleotides are added sequentially, with each nucleotide addition requiring several cycles of chemical steps to add and deprotect the growing oligonucleotide ("oligo") in preparation for the subsequent step. At the end of the sequential addition of nucleotides, the oligo is released from the solid-phase support, further deprotection is performed, and the crude oligonucleotide is then further purified by column chromatography.

[0003] Although this method may be considered conventional and can be automated, this methodology has some drawbacks, especially when the goal is to prepare oligonucleotides and polynucleotides on a large scale, as may be necessary for oligonucleotide therapeutics, such as antisense molecules, including gapmers, siRNAs, miRNAs and aptamers, and polynucleotide therapeutics, such as therapeutic mRNAs.These drawbacks include the limitations of scaling up solid support chemistry, which limits batch size, and the practical limitations of using chromatography to purify large amounts of oligonucleotide.These limitations make scaling up expensive and time-consuming, and require multiple rounds of synthesis.In addition, errors accumulate with the length of the oligonucleotide or polynucleotide that is synthesized, which imposes further practical limitations on the scaling up of longer oligonucleotide and polynucleotide products.

[0004] Thus, there is a need to both reduce or ideally eliminate both chemical and solid-supported synthesis to generate oligonucleotides and longer polynucleotides while minimizing errors in sequence, and to perform synthesis efficiently, in a cost-effective manner, and that can be operated on a larger scale.

[0005] WO2018 / 011067 discloses a novel ligation method in which a pool of oligonucleotides is ligated together in a directed manner using complementary templates and ligation methods. WO2019 / 121500 discloses a novel method of using enzymes, particularly single-stranded ligases and transferases, in the production of modified oligonucleotides that can be used in ligation methods.

[0006] The method for adding each nucleotide to a chain using single strand ligase and transferase is complicated and must be performed sequentially with one nucleotide added at a time, and a deprotection step is required in each round to facilitate addition, which is time consuming.The individual oligonucleotides in the pool must then be ligated to each other in the correct orientation using a template to create the final oligonucleotide product.Scaling up this method to larger polynucleotides means that it is necessary to create multiple short oligonucleotides by adding one nucleotide at a time, which then must be ligated in the correct orientation to create the final product.In essence, in the creation of smaller oligonucleotides that are ligated to create the final product, every single nucleotide of the final product must be added one at a time, which reduces efficiency and requires a long production process.Therefore, there is a higher risk of introducing errors.Therefore, there is a need to provide alternative and / or improved methods that reduce complexity to increase the efficiency of the creation of oligonucleotides and larger polynucleotides while minimizing errors in the product sequence. Summary of the Invention

[0007] In a first aspect of the present invention there is provided a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising the steps of: a) contacting a template polynucleotide comprising a sequence complementary to a single stranded polynucleotide product with a pool of at least two segment polynucleotides under conditions that allow annealing of the at least two segment polynucleotides to the template polynucleotide to produce a template polynucleotide having at least two annealed segment polynucleotides, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; b) extending at least one of the annealed segment polynucleotides using the pool of nucleoside triphosphates and a polymerase to fill in at least one sequence gap and generate at least one extended segment polynucleotide; c) ligating one or more segment polynucleotide(s) and / or one or more extended segment polynucleotide(s) using a ligase to form a single-stranded polynucleotide product that is linked in a duplex fashion to the template polynucleotide; and d) changing the conditions to denature the duplex comprising the single-stranded polynucleotide product and the template polynucleotide, thereby producing a single-stranded polynucleotide product. A method is provided, comprising:

[0008] In a second aspect of the invention, there is provided a method for making a double-stranded polynucleotide product comprising annealing two complementary single-stranded polynucleotide products, at least one of which was made by a method for making a single-stranded polynucleotide disclosed herein, and optionally both of which were made by a method for making a single-stranded polynucleotide disclosed herein.

[0009] In a third aspect of the invention there is provided a method for producing a double stranded polynucleotide product comprising the steps of: a) contacting a template polynucleotide comprising a sequence complementary to a single stranded polynucleotide product with a pool of at least two segment polynucleotides under conditions that allow annealing of the at least two segment polynucleotides to the template polynucleotide to produce a template polynucleotide having at least two annealed segment polynucleotides, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; b) extending at least one of the annealed segment polynucleotides using the pool of nucleoside triphosphates and a polymerase to fill in at least one sequence gap to generate at least one extended segment polynucleotide; c) ligating one or more segment polynucleotide(s) and / or one or more extended segment polynucleotide(s) using a ligase to form a single-stranded polynucleotide product linked in a duplex fashion to the template polynucleotide; d) changing conditions to denature the duplex comprising the single-stranded polynucleotide product and the template polynucleotide, thereby producing a single-stranded polynucleotide product; and e) using the single-stranded polynucleotide product as a template polynucleotide in step a) and repeating steps a) to c) to generate a double-stranded polynucleotide product. A method is provided, comprising: [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic example showing the generation of an oligonucleotide or polynucleotide product using a polymerase, including ligating segment oligonucleotides or polynucleotides to form a product and altering conditions to remove impurities. [Diagram 2] FIG. 2 is a schematic example of a multiple template configuration. [Figure 3a] FIG. 3a shows an HPLC trace showing the gap-filling reaction starting materials: one template and two segment oligonucleotides (Primer N, which acts as a primer for the polymerase, and 3′ Block 1, which acts as a stopper). [Figure 3b] Figure 3b shows an HPLC trace showing product formation after a gap-filling reaction. Primer N+5 shows successful extension of primer N using polymerase and nucleoside triphosphates to fill a 5 bp gap. [Figure 4] FIG. 4 is a schematic example showing a template with an annealed 5′ primer and a template with a hairpin loop acting as a 5′ primer separately. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] definition As used herein, "polynucleotide" refers to a polymer of two or more nucleotide monomers, i.e., nucleotide residues, connected to each other via covalent bonds. A single polynucleotide molecule can, for example, contain 14 or more nucleotide monomers in a chain structure. DNA and RNA are examples of polynucleotides. Polynucleotides include oligonucleotides. Polynucleotides can contain an infinite number of nucleotides. Polynucleotides are useful in therapy, for example, in the creation of therapeutic mRNA (which can be used as mRNA vaccines), antisense oligonucleotides, siRNA, miRNA, aptamers, CRISPR guide RNA, and oligonucleotides that recruit and guide DNA and RNA editing enzymes, such as A-to-I RNA base editing oligonucleotides (AIMer).

[0012] As used herein, the term "therapeutic polynucleotide" refers to a polynucleotide that has therapeutic use, for example, in the prevention or treatment of a human or animal condition or disease. Such polynucleotides typically contain one or more modified nucleotide residues or linkages. Therapeutic polynucleotides act through one of several different mechanisms, including, but not limited to, antisense, splice switching or exon skipping, immunostimulation, RNA interference (RNAi) via microRNA (miRNA) and small interfering RNA (siRNA), and the like, and the recruitment and induction of DNA and RNA editing enzymes. Therapeutic polynucleotides may be aptamers. Therapeutic polynucleotides usually, but not always, have a defined sequence. Therapeutic polynucleotides include therapeutic oligonucleotides.

[0013] The terms "polynucleotide" and "therapeutic polynucleotide" encompass "oligonucleotides" and "therapeutic oligonucleotides," respectively.

[0014] As used herein, the term "oligonucleotide", or "oligo" for short, refers to a polymer of nucleotide residues. The term "oligonucleotide" is usually used for polynucleotide sequences that are shorter than the term "polynucleotide", generally ranging from 3 to 30 nucleotides. These can be deoxyribonucleotides (the resulting oligonucleotide is DNA), ribonucleotides (the resulting oligonucleotide is RNA), modified nucleotides, or mixtures thereof.

[0015] A polynucleotide or oligonucleotide may be composed entirely of nucleotide residues found in nature (i.e., "natural nucleotides" or "naturally occurring nucleotides"), or may contain at least one modified nucleotide, or at least one modified linkage between nucleotides. Examples of naturally occurring nucleotides include deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, deoxyuridine monophosphate, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, thymidine monophosphate, and uridine monophosphate. A modified nucleotide is not a naturally occurring nucleotide (i.e., it is a non-natural nucleotide). A modified nucleotide may be a modified, e.g., chemically modified, naturally occurring nucleotide. A modified nucleotide may include a modified backbone, sugar, and / or nucleobase. It is recognized that certain modifications, such as 2'OMe or C5 pyrimidine modifications, occur sporadically in nature, i.e., naturally occurring nucleotides, but are considered modified nucleotides in this disclosure. A polynucleotide (including an oligonucleotide) may be single-stranded or double-stranded. A polynucleotide or oligonucleotide of the disclosure may be conjugated to another molecule, such as N-acetylgalactosamine (GalNAc) or a plurality thereof (GalNAc cluster).

[0016] As used herein, the term "therapeutic oligonucleotide" refers to an oligonucleotide that has therapeutic use, for example, in the prevention or treatment of a human or animal condition or disease. Such oligonucleotides typically contain one or more modified nucleotide residues or linkages. Therapeutic oligonucleotides act through one of several different mechanisms, including, but not limited to, antisense, splice switching or exon skipping, immunostimulation, and RNA interference (RNAi) via microRNA (miRNA) and small interfering RNA (siRNA), etc. Therapeutic oligonucleotides may be aptamers. Therapeutic oligonucleotides usually, but not always, have a defined sequence. Therapeutic oligonucleotides are an example of a therapeutic polynucleotide.

[0017] As used herein, the term "template" refers to a polynucleotide or oligonucleotide that comprises a sequence that is complementary to a single-stranded polynucleotide or oligonucleotide product. The template may comprise a sequence that is 100% complementary to the sequence of the target (or product) polynucleotide or oligonucleotide. The template may consist of a sequence that is 100% complementary to the sequence of the target (or product) polynucleotide or oligonucleotide. The template may be a longer sequence compared to the product sequence. The template may include a sequence that is not used to generate the polynucleotide or oligonucleotide product. If the template is longer in terms of sequence compared to the product sequence, a stopper may be used to control the generation and / or length of the polynucleotide or oligonucleotide product. If the template is longer in terms of sequence compared to the product sequence, a primer may be used to control the generation and / or length of the polynucleotide or oligonucleotide product. If the template is longer in terms of sequence compared to the product sequence, it may include a hairpin loop. The hairpin loop may act as a primer. The template may include one of the segments in a hairpin loop. In such cases, the product can be released from the template through cleavage using nuclease, nickase, DNA enzyme, or chemical methods. The template can be a shorter sequence compared to the product sequence. If the template is a shorter sequence compared to the product sequence, at least one segment will protrude from the template when the segment is annealed to the template. The template can include or consist of a sequence that is less than 100% complementary. The template sequence can be such that each complementary nucleotide is complementary to the unmodified form of the modified nucleotide in the target sequence. Unless otherwise specified, as used herein, the term "complementary" means 100% complementary.

[0018] As used herein, the term "product" refers to a desired polynucleotide or oligonucleotide having a particular sequence and set of modifications, also referred to herein as a "target polynucleotide" or "target oligonucleotide." "Product sequence" is used interchangeably with "target polynucleotide sequence" and "target oligonucleotide sequence" and refers to the base sequence of the product.

[0019] As used herein, the term "pool of polynucleotides or oligonucleotides" refers to a group of polynucleotides or oligonucleotides, respectively, that may vary in sequence, may be shorter than the target sequence, and may not have the same sequence as the target sequence. The pool of polynucleotides or oligonucleotides may be the product of polynucleotide or oligonucleotide synthesis. The pool of polynucleotides or oligonucleotides may include at least two segment polynucleotides or oligonucleotides. The at least two segment polynucleotides or oligonucleotides may not be random (i.e., not a group of randomly selected segment polynucleotides or oligonucleotides, but specifically designed and selected for the purpose of forming a final polynucleotide product according to the method of the present invention). The at least two segment polynucleotides or oligonucleotides may be segments of a polynucleotide or oligonucleotide product. The at least two segment polynucleotides or oligonucleotides may differ in sequence. The pool of polynucleotides or oligonucleotides may include a segment of a product sequence. The pool of polynucleotides or oligonucleotides may consist of a segment of a product sequence. The pool of polynucleotides or oligonucleotides may be engineered to specifically include a segment of a polynucleotide or oligonucleotide product. At least one segment of a product sequence may contain at least one modified nucleotide residue. The pool of polynucleotides or oligonucleotides can be the product of polynucleotide or oligonucleotide synthesis using polymerase, such as DNA polymerase or RNA polymerase.One or more or all of the segment polynucleotides or oligonucleotides can be made using chemical synthesis, such as solid-supported or liquid-phase synthesis, for example, via phosphoramidite chemistry, or can be made using enzymatic synthesis, or a combination thereof.Enzymatic synthesis can involve the use of polymerase, single-stranded ligase and / or transferase, or a combination thereof.The pool of polynucleotides or oligonucleotides can be the product of polynucleotide or oligonucleotide synthesis using chemical synthesis, such as via phosphoramidite chemistry, e.g., solid-supported or solution-phase synthesis, or using enzymatic synthesis or a combination thereof. Enzymatic synthesis can involve the use of polymerases, single-stranded ligases and / or transferases, or a combination thereof.

[0020] As used herein, the term "annealing" refers to the hybridization of complementary polynucleotides or oligonucleotides in a sequence-specific manner, e.g., the pairing of two single-stranded polynucleotides or oligonucleotides, via hydrogen bonds of Watson and Crick base pairing to form a double-stranded polynucleotide or oligonucleotide ("duplex"). "Conditions that allow annealing" refer to the T of the hybridized complementary polynucleotides or oligonucleotides. m For example, the temperature for annealing will depend on the T of the polynucleotides or oligonucleotides to be hybridized, as will be readily apparent to one of skill in the art. m Alternatively, the temperature for annealing can be less than the T m The temperature for annealing can be within the vicinity, e.g., + / - 1, 2 or 3°C. The temperature for annealing is generally selected from the T m It is not more than 10°C higher than that.

[0021] As used herein, the term "denaturing" in the context of double-stranded polynucleotides or oligonucleotides is used to mean that the complementary strands are no longer annealed, i.e., Watson and Crick base pairing is broken and the strands are dissociated. Denaturing is performed as a result of changing conditions, for example, by increasing temperature, changing pH, or changing the salt concentration of a buffer solution. Conditions for denaturing are well known to those skilled in the art. Denaturing a double-stranded polynucleotide or oligonucleotide as described herein (i.e., denaturing the duplex) results in a single-stranded product, or impurity polynucleotide or oligonucleotide, and a single-stranded template polynucleotide or oligonucleotide.

[0022] As used herein, the term "impurity" or "impurities" refers to polynucleotides or oligonucleotides that do not have the desired product sequence. These polynucleotides or oligonucleotides may include polynucleotides or oligonucleotides that are shorter than the product (e.g., 1, 2, 3, 4, or 5 nucleotide residues or more shorter) or longer than the product (e.g., 1, 2, 3, 4, or 5 nucleotide residues or more longer). When the production method includes a step of forming linkages between segments, the impurities include polynucleotides or oligonucleotides that remain when one or more linkages cannot be formed. The impurities also include polynucleotides or oligonucleotides that incorporate incorrect nucleotides, resulting in mismatches when compared to the template. The impurities may have one or more of the characteristics described above. The terms "impurity" and "impurity polynucleotide" are used interchangeably herein.

[0023] As used herein, the term "segment" refers to a smaller portion of a longer polynucleotide or oligonucleotide, particularly a smaller portion of a product or target polynucleotide or oligonucleotide. For a given product, when all of its segments are annealed to its template, gaps are filled by polymerase extension, and ligated together, a product is formed. A segment may act as a primer for polymerase. A segment may act as a stopper for polymerase. A segment may be part of a hairpin loop in the template that is later cut from the template after polymerase extension and becomes part of the product.

[0024] As used herein, the term "enzymatic ligation" means that a linkage between two adjacent nucleotides is formed enzymatically, i.e., by an enzyme. This linkage may be a naturally occurring phosphodiester bond (PO) or a modified linkage, including, but not limited to, phosphorothioate (PS), phosphoramidate (PA), or phosphorodithioate (PS2).

[0025] As used herein, the term "enzymatic synthesis" refers to the creation of polynucleotides and oligonucleotides, including segments and end products, using enzymes such as polymerases, ligases, transferases, phosphatases, and nucleases, such as endonucleases. These enzymes can be wild-type enzymes or mutant enzymes or engineered enzymes. Mutant or engineered enzymes that can act on modified nucleotide or oligonucleotide substrates are within the scope of this disclosure.

[0026] As used herein, the term "polymerase" refers to an enzyme that catalyzes the binding, i.e., covalent, of a nucleotide to the 3'-OH of another nucleotide or oligonucleotide or polynucleotide, for example, by forming a phosphodiester bond between the 3' end of one nucleotide or oligonucleotide or polynucleotide and the 5' end of another nucleotide. Thus, the polymerase activity is 5'→3'. Polymerases can include DNA and / or RNA polymerases. Polymerases can be wild-type enzymes, mutant enzymes, or engineered enzymes.

[0027] Those skilled in the art will understand that the "pool of nucleotides" used in this disclosure is a substrate for polymerase. Thus, the "pool of nucleotides" in this context refers to a pool of nucleoside triphosphates (NTPs) or their analogs that become nucleotides when incorporated into polynucleotide or oligonucleotide products. Thus, the terms "pool of nucleotides" and "pool of nucleoside triphosphates" are used interchangeably herein. Nucleoside triphosphates can be considered to be molecular precursors of both DNA and RNA. The pool of nucleoside triphosphates can include one or more of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, deoxyuridine triphosphate, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, thymidine triphosphate, and uridine triphosphate. The pool of nucleoside triphosphates may include one or more of modified deoxyadenosine triphosphate, modified deoxycytidine triphosphate, modified deoxyguanosine triphosphate, modified deoxythymidine triphosphate, modified deoxyuridine triphosphate, modified adenosine triphosphate, modified cytidine triphosphate, modified guanosine triphosphate, modified thymidine triphosphate, and modified uridine triphosphate. The pool of nucleoside triphosphates may include nucleoside triphosphate analogs. Examples of nucleoside alpha thiotriphosphates include 2'-deoxyadenosine-5'-(α-thio)-triphosphate, 2'-deoxycytidine-5'-(α-thio)-triphosphate, 2'-deoxyguanosine-5'-(α-thio)-triphosphate, 2'-deoxythymidine-(α-thio)-triphosphate, 2'-deoxyuridine-(α-thio)-triphosphate, 2'-adenosine-5'-(α-thio)-triphosphate, 2'-cytidine-5'-(α-thio)-triphosphate, 2'-guanosine-5'-(α-thio)-triphosphate, 2'-thymidine-(α-thio)-triphosphate, 2'-uridine-(α-thio)-triphosphate, and modified base variants thereof.The pool of nucleoside triphosphates was composed of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, deoxyuridine triphosphate, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, thymidine triphosphate, uridine triphosphate, modified deoxyadenosine triphosphate, modified deoxycytidine triphosphate, modified deoxyguanosine triphosphate, modified deoxythymidine triphosphate, modified deoxyuridine triphosphate, modified adenosine triphosphate, modified cytidine triphosphate, modified guanosine triphosphate, modified thymidine triphosphate, modified uridine triphosphate. , 2'-deoxyadenosine-5'-(α-thio)-triphosphate, 2'-deoxycytidine-5'-(α-thio)-triphosphate, 2'-deoxyguanosine-5'-(α-thio)-triphosphate, 2'-deoxythymidine-(α-thio)-triphosphate, 2'-deoxyuridine-(α-thio)-triphosphate, 2'-adenosine-5'-(α-thio)-triphosphate, 2'-cytidine-5'-(α-thio)-triphosphate, 2'-guanosine-5'-(α-thio)-triphosphate, 2'-thymidine-(α-thio)-triphosphate, and 2'-uridine-(α-thio)-triphosphate. The pool of nucleoside triphosphates may include one or more of adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, uridine triphosphate, modified adenosine triphosphate, modified cytidine triphosphate, modified guanosine triphosphate, modified uridine triphosphate, 2'-adenosine-5'-(α-thio)-triphosphate, 2'-cytidine-5'-(α-thio)-triphosphate, 2'-guanosine-5'-(α-thio)-triphosphate, and 2'-uridine-(α-thio)-triphosphate. The pool of nucleoside triphosphates can include one or more of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, modified deoxycytidine triphosphate, modified deoxyguanosine triphosphate, modified deoxythymidine triphosphate, 2'-deoxyadenosine-5'-(α-thio)-triphosphate, 2'-deoxycytidine-5'-(α-thio)-triphosphate, 2'-deoxyguanosine-5'-(α-thio)-triphosphate, and 2'-deoxythymidine-(α-thio)-triphosphate.The pool of nucleoside triphosphates may include adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and modified uridine triphosphate. The pool of nucleoside triphosphates may include adenosine triphosphate, cytidine triphosphate, guanosine triphosphate, and N1-methyl-pseudouridine triphosphate. The pool of nucleoside triphosphates may include 2'-adenosine-5'-(α-thio)-triphosphate, 2'-cytidine-5'-(α-thio)-triphosphate, 2'-guanosine-5'-(α-thio)-triphosphate, and 2'-modified uridine-(α-thio)-triphosphate.

[0028] Polymerases capable of binding unmodified nucleotides to other unmodified nucleotides, polymerases capable of binding unmodified nucleotides to modified nucleotides (i.e., modified 5' nucleotides to unmodified 3' nucleotides and / or unmodified 5' nucleotides to modified 3' nucleotides), and polymerases capable of binding modified nucleotides to other modified nucleotides are within the scope of the present disclosure. Optionally, the polymerase can bind unmodified nucleotides to other unmodified nucleotides. The unmodified nucleotides can then be modified. Examples of modifications of nucleotides are disclosed herein and include modifications selected from the group including sugar moiety modifications, nucleobase modifications, and backbone modifications.

[0029] The modification may be at the 2' position of the sugar moiety, optionally selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE, and 2'-amino. The oligonucleotide may comprise PMO, LNA, c-Et, PNA, BNA, or L-ribonucleic acid. The modification may be at the nucleobase, optionally selected from the group consisting of 5-methylpyrimidine, 7-deazaguanosine, and abasic nucleotides. The modification may be at the backbone, optionally selected from the group consisting of phosphorothioate, phosphorodithioate, phosphoroamidate, and phosphorodiamidate.

[0030] In addition, exemplary engineered DNA and RNA polymerases that can incorporate modified nucleotides include those disclosed in "Engineering and application of polymerases for synthetic genetics," Houlihan et al., Current Opinion in Biotechnology 2017, 48;168-179. For example, DNA and RNA polymerases can be engineered to accept 2' sugar modifications, including polymerases with mutations in the polymerase thumb subdomain of Thermococcus gogonarius (Tgo) replicative DNA polymerase, optionally including E664K and Y409G mutations. Such polymerases provide for the inclusion of pseudouridine, 5-methyl-C, 2'-fluoro, or 2-azido modified NTPs, or combinations thereof, primed from, for example, DNA, RNA, locked nucleic acid, or 2'-OMe RNA modified nucleotides.

[0031] Further exemplary RNA polymerases engineered to accept 2' sugar modifications include T7 RNA polymerase. For example, T7 RNA polymerase containing the Y639F mutation can facilitate the inclusion of, for example, 2' fluoropyrimidines and 2' aminopyrimidines.

[0032] A variant of the Stoffel fragment of Taq polymerase (SM19) can be engineered to accept 2' sugar modifications. For example, the introduction of a negatively charged amino acid at position 614 and the mutation E615G achieves the inclusion of 2' sugar modifications. SM19 can be further evolved into polymerases SFM4-3 and SFM4-9. For example, SFM4-3 can transcribe a fully modified 2'OMe 60 nucleotide sequence.

[0033] The thermophilic RNA polymerase from the marine cyanophage Syn5 can be engineered to accept 2' sugar modifications.

[0034] Tgo polymerase containing the Y409G, I521L, F545L, and E664K mutations can synthesize DNA and RNA with regioisomeric 2'-5' linkages by incorporation of 3'deoxy or 3'OMe nucleotides.

[0035] As used herein, the term "ligase" refers to an enzyme that catalyzes the joining, i.e., covalent bonding, of two polynucleotide or oligonucleotide molecules, for example by the formation of a phosphodiester bond between the 3' end of one polynucleotide or oligonucleotide (or segment) and the 5' end of the same or another polynucleotide or oligonucleotide (or segment). These enzymes are often referred to as DNA ligases or RNA ligases and utilize a cofactor: ATP (eukaryotic, viral and archaeal DNA ligases) or NAD (prokaryotic DNA ligases). Despite their occurrence in all organisms, DNA ligases exhibit a wide variety of amino acid sequences, molecular sizes and properties (Nucleic Acids Research, 2000, Vol. 28, No. 21, 4051-4058). They are usually members of the enzyme class EC6.5 defined by the International Union of Biochemistry and Molecular Biology, i.e., ligases used to form phosphate ester bonds. Ligases capable of joining an unmodified polynucleotide or oligonucleotide to another unmodified polynucleotide or oligonucleotide, an unmodified polynucleotide or oligonucleotide to a modified polynucleotide or oligonucleotide (i.e., a modified 5' polynucleotide or oligonucleotide to an unmodified 3' polynucleotide or oligonucleotide and / or an unmodified 5' polynucleotide or oligonucleotide to a modified 3' polynucleotide or oligonucleotide), and a modified polynucleotide or oligonucleotide to another modified polynucleotide or oligonucleotide are within the scope of this disclosure.

[0036] As used herein, the term "single-stranded ligase" or "ss ligase" refers to an enzyme, e.g., an RNA ligase, that can catalyze the ATP-dependent ligation of (i) 5' phosphorylated single-stranded RNA to the 3'-OH of a single-stranded acceptor RNA strand, and (ii) the ligation of a single residue (including modified residues), e.g., nucleotide-3',5'-bisphosphate, 3',5'-bisthiophosphate, or 3'-phosphate-5'thiophosphate, to the 3' end of an RNA or modified polynucleotide or oligonucleotide (Modified Oligoribonucleotides: 17(11), 2077-2081, 1978). One example of an ss ligase is T4 RNA ligase, which has also been shown to function on DNA substrates under certain conditions (Nucleic Acids research 7(2), 453-464, 1979). The natural function of T4 RNA ligase in Escherichia coli infected with T4 bacteriophage is to repair single-strand breaks in bacterial tRNA caused by bacterial defense mechanisms against viral attack. ss ligases capable of joining unmodified nucleotides to unmodified polynucleotides or oligonucleotides, ss ligases capable of joining unmodified nucleotides to modified polynucleotides or oligonucleotides, ss ligases capable of joining modified nucleotides to unmodified polynucleotides or oligonucleotides, and ss ligases capable of joining modified nucleotides to modified polynucleotides or oligonucleotides are within the scope of the present disclosure. The ss ligase according to the present disclosure is a ligase that does not require a template polynucleotide or oligonucleotide for ligation to occur, i.e., the ligation activity of the ligase is template-independent.

[0037] As used herein, "junction nucleotide" refers to the nucleotide at the end of a polynucleotide or oligonucleotide that is connected to another polynucleotide or oligonucleotide.For example, when two segments, namely, a 5' segment and a 3' segment, are ligated together, the two junction nucleotides are 1) the nucleotide at the 3' end of the 5' segment and 2) the nucleotide at the 5' end of the 3' segment.

[0038] As used herein, "transferase" refers to an enzyme that catalyzes the non-template-dependent attachment of one nucleotide to another nucleotide or oligonucleotide. Transferases described herein include terminal nucleotidyl transferase (TdT), also known as DNA nucleotidyl exotransferase (DNTT) or terminal transferase. TdT is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells that allows for VDJ antibody gene junctional diversity. TdT catalyzes the addition of nucleotides to the 3' end of a DNA molecule. Transferases described herein include non-naturally occurring TdT or mutant TdT. Transferases capable of attaching unmodified nucleotides to unmodified oligonucleotides, transferases capable of attaching unmodified nucleotides to modified oligonucleotides, transferases capable of attaching modified nucleotides to unmodified oligonucleotides, and transferases capable of attaching modified nucleotides to modified oligonucleotides are within the scope of this disclosure.

[0039] As used herein, a "thermostable ligase," "thermostable polymerase," or "thermostable transferase" is a ligase, polymerase, or transferase, respectively, that is active at elevated temperatures, i.e., above human body temperature, i.e., above 37° C. A thermostable ligase, thermostable polymerase, or thermostable transferase can be active, for example, between 40° C. and 65° C., or between 40° C. and 90° C., etc.

[0040] As used herein, the term "primer" refers to a polynucleotide or oligonucleotide sequence used as a starting point for synthesizing a segmented polynucleotide or oligonucleotide of the present disclosure. A polymerase may require a primer, for example, a DNA polymerase may require a primer. A primer may comprise at least three nucleotides. It is within the scope of the present disclosure to use a segmented polynucleotide or oligonucleotide as a primer. At least one segmented polynucleotide or oligonucleotide may act as a primer. A primer may bind to a template before a polymerase catalyzes the binding of nucleotides. A primer may not be removed. A primer may form part of a polynucleotide or oligonucleotide template. For example, a template may include a hairpin loop that includes a primer. A primer may form part of a polynucleotide or oligonucleotide product.

[0041] As used herein, the term "stopper", also known as "block" or "3'-flanking oligonucleotide", refers to a polynucleotide or oligonucleotide sequence that stops or prevents a polymerase from binding additional nucleotides. The stopper may terminate polymerase elongation. The stopper, for example, one of at least two segment polynucleotides or oligonucleotides, may include a 5' phosphate, a 5' thiophosphate (which may produce a phosphorothioate bond), a 5' amidophosphate (which may produce a phosphoroamidate bond), a 5' diamidate phosphoric acid (which may produce a phosphorodiamidate bond), a 5' amidothiophosphate, a 5' amidodithiophosphate, a 5' diamidothiophosphate, or a 5' dithiophosphate (which may produce a phosphorodithioate bond). A 5' phosphate, 5' thiophosphate, 5' amidophosphate, 5' diamidophosphate, 5' dithiophosphate, 5' amidothiophosphate, 5' amidodithiophosphate, or 5' diamidothiophosphate may be required for ligation involving a ligase. One or more of the at least two segment polynucleotides or oligonucleotides may act as a stopper.

[0042] The methods of the present disclosure can be used to generate RNA and / or DNA (including modifications thereto), including non-replicating mRNA and self-amplifying RNA derived from viruses. Such RNA has utility, for example, in vaccine production.

[0043] The term "RNA" is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer made up of nucleotide monomers. These nucleotides are usually adenosine monophosphate, uridine monophosphate, guanosine monophosphate and cytidine monophosphate monomers or their analogs, which are connected to each other along a so-called backbone. The backbone is formed by a phosphodiester bond between the sugar, i.e. the ribose, of the first adjacent monomer and the phosphate moiety of the second adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar / phosphate backbone, is called the RNA sequence. The term "RNA" generally refers to a molecule or molecular species selected from the group consisting of long RNA, coding RNA, non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotide, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA, riboswitch, immunostimulatory RNA (isRNA), ribozyme, aptamer, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA). Optionally, any type of therapeutic RNA is within the context of this disclosure. "Therapeutic RNA" should be understood as referring to RNA suitable for use for medical purposes in the human or animal body, e.g., it has clinical grade, in particular with respect to parameters such as purity, integrity, and with respect to the underlying production method, which must comply with the conditions of current Good Manufacturing Practice (cGMP). Therapeutic RNA may have therapeutic applications, e.g., in the prevention or treatment of conditions or diseases.

[0044] The term "messenger RNA" (mRNA) refers to a type of RNA molecule. In vivo transcription of DNA usually results in so-called premature RNA, which must be processed into so-called messenger RNA, usually abbreviated as mRNA. The processing of premature RNA, for example in eukaryotes, includes a variety of different post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, export from the nucleus or mitochondria, etc. The totality of these processes is also called mRNA maturation. Mature mRNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mature mRNA includes a 5' cap, a 5' untranslated region (5' UTR), an open reading frame, a 3' untranslated region (3' UTR), and a homopolymeric tail, such as a polyA or polyC sequence. In the context of the present disclosure, mRNA can be an artificial molecule, i.e. a molecule that does not exist in nature. This means that an mRNA in the context of the present disclosure may contain a combination of a 5'UTR, an open reading frame, a 3'UTR and a polyA sequence that does not exist in that combination in nature.

[0045] Depending on the intended therapeutic use of the protein encoded by the therapeutic mRNA, the dosage and duration of treatment of the therapeutic mRNA may vary by several orders of magnitude. For vaccines, expression of antigens in the nanogram or microgram range may be sufficient to induce the required immune response. However, for growth factors, hormones, or antibodies, therapeutic doses may range from micrograms to milligrams, or potentially up to gram amounts of protein. Dose-dependent toxicity of mRNA is likely to be a limiting factor for scale-up to achieve such large protein amounts, and therefore modifications that result in increased mRNA stability without modification-specific toxicity are beneficial (Aditham et al., ACS Chem.Biol., December 2021, https: / / doi.org / 10.1021 / acschembio.1c00569).

[0046] For any application of mRNA in a therapeutic context, it is desirable to use mRNA with a defined sequence and structure that can be reproduced in a reliable manner. For example, the 5'UTR (e.g., containing a cap structure) and 3'UTR (e.g., containing a homopolymeric tail such as a polyA tail) of an mRNA are known to be involved in regulating mRNA stability and translation efficiency. Thus, the 5' cap structure and 3' tail are important features for efficient translation of mRNA and protein synthesis in eukaryotic cells. Thus, mRNA production methods can control for such important functional features. The inclusion of a 3' terminal PS linkage in the polyA tail has been shown to increase protein production in human HeLa cell lines by 2-4 fold, primarily by stabilizing the mRNA (Aditham et al., ACS Chem.Biol., December 2021).

[0047] The mRNA may have a modified cap: 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NImpNp (cap 1), 7mG(5')-ppp(5')NImpN2mp (cap 2), or m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0048] In addition, non-capped RNA typically contains 5'-terminal triphosphate group, which is known to stimulate the innate immune system.Therefore, non-capped RNA may generate undesirable immune response in subjects.Therefore, the mRNA product as medicine must be controlled for the presence of non-capped 5'-triphosphate RNA.

[0049] While conventional mRNA vaccines code for the antigen of interest and contain 5' and 3' UTRs, self-amplifying RNA codes for not only the antigen but also the viral replication machinery that allows for intracellular RNA amplification and abundant protein expression. Messenger RNA molecules are typically produced by RNA in vitro transcription of a suitable DNA template. The 5' cap structure and 3' homopolymer tail (e.g., polyA tail) are typically introduced during RNA in vitro transcription, e.g., can be encoded within the DNA template, or can be encoded via enzymatic methods after RNA in vitro transcription.

[0050] The disclosed method can be used to prepare self-replicating RNA by in vitro transcription. For example, a DNA-dependent RNA polymerase (e.g., bacteriophage T7, T3 or SP6 RNA polymerase) can be used to transcribe self-replicating RNA from a DNA template.

[0051] Appropriate capping and poly A tailing reactions via enzymatic methods can be used as needed after RNA production using the methods of the present disclosure or can be encoded within the DNA template.

[0052] Polymerases may have strict requirements regarding the nucleotides they incorporate (i.e., their nucleoside triphosphate substrates), which may be consistent with the requirements of the encoded replicase to ensure that the transcribed RNA serves as a substrate for that self-encoded replicase.

[0053] The method of the present disclosure can be used to prepare non-replicative mRNA.For example, DNA or RNA polymerase can be used to transcribe non-replicative mRNA from DNA or RNA template.Polymerase that can be utilized is described herein.Exemplary polymerase includes DNA polymerase I or T7 RNA polymerase, which can be further mutated as described herein.

[0054] In the methods of the disclosure, the RNA can be modified and / or stabilized RNA.

[0055] "Stabilized RNA" is defined as RNA that exhibits increased resistance to in vivo degradation and / or increased stability in vivo and / or increased translatability in vivo.

[0056] Stabilization can be achieved, for example, by the modified phosphate backbone of the RNA produced.Backbone modification is the modification that the phosphate of the backbone of the nucleotide contained in RNA is chemically modified.The nucleotide that can be used in this context contains, for example, phosphorothioate modified phosphate backbone, and optionally, at least one of the phosphate oxygens contained in phosphate backbone is replaced with sulfur atom.

[0057] The stabilized RNA may further comprise, for example, phosphate analogs, such as alkyl and aryl phosphonates, or alkyl phosphotriesters, etc. Such backbone modifications typically include, but are not limited to, modifications from the group consisting of methyl phosphonates, phosphoramidates, and phosphorothioates.

[0058] A "modified RNA" contains one or more modified nucleotides.

[0059] As used herein, the term "modified nucleotide residue" or "modified polynucleotide or oligonucleotide" refers to a nucleotide residue or polynucleotide or oligonucleotide that contains at least one aspect of the chemical nature of the nucleotide residue or polynucleotide or oligonucleotide that is different from that of naturally occurring nucleotide residue or polynucleotide or oligonucleotide. Such modifications, such as sugar moiety modification, nucleic acid base modification, and / or backbone modification, can be made at any part of the nucleotide residue. The modified nucleotide residue can form a part of a modified polynucleotide or oligonucleotide. The modified polynucleotide or oligonucleotide can be DNA or RNA.

[0060] Those skilled in the art will appreciate that many synthetic derivatives of nucleotides exist.

[0061] Additionally, any nucleotide other than G, C, U, T, A may be considered a "modified nucleotide." Examples of nucleotide modifications are disclosed herein.

[0062] The polynucleotide or oligonucleotide may comprise a PMO, an LNA, a c-Et, a PNA, a BNA, or an L-ribonucleic acid.

[0063] A backbone modification in the context of the present disclosure is a modification in which the phosphate of the backbone of a nucleotide contained in a nucleic acid is chemically modified. A sugar modification in the context of the present disclosure is a chemical modification of the sugar of one or more nucleotides (nucleotide(s)). A base modification in the context of the present disclosure is a chemical modification of the base portion of one or more nucleotides (nucleotide(s)). In this context, the nucleotide modification is selected from nucleotide analogs that are applicable to transcription and / or translation.

[0064] Modified nucleoside triphosphates known in the art include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 4-amino-6-chloropurine riboside ... '-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate '-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 06-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, xanthosine-5'-triphosphate.

[0065] Base-modified nucleotides known in the art include 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 5-methyl-uridine, 5-methyl-uridine, 5-carboxymethyl-uridine, 1-carboxymethyl-uridine, 5-propynyl-uridine, 5-methyl ... uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- Dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine , 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine, 5'-0-(1-thiophosphate)-guanosine, 5'-0-(1-thiophosphate)-uridine, 5'-0-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-amino-cytidine, alpha-amino-cytidine, pseudo-iso ... Allyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thiouridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro- pseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 2'-O-methyluridine, pseudouridine (y), N1-methylpseudouridine, 5-methylcytosine, and 5-methoxyuridine.

[0066] Those skilled in the art will understand that in vitro transcribed (IVT) mRNA elicits a strong immune response and suppresses protein production when transfected. Therefore, 100% replacement of uridine with pseudouridine or N1-methylpseudouridine is widely used in therapeutic mRNA to reduce immunotoxicity by blocking Toll-like receptor recognition, thereby increasing translation efficiency.

[0067] The polynucleotide or oligonucleotide of the present disclosure can include at least one coding sequence, where at least one coding sequence is a pseudouridine modified coding sequence, i.e., in the coding sequence, all uridines are replaced with pseudouridine. The polynucleotide or oligonucleotide can include at least one or more, or all uridines are replaced with pseudouridine, nucleic acid sequence. The polynucleotide or oligonucleotide can include at least one coding sequence, where at least one coding sequence is a N1-methylpseudouridine modified coding sequence, i.e., in the coding sequence, all uridines are replaced with N1-methylpseudouridine. N1-methylpseudouridine and 1-methylpseudouridine can be used interchangeably. The polynucleotide or oligonucleotide can include at least one or more, or all uridines are replaced with N1-methylpseudouridine, nucleic acid sequence. The polynucleotide or oligonucleotide can include at least one coding sequence, where at least one coding sequence is a codon modified coding sequence. At least one coding sequence may be a codon-modified coding sequence, wherein the amino acid sequence encoded by the at least one codon-modified coding sequence is unmodified compared to the amino acid sequence encoded by the corresponding wild-type coding sequence, i.e., the amino acid sequences are identical.

[0068] The term "codon-modified coding sequence" refers to a coding sequence in which at least one codon (a triplet of nucleotides encoding one amino acid) differs compared to the corresponding wild-type coding sequence. Advantageously, the codon-modified coding sequence may exhibit increased resistance to in vivo degradation and / or increased stability in vivo and / or increased translatability in vivo. Codon modification may take advantage of the degeneracy of the genetic code, where multiple codons can code for the same amino acid and can be used interchangeably to optimize the coding sequence for in vivo applications.

[0069] Modifications on the sugar moiety can include modifications at the 2' position of the sugar moiety, bicyclic sugars or 4'-CH(CH3)-O-2' groups, and combinations thereof. For example, modifications at the 2' position of the sugar moiety can include 2'-F, 2'-OMe, 2'-MOE, and / or 2'-amino.

[0070] Examples of modified nucleobases include cytosine, e.g., 5-methylcytosine, 5-methylpyrimidine, 7-deazaguanosine, and abasic nucleotides. Further examples of modified nucleobases include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyl adenosine); ms2i6A (2-methylthio-N6 isopentenyl adenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine);ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine);g6A(N6-glycinylcarbamoyladenosine);t6A(N6-threonylcarbamoyladenosine);ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine);m6t6A(N6-methyl-N6-threonylcarbamoyladenosine);hn6A(N6.-hydroxynorvalylcarbamoyladenosine);ms2hn 6A(2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine);Ar(p)(2'-O-ribosyladenosine (phosphate);I(inosine);mil(1-methylinosine);m'lm(l,2'-O-dimethylinosine);m3C(3-methylcytidine);Cm(2T-0-methylcytidine);s2C(2-thiocytidine);ac4C(N4-acetylcytidine);f5C(5-fonnylcytidine);m5Cm(5,2-0-dimethylcytidine);ac 4Cm (N4 acetyl-2TOmethylcytidine); k2C (lysidine); mlG (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine);OHyW (hydroxywybutosine);OHyW* (undermodified hydroxywybutosine);imG (wybutosine);mimG (methylguanosine);Q (queuosine);oQ (epoxyqueuosine);galQ (galactosyl-queuosine);manQ (mannosyl-queuosine);preQo (7-cyano-7-deazaguanosine);preQi (7-aminomethyl-7-deazaguanosine);G (archaeosine);D (dihydrouridine);m5Um (5,2'-O-dimethyluridine);s4U (4-thiouridine);m5s2U(5-methyl-2-thiouridine);s2Um(2-thio-2'-O-methyluridine);acp3U(3-(3-amino-3-carboxypropyl)uridine);ho5U(5-hydroxyuridine);mo5U(5-methoxyuridine);cmo5U(uridine 5-oxyacetic acid);mcmo5U(uridine 5-oxyacetic acid methyl ester);chm5U(5-(carboxyhydroxymethyl)uridine);mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm5U(5 -Methoxycarbonylmethyluridine;mcm5Um(S-Methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U(5-Methoxycarbonylmethyl-2-thiouridine);nm5s2U(5-Aminomethyl-2-thiouridine);mnm5U(5-Methylaminomethyluridine);mnm5s2U(5-Methylaminomethyl-2-thiouridine);mnm5se2U(5-Methylaminomethyl-2-selenouridine);ncm5U(5-Carbamoylmethyluridine);ncm5Um(5-Carbamoylmethyl-2'-O- methyluridine);cmnm5U(5-carboxymethylaminomethyluridine);cnmm5Um(5-carboxymethylaminomethyl-2-LO methyluridine);cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine);m62A(N6,N6-dimethyladenosine);Tm(2'-O-methylinosine);m4C(N4-methylcytidine);m4Cm(N4,2-O-dimethylcytidine);hm5C(5-hydroxymethylcytidine);m3U(3-methyluridine);cm5U(5-carboxymethyluridine);m6Am(N6,T-0-dimethyladenosine);rn62Am(N6,N6,0-2-trimethyladenosine);m2'7G(N2,7-dimethylguanosine);m2'2'7G(N2,N2,7-trimethylguanosine);m3Um(3,2T-0-dimethyluridine);m5D(5-methyldihydrouridine);￡5Cm(5-formyl-2'-O-methylcytidine);mlGm(l,2'-O-dimethylguanosine);m'Am(1,2-O-dimethyladenosine tm5s2U (S-taurinomethyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine); and ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Cl-C6)-alkyluracil, 5-methyluracil, 5 -(C2-C6)-alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Cl-C6)-alkylcytosine, 5-methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine , 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, and 7-deaza-8-substituted purine. For example, the polynucleotide may contain one or more modified pyrimidine nucleobases, such as pseudouridine and / or 5-methylcytosine residues;

[0071] Modifications in the backbone can include phosphorothioates, phosphoramidates, phosphorodiamidates, and phosphorodithioates. At least one or each internucleoside linkage can be a modified internucleoside linkage.

[0072] Polynucleotides of the disclosure may contain only phosphodiester linkages between nucleosides, but in other examples may contain phosphoramidate, phosphorothioate, phosphorodiamidate, and phosphorodithioate, and / or methylphosphonate linkages.

[0073] Modifications may include N1-methylpseudouridine.

[0074] Pseudouridine, N1-methylpseudouridine, 5-methylcytosine, and 5-methoxyuridine can be used in place of uridine in the nucleoside triphosphate pool for incorporation using polymerases.

[0075] As used herein, the term "gapmer" refers to an oligonucleotide having an inner "central region" flanked by two outer "wing regions" (5' wing and 3' wing), where the central region contains a number of nucleotides that support RNase H cleavage, and each wing region contains one or more nucleotides that are chemically distinct from the nucleotides in the central region. Gapmers are antisense polynucleotides. Gapmers include a central region, a 5' wing region located at the 5' end of the central region, and a 3' wing region located at the 3' end of the central region.

[0076] As used herein, the term "support material" refers to a high molecular weight compound or material, e.g., a template or primer, that increases the molecular weight of a polynucleotide or oligonucleotide, thereby allowing the polynucleotide or oligonucleotide to be retained, e.g., when impurities and / or products are separated from a reaction mixture.

[0077] As used herein, the "percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "identity" value expressed as a percentage calculated by the BLASTN algorithm when the subject nucleic acid sequence has 100% query coverage with respect to the query nucleic acid sequence after pairwise BLASTN alignment is performed. Such pairwise BLASTN alignment between the query nucleic acid sequence and the subject nucleic acid sequence is performed by using the default settings of the BLASTN algorithm available at the National Center for Biotechnology Institute website with the filter for low complexity regions turned off. Importantly, the query nucleic acid sequence may be described by the nucleic acid sequence identified in one or more claims in this application. The query sequence may be 100% identical to the subject sequence, or may contain up to a certain integer number of nucleotide changes compared to the subject sequence such that the identity % is less than 100%. For example, the query sequence is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence.

[0078] As used herein, the term "about" when referring to a measurable value, e.g., amount, duration in time, etc., is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1%, from the stated value as appropriate for practicing the embodiments disclosed herein.

[0079] Embodiment The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) contacting a template polynucleotide comprising a sequence complementary to a single stranded polynucleotide product with a pool of at least two segment polynucleotides under conditions that allow annealing of the at least two segment polynucleotides to the template polynucleotide to produce a template polynucleotide having at least two annealed segment polynucleotides, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; b) extending at least one of the annealed segment polynucleotides using the pool of nucleoside triphosphates and a polymerase to fill in at least one sequence gap to generate at least one extended segment polynucleotide; and c) ligating one or more segment polynucleotide(s) and / or one or more extended segment polynucleotide(s) using a ligase to form a single-stranded polynucleotide product that is linked in a duplex fashion to the template polynucleotide; and d) changing the conditions to denature the duplex comprising the single-stranded polynucleotide product and the template polynucleotide, thereby generating single-stranded polynucleotide products, and optionally separating the single-stranded polynucleotide products. The present invention provides a method comprising:

[0080] Steps (b) and (c) may be performed simultaneously. In certain circumstances, steps (a), (b) and (c) may be performed simultaneously. In certain embodiments, the polymerase or the pool of nucleoside triphosphates described in step b) is present in step (a). In certain embodiments, the polymerase described in step b) is present in step (a), and once at least two segment polynucleotides have annealed to the template polynucleotide, the pool of nucleoside triphosphates is added to allow the polymerase to extend at least one of the annealed segment polynucleotides to fill at least one sequence gap. In an alternative embodiment, the pool of nucleoside triphosphates described in step b) is present in step (a), and once at least two segment polynucleotides have annealed to the template polynucleotide, the polymerase is added to extend at least one of the annealed segment polynucleotides to fill at least one sequence gap.

[0081] Step d) may further include changing the conditions to denature the duplex comprising the impurity polynucleotide and the template polynucleotide, and changing the conditions to separate any impurity polynucleotide(s) prior to denaturing the duplex comprising the single-stranded polynucleotide product and the template polynucleotide.

[0082] In some embodiments, at least one segment polynucleotide comprises at least one modified nucleotide residue. In some embodiments, the pool of nucleoside triphosphates consists of (i) naturally occurring nucleoside triphosphates, (ii) modified nucleoside triphosphates, or (iii) naturally occurring nucleoside triphosphates and modified nucleoside triphosphates.

[0083] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. The present invention provides a method comprising:

[0084] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. wherein at least one of the at least two segment polynucleotides comprises at least one modified nucleotide residue and / or the pool of nucleoside triphosphates comprises at least one modified nucleotide.

[0085] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; and g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. The present invention provides a method comprising:

[0086] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; and g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. wherein at least one of the at least two segment polynucleotides comprises at least one modified nucleotide residue and / or the pool of nucleoside triphosphates comprises at least one modified nucleotide.

[0087] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) changing conditions to denature the annealed template and any impurities and to separate the impurities; and g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. The present invention provides a method comprising:

[0088] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) changing conditions to denature the annealed template and any impurities and to separate the impurities; and g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. wherein at least one of the at least two segment polynucleotides comprises at least one modified nucleotide residue and / or the pool of nucleoside triphosphates comprises at least one modified nucleotide.

[0089] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; and g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products and, optionally, separate the single-stranded polynucleotide products. The present invention provides a method comprising:

[0090] The pool of nucleoside triphosphates can include at least one modified nucleoside triphosphate.

[0091] Step d) may be further divided into three separate steps: d1) providing a pool of nucleoside triphosphates, d2) providing a polymerase, and d3) extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and a polymerase to fill at least one sequence gap.

[0092] The present disclosure provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; d) providing a pool of nucleoside triphosphates; e) providing a polymerase; f) extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and a polymerase to fill at least one sequence gap; g) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; h) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; and i) changing conditions to denature the annealed template and the single-stranded polynucleotide products, and optionally separating the single-stranded polynucleotide products; The present invention provides a method comprising:

[0093] Two or more of steps a)-i) may be performed simultaneously, and optionally steps f) and g) may be performed simultaneously. Two or more of steps a)-i) may be performed sequentially, and optionally steps f) and g) may be performed sequentially.

[0094] In any of the methods described above, the method may further comprise a further step (step j) of reusing the template. The method may further comprise another step (step k) of repeating the previous steps (steps a)-i) or steps a)-j)) with the reused template.

[0095] The present disclosure also provides a method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) a sequence complementary to the single stranded polynucleotide product; i) a hairpin loop that acts as a primer for the polymerase; ii) a hairpin loop that acts as a stopper for the polymerase, or iii) hairpin loops at both ends of a template polynucleotide, one of which acts as a primer for polymerase and the other hairpin loop is active as a stopper for polymerase; Providing a template polynucleotide comprising: b) contacting the template polynucleotide with the pool of at least one segment polynucleotide under conditions that allow annealing of at least one segment polynucleotide to the template polynucleotide to produce a template polynucleotide with at least one annealed segment polynucleotide, wherein at least one sequence gap is formed between the annealed segment polynucleotide and an end of the hairpin loop; c) extending the ends of the annealed segment polynucleotides or hairpin loops using a pool of nucleoside triphosphates and a polymerase to fill in at least one sequence gap and generate at least one extended segment polynucleotide or extended hairpin polynucleotide; d) ligating one or more segment polynucleotide(s) and / or one or more extended segment / hairpin polynucleotide(s) using a ligase to form single-stranded polynucleotide products that are linked in a duplex fashion to the template polynucleotide; e) cleaving the single-stranded polynucleotide product from the template polynucleotide; and f) changing conditions to denature a duplex comprising the single-stranded polynucleotide product and the template polynucleotide, thereby generating a single-stranded polynucleotide product. The cleavage step can be carried out by a nuclease, a nickase, a DNA enzyme, or a chemical method.

[0096] The present disclosure also provides a method for generating a double-stranded polynucleotide product comprising annealing two complementary single-stranded polynucleotide products, where at least one of the two complementary single-stranded polynucleotide products was generated by a method of the present disclosure, and optionally, both of the two complementary single-stranded polynucleotide products were generated by a method of the present disclosure.

[0097] The present disclosure also provides a method for generating a double-stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) contacting a template polynucleotide comprising a sequence complementary to a single stranded polynucleotide product with a pool of at least two segment polynucleotides under conditions that allow annealing of the at least two segment polynucleotides to the template polynucleotide to produce a template polynucleotide having at least two annealed segment polynucleotides, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; b) extending at least one of the annealed segment polynucleotides using the pool of nucleoside triphosphates and a polymerase to fill in at least one sequence gap to generate at least one extended segment polynucleotide; c) ligating one or more segment polynucleotide(s) and / or one or more extended segment polynucleotide(s) using a ligase to form a single-stranded polynucleotide product linked in a duplex fashion to the template polynucleotide; d) optionally changing conditions to denature the duplex comprising the single-stranded template polynucleotide and the impurity polynucleotides and separate one or more of any impurity polynucleotide(s) from the template polynucleotide; e) changing conditions to denature the duplex comprising the single-stranded polynucleotide product and the template polynucleotide, thereby producing a single-stranded polynucleotide product; and f) using the single-stranded polynucleotide product as a template polynucleotide in step a) and repeating steps a) to c) or steps a) to d) to generate a double-stranded polynucleotide product. The present invention provides a method comprising:

[0098] Without being bound by theory, these methods have been found to reduce the complexity of polynucleotide or oligonucleotide product production by allowing the segment polynucleotide and / or oligonucleotide production step and the ligation method step to use the same template.Simultaneous segment polynucleotide and / or oligonucleotide extension and ligation can further be performed in the production of polynucleotide and / or oligonucleotide product.These methods can facilitate the control of chirality of the final product, reduce the number of ligation steps, and reduce the overall reaction time.

[0099] The present disclosure also provides a method for producing a double-stranded polynucleotide or oligonucleotide product, in which two complementary single-stranded polynucleotides or oligonucleotides produced by the methods of the present disclosure are mixed under conditions that allow for annealing.

[0100] Alternatively, the disclosure also provides methods for generating a double-stranded polynucleotide or oligonucleotide product, in which a single-stranded polynucleotide or oligonucleotide product acts and / or is used as a template.

[0101] For example, the disclosure provides a method for generating a double-stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one annealed segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products, and optionally to separate the single-stranded polynucleotide products; and h) using the single stranded polynucleotide product as a template in step a) and repeating steps a) through e) or steps a) through f) to generate a double stranded polynucleotide product. The present invention provides a method comprising:

[0102] Optionally, the double-stranded polynucleotide product is purified.

[0103] For example, the disclosure provides a method for generating a double-stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides; d) providing a pool of nucleoside triphosphates and a polymerase and extending at least one segment polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; g) changing conditions to denature the annealed templates and the single-stranded polynucleotide products, and optionally to separate the single-stranded polynucleotide products; and h) using the single stranded polynucleotide product as a template in step a) and repeating steps a) through e) or steps a) through f) to generate a double stranded polynucleotide product. wherein at least one segment polynucleotide comprises at least one modified nucleotide residue and / or the pool of nucleoside triphosphates comprises at least one modified nucleotide.

[0104] Optionally, the double-stranded polynucleotide product is purified.

[0105] In some embodiments, at least one segment polynucleotide comprises at least one modified nucleotide residue. In some embodiments, at least two segment polynucleotides comprise at least one modified nucleotide residue. In some embodiments, all segment polynucleotides comprise at least one modified nucleotide residue.

[0106] In certain embodiments, at least one nucleoside triphosphate in the pool of nucleoside triphosphates of step (d) is modified. In certain embodiments, A, T, C, G and / or U in the pool of nucleoside triphosphates of step (d) are modified. In certain embodiments, all nucleoside triphosphates in the pool of nucleoside triphosphates of step (d) are modified.

[0107] In some embodiments, the ligase used in step I is used to ligate the 3' and / or 5' ends of each segment polynucleotide or extended segment polynucleotide to each adjacent segment polynucleotide or extended segment polynucleotide to form a product polynucleotide chain. The ligase used in step (e) can be an RNA and / or a DNA ligase.

[0108] In some embodiments, the pool of polynucleotides is produced by enzymatic synthesis, chemical synthesis, optionally solid-supported synthesis or liquid phase synthesis, or a combination thereof. The pool of polynucleotides can be produced by enzymatic synthesis using single-stranded ligase, transferase, polymerase, or a combination thereof.

[0109] In some embodiments, the modification is selected from the group consisting of a modified sugar moiety, a modified nucleobase, and a modified backbone. In some embodiments, at least one modified nucleotide comprises a modified sugar moiety, a modified nucleobase, and / or a modified backbone. In other words, at least one modified nucleotide comprises a modified sugar moiety, a modified nucleobase, and / or a modified backbone.

[0110] The polynucleotides or oligonucleotides used in the methods of the invention may be modified in a sugar manner, i.e., modified form of the ribosyl moiety, e.g., 2'-O-modified RNA, e.g., 2'-O-alkyl or 2'-O-(substituted) alkyl, e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(3-amino)propyl, 2'-O-(3-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkoxy)methyl (Arai K. et al., Bioorg. Med. Chem. 2011, 21, 6285), such as 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl, such as 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halo, such as 2'-F, FANA (2'-F arabinosyl nucleic acid); carba sugar and aza sugar modifications; 3'-O-alkyl, such as 3'-O-methyl, 3'-O-butyryl, 3'-O-propargyl; and derivatives thereof.

[0111] In certain embodiments of the invention, the sugar modification is selected from the group consisting of 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), and 2'-amino. In further embodiments, the modification is 2'-MOE.

[0112] Other sugar modifications include "bridged" or "bicyclic" nucleic acids (BNA), such as locked nucleic acids (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acids (ENA), tricyclic DNA; unlocked nucleic acids (UNA); cyclohexenyl nucleic acids (CeNA), altriol nucleic acids (ANA), hexitol nucleic acids (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA); morpholinos (e.g., as in PMO, PPMO, PMOPlus, PMO-X); and derivatives thereof.

[0113] The polynucleotides or oligonucleotides used in the methods of the present disclosure may include other modifications, such as peptide nucleic acids (PNAs), boron modified PNAs, pyrrolidine-based oxypeptide nucleic acids (POPNAs), glycol or glycerol-based nucleic acids (GNAs), threose-based nucleic acids (TNAs), acyclic threoninol-based nucleic acids (aTNAs), oligonucleotides with incorporated bases and backbones (ONIBs), pyrrolidine amide oligonucleotides (POMs); and derivatives thereof.

[0114] In certain embodiments of the invention, the modified oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA), such as (S)-cEt-BNA, or an L-ribonucleic acid, also known as SPIEGELMER.

[0115] In further embodiments, the modification is in the nucleic acid base. Base modifications include modified forms of natural purine and pyrimidine bases (e.g., adenine, uracil, guanine, cytosine, and thymine), such as inosine, hypoxanthine, orotic acid, agmatidine, lysidine, 2-thiopyrimidines (e.g., 2-thiouracil, 2-thiothymine), G-clamp and derivatives thereof, 5-substituted pyrimidines (e.g., 5-methylcytosine, 5-methyluracil, 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Superaminomethylcytosine ... T), 2,6-diaminopurine, 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases such as 2,6-difluorotoluene, or missing bases such as abasic sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is replaced with a nitrogen (azaribose)). Examples of Super A, Super G, and Super T derivatives can be found in US6683173. cPent-G, cPent-AP, and Pr-AP have been shown to suppress immunostimulatory effects when incorporated into siRNA (Peacock H. et al., J. Am. Chem. Soc. (2011), 133, 9200).

[0116] In some embodiments of the invention, the nucleobase modification is selected from the group consisting of 5-methylpyrimidine, 7-deazaguanosine, and abasic nucleotides, hi some embodiments, the modification is 5-methylcytosine.

[0117] Polynucleotides or oligonucleotides used in the methods of the disclosure may include backbone modifications, such as modified forms of phosphodiesters present in RNA, such as phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrugs, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methyl boranophosphonothioate, and derivatives thereof. Other modifications include phosphoramidites, phosphoramidates, '3'→P5' phosphoramidates, phosphorodiamidates, phosphorothiodiamidates, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamide nucleic acid (TANA), and derivatives thereof.

[0118] In a further embodiment, the modification is in the backbone and is selected from the group consisting of phosphorothioate (PS), phosphoramidate (PA), phosphorodiamidate, and phosphorodithioate (PS2). In an embodiment of the present invention, the modified oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). The PMO has a backbone of methylene morpholine rings with phosphorodiamidate linkages. In an embodiment of the present invention, the product has a phosphorothioate (PS) backbone. In an embodiment of the present invention, the product has at least one phosphorothioate (PS) bond in the backbone.

[0119] In certain embodiments of the invention, an oligonucleotide comprises a combination of two or more of the modifications disclosed herein.Those of skill in the art will appreciate that there are many synthetic derivatives of oligonucleotides and their constituent nucleotides.

[0120] The modifications can include modifications at the 2' position of the sugar moiety, bicyclic sugars or 4'-CH(CH3)-O-2' groups, and combinations thereof. The modifications at the 2' position of the sugar moiety can include 2'-MOE. The modified nucleobase can include cytosine, optionally 5-methylcytosine. The modifications at the nucleobase can be selected from the group including 5-methylpyrimidine, 7-deazaguanosine, and abasic nucleotides. The modifications at the backbone can be selected from the group including phosphorothioates, phosphoramidates, phosphorodiamidates, and phosphodithioates.

[0121] In embodiments, each polynucleotide segment can be composed of linked 2'-deoxynucleotides or deoxynucleosides. In embodiments, at least one or each internucleoside linkage can be a modified internucleoside linkage.

[0122] In some embodiments, the single-stranded polynucleotide product may be a DNA polynucleotide product, an RNA polynucleotide product, or a combination thereof. The RNA polynucleotide product may be an mRNA. In some embodiments, the nucleotide modification may include the substitution of one or more uracil residues. At least one modified nucleotide may include 1-methyl-pseudouridine, 5-methoxy-uracil, 1-ethyl-pseudouracil, pseudouracil, 1-methylpseudouracil, 5-methyl-cytidine, 5-methyl-cytosine, N6-methyladenosine, or 7-methylguanosine, or a combination thereof. In some embodiments, the single-stranded polynucleotide product is an mRNA in which each U residue is an N1-methyl-pseudouridine. In some embodiments, the single-stranded polynucleotide product is an mRNA in which each U residue is an N1-methyl-pseudouridine and the backbone is a phosphorothioate backbone.

[0123] In some embodiments, the polymerase used in step (d) lacks 5' to 3' exonuclease activity. In some embodiments, the polymerase used in step (d) lacks 3' to 5' exonuclease activity. In some embodiments, the polymerase used in step (d) lacks 5' to 3' exonuclease activity and lacks 3' to 5' exonuclease activity.

[0124] In embodiments, the polymerase used in step (d) may be a DNA polymerase, an RNA polymerase, or a combination thereof.

[0125] The steps of the method can be performed sequentially. The steps of the method can be reordered without rendering the claims infeasible. When the steps of providing a pool of nucleoside triphosphates, providing a polymerase, and extending at least one segmented polynucleotide using the pool of nucleoside triphosphates and the polymerase to fill at least one sequence gap are performed as separate steps, the pool of nucleoside triphosphates can be provided in any preceding step, so long as the polymerase is added after the segmented polynucleotide or oligonucleotide is annealed to the template. Alternatively, the polymerase can be provided in any preceding step, so long as the pool of nucleoside triphosphates is added after the segmented polynucleotide or oligonucleotide is annealed to the template. In some embodiments, two or more steps are performed simultaneously. In some embodiments, two or more steps of steps a)-g) are performed simultaneously. In some embodiments, two or more steps are performed sequentially. In some embodiments, two or more steps a)-g) are performed sequentially. In some embodiments, method steps (d) and (e) are performed simultaneously. In other embodiments, method steps (d) and (e) are performed sequentially.

[0126] In embodiments, the template polynucleotide or oligonucleotide may consist of a sequence complementary to the sequence of the single-stranded polynucleotide or oligonucleotide product.

[0127] In some embodiments, the template polynucleotide is part of a hairpin loop. The terms hairpin loop and stem loop can be used interchangeably. The hairpin loop can be asymmetric. The hairpin loop can be a DNA hairpin loop. The hairpin loop can be an RNA hairpin loop. When the template polynucleotide comprises a hairpin loop, the polymerase may not require a primer to initiate polymerization. The single-stranded polynucleotide product can be released from the template and hairpin loop using an enzyme that introduces a single-strand break between the hairpin loop and the product polynucleotide strand (e.g., using a nickase), followed by denaturation of the annealed template polynucleotide and product polynucleotide strands.

[0128] In embodiments, the single stranded polynucleotide product can be 3-40 nucleotides, 3-35 nucleotides, or 3-30 nucleotides in length, optionally 10-35 nucleotides, 10-30 nucleotides, 3-15 nucleotides, 13-35 nucleotides, 15-35 nucleotides, 13-30 nucleotides, 15-30 nucleotides, 13-25 nucleotides, 15-25 nucleotides, 13-20 nucleotides, 15-20 nucleotides, 17-25 nucleotides, 20-25 nucleotides, or 20-30 nucleotides in length. In some embodiments, the product is 20 nucleotides in length, (i) a 5' segment that is 7 nucleotides long, a central segment that is 6 nucleotides long, and a 3' segment that is 7 nucleotides long; (ii) a 5' segment that is 6 nucleotides long, a central segment that is 8 nucleotides long, and a 3' segment that is 6 nucleotides long; (iii) a 5' segment that is 5 nucleotides long, a central segment that is 10 nucleotides long, and a 3' segment that is 5 nucleotides long; (iv) a 5' segment that is 4 nucleotides long, a central segment that is 12 nucleotides long, and a 3' segment that is 4 nucleotides long; or (v) A three segment polynucleotide comprising a 5' segment that is 3 nucleotides long, a central segment that is 14 nucleotides long, and a 3' segment that is 3 nucleotides long. The single stranded polynucleotide product can be a gapmer antisense polynucleotide comprising a central region, a 5' wing region located at the 5' end of the central region, and a 3' wing region located at the 3' end of the central region. This is particularly useful in therapeutic oligonucleotide production where it is important to generate highly sequence specific oligonucleotide products.

[0129] In some embodiments, at least two segment polynucleotides comprise: (i) a 5' segment that is 7 nucleotides in length and a 3' segment that is 7 nucleotides in length; (ii) a 5' segment that is 6 nucleotides in length and a 3' segment that is 6 nucleotides in length; (iii) a 5' segment that is 5 nucleotides in length and a 3' segment that is 5 nucleotides in length; (iv) a 5' segment that is 4 nucleotides in length and a 3' segment that is 4 nucleotides in length; or (v) a 5' segment that is 3 nucleotides long and a 3' segment that is 3 nucleotides long Includes.

[0130] In embodiments, the single stranded polynucleotide products can be 30-20,000 nucleotides in length, optionally 30-10,000 nucleotides in length, 30-5,000 nucleotides in length, 30-4,000 nucleotides in length, 30-3,000 nucleotides in length, 30-2,000 nucleotides in length, 30-1,000 nucleotides in length, 30-500 nucleotides in length, 30-400 nucleotides in length, 30-300 nucleotides in length, 30-200 nucleotides in length, 30-100 nucleotides in length, 30-50 nucleotides in length, or 30-40 nucleotides in length. These products are useful, for example, in therapeutic mRNA production where it is important to generate highly sequence specific polynucleotide products.

[0131] In further embodiments, the single stranded polynucleotide product is greater than 30 nucleotides in length. In another embodiment, the single stranded polynucleotide product is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides.In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or more than 5000 nucleotides.

[0132] In embodiments, the template has properties that allow it to be separated from the product and reused for a subsequent reaction. The method can include a final step of reusing the template. The method can include step j) of reusing the template. The method can include a final step of reusing the template for use in a subsequent reaction. The method can include step j) of reusing the template for use in a subsequent reaction. The method can include a final step of reusing the template and repeating the method. The method can include a final step of reusing the template and repeating one or more steps of the method. The method can include step j) of reusing the template and step k) of repeating steps a) to i). The method can include step j) of reusing the template and step k) of repeating steps a) to k).

[0133] The processes described herein can be semi-continuous or continuous.

[0134] Methods are provided in which products are made on a gram or kilogram scale or larger scale and / or are performed in a reaction volume of at least 1 L. Methods are provided in which products are made on a gram or kilogram scale or larger scale. Methods are provided in which products are made in a reaction volume of at least 200 mL. Methods are provided in a reaction volume of at least 500 mL. Methods are provided in a reaction volume of at least 1 L. Methods are provided in a reaction volume of at least 2 L. Methods are provided in a reaction volume of at least 5 L.

[0135] The resulting single-stranded polynucleotide or oligonucleotide product can be at least 90% pure, at least 95% pure, or at least 98% pure.

[0136] The method can be used to generate therapeutic polynucleotides or oligonucleotides. In some embodiments, the method is for generating single-stranded therapeutic polynucleotides or oligonucleotides. In some embodiments, the method is for generating double-stranded therapeutic polynucleotides or oligonucleotides. These methods are useful, for example, in therapeutic oligonucleotide generation, where it is important to generate highly sequence-specific oligonucleotide products. The products are also useful, for example, in therapeutic mRNA generation, where it is important to generate highly sequence-specific polynucleotide products.

[0137] Methods are provided in which the denaturation of template-impurity duplexes and / or template-product duplexes results from an increase in temperature. In some embodiments, the denaturation can be the result of changing the pH. In some embodiments, the denaturation can be by changing the salt concentration in a buffer solution.

[0138] Methods are provided in which the segment oligonucleotides are 3-15 nucleotides long. In embodiments, the segments can be 5-10 nucleotides long. In embodiments, the segments can be 5-8 nucleotides long. In embodiments, the segments can be 4, 5, 6, 7, or 8 nucleotides long. In embodiments, there are three segment oligonucleotides that, when ligated together, form an oligonucleotide that is 20 nucleotides long (a "20-mer"), i.e., a 5' segment that is 7 nucleotides long, a central segment that is 6 nucleotides long, and a 3' segment that is 7 nucleotides long. In embodiments, there are three segment oligonucleotides that, when ligated together, form an oligonucleotide that is 20 nucleotides long (a "20-mer"), i.e., a 5' segment that is 6 nucleotides long, a central segment that is 8 nucleotides long, and a 3' segment that is 6 nucleotides long. In embodiments, there are three segment oligonucleotides that, when ligated together, form an oligonucleotide that is 20 nucleotides long (a "20-mer"), i.e., a 5' segment that is 5 nucleotides long, a central segment that is 10 nucleotides long, and a 3' segment that is 5 nucleotides long. In some embodiments, there are three segmented oligonucleotides that, when ligated together, form an oligonucleotide that is 20 nucleotides long (a "20-mer"): a 5' segment that is 4 nucleotides long, a central segment that is 12 nucleotides long, and a 3' segment that is 4 nucleotides long. In some embodiments, there are four segmented oligonucleotides that, when ligated together, form an oligonucleotide that is 20 nucleotides long (a "20-mer"): a 5' segment that is 5 nucleotides long, a 5' central segment that is 5 nucleotides long, a central 3' segment that is 5 nucleotides long, and a 3' segment that is 5 nucleotides long.

[0139] Methods are provided in which the product is between 3 and 40 nucleotides in length. In embodiments, the product may be between 13 and 40 nucleotides in length. In embodiments, the product may be between 15 and 40 nucleotides in length. In embodiments, the product may be between 13 and 35 nucleotides in length. In embodiments, the product may be between 15 and 35 nucleotides in length. In embodiments, the product may be between 15 and 30 nucleotides in length. In embodiments, the product may be 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In certain embodiments of the invention, the product is 20 nucleotides in length, i.e., a "20-mer." In certain embodiments of the invention, the product is 21 nucleotides in length, i.e., a "21-mer." In certain embodiments of the invention, the product is 22 nucleotides in length, i.e., a "22-mer." In certain embodiments of the invention, the product is 23 nucleotides long, i.e., a "23-mer." In certain embodiments of the invention, the product is 24 nucleotides long, i.e., a "24-mer." In certain embodiments of the invention, the product is 25 nucleotides long, i.e., a "25-mer." In certain embodiments of the invention, the product is 26 nucleotides long, i.e., a "26-mer." In certain embodiments of the invention, the product is 27 nucleotides long, i.e., a "27-mer." In certain embodiments of the invention, the product is 28 nucleotides long, i.e., a "28-mer." In certain embodiments of the invention, the product is 29 nucleotides long, i.e., a "29-mer." In certain embodiments of the invention, the product is 30 nucleotides long, i.e., a "30-mer." Such products have utility, for example, in gapmer generation. In certain embodiments, such 3-40 nucleotide long single-stranded products are therapeutic antisense gapmers. In certain embodiments, such 3-40 nucleotide long products are therapeutic double-stranded products, e.g., siRNAs and miRNAs.In certain embodiments, such 3-40 nucleotide long products are oligonucleotides that recruit and guide DNA and / or RNA editing enzymes, such as RNA base-modified oligonucleotides, such as AImers.

[0140] Methods are provided in which the product is up to 10,000 nucleotides in length. In embodiments, the single stranded polynucleotide product can be 10-10,000 nucleotides in length, optionally 10-5,000 nucleotides in length, 10-1,000 nucleotides in length, 10-500 nucleotides in length, 10-400 nucleotides in length, 10-300 nucleotides in length, 10-200 nucleotides in length, 10-100 nucleotides in length, 10-50 nucleotides in length, or 10-40 nucleotides in length. Such products have utility, for example, as therapeutic mRNA polynucleotides.

[0141] Methods are provided in which the property that allows the template to be separated from the product is that the template adheres to a support material. In some embodiments, the support material is a soluble support material. In some embodiments, the soluble support material is selected from the group consisting of polyethylene glycol, soluble organic polymers, DNA, proteins, dendrimers, polysaccharides, oligosaccharides, and carbohydrates. In some embodiments, the support material is polyethylene glycol (PEG). In some embodiments, the support material is an insoluble support material. In some embodiments, the support material is a solid support material. In some embodiments, the solid support material is selected from the group consisting of glass beads, polymer beads, fibrous supports, membranes, streptavidin-coated beads, and cellulose. In some embodiments, the solid support material is streptavidin-coated beads. In some embodiments, the solid support material is itself part of the reaction vessel, for example, the reaction wall.

[0142] Methods are provided in which repeated copies of a template are attached in a sequential manner to a support material via a single attachment point. The repeated copies of the template may be separated by a linker, for example as shown in Figure 2. The repeated copies of the template may be direct repeats, i.e., they are not separated by a linker.

[0143] In embodiments, the template is attached to the support material at multiple attachment points.

[0144] Methods are provided in which the property that allows the template to be separated from the product is the molecular weight of the template. For example, repeated copies of a template sequence can be present in a single polynucleotide or oligonucleotide, with or without linker sequences.

[0145] Methods are provided in which the template, or the template and support material, are reused for use in subsequent reactions, for example as described in more detail below.Methods are provided in which the reactions are carried out using continuous or semi-continuous flow processes.

[0146] In some embodiments, the method is for large-scale production of polynucleotide or oligonucleotide, optionally therapeutic polynucleotide or oligonucleotide. In the context of the present disclosure, large-scale production of polynucleotide or oligonucleotide means production at 1 liter or more scale, for example, the method is carried out in 1 L or more reactor. Alternatively or additionally, in the context of the present disclosure, large-scale production of polynucleotide or oligonucleotide means production of product at gram scale, particularly production of 10 grams or more of product. In some embodiments, the product is produced at gram scale or kilogram scale, and / or the method is carried out in at least 1 L reactor. In some embodiments, the amount of polynucleotide or oligonucleotide product produced is gram scale. In some embodiments of the present disclosure, the amount of product produced is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 grams or more. In some embodiments, the amount of oligonucleotide product produced is 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 grams or more. In some embodiments, the amount of polynucleotide or oligonucleotide product produced is 500 grams or more. In some embodiments, the polynucleotide or oligonucleotide product produced is in kilogram scale. In some embodiments, the amount of polynucleotide or oligonucleotide product produced is 1 kg or more. In some embodiments, the amount of polynucleotide or oligonucleotide product produced is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 kg or more. In embodiments, the amount of polynucleotide or oligonucleotide product produced is 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kg or more.

[0147] In some embodiments, the amount of product produced is between 10 grams and 100 kg. In some embodiments, the amount of product produced is between 10 grams and 50 kg. In some embodiments, the amount of product produced is between 100 grams and 100 kg. In some embodiments, the amount of product produced is between 100 grams and 50 kg. In some embodiments, the amount of product produced is between 500 grams and 100 kg. In some embodiments, the amount of product produced is between 500 grams and 50 kg. In some embodiments, the amount of product produced is between 1 kg and 50 kg. In some embodiments, the amount of product produced is between 10 kg and 50 kg.

[0148] In embodiments, polynucleotide or oligonucleotide production is carried out at a 2, 3, 4, 5, 6, 7, 8, 9, 10 liter or greater scale, such as in a 2, 3, 4, 5, 6, 7, 8, 9, or 10 L reactor. In embodiments, polynucleotide or oligonucleotide production is carried out at a 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 liter or greater scale, such as in a 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 L reactor. In embodiments, polynucleotide or oligonucleotide production is performed at a scale of 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 liters or more, e.g., in 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 L reactors.

[0149] In embodiments, the reactor volume is about 10,000L, about 5000L, about 2000L, about 1000L, about 500L, about 125L, about 50L, about 20L, about 10L, or about 5L.

[0150] In several embodiments, the reactor volume is 5 to 10,000 L, 10 to 5000 L, 20 to 2000 L, or 50 to 1000 L.

[0151] Methods are provided for making polynucleotides using polymerases and ligases. Two or more complementary polynucleotide or oligonucleotide sequences, such as a template polynucleotide to which segments are attached, are used. A polymerase is used to fill each gap between the complementary polynucleotide or oligonucleotide sequences. The adjacent sequences are fused together using a ligase. Advantageously, the entire method is fully enzymatic, which can reduce the complexity and increase the efficiency of the method. The two or more complementary polynucleotide or oligonucleotide sequences can include modified nucleotides. Alternatively, or in addition, the nucleotides introduced by the polymerase can include modified nucleotides. This allows for the creation of therapeutic polynucleotides and oligonucleotides that can be short oligonucleotides (e.g., 10-30 nucleotides in length) or long polynucleotides (e.g., 30-10,000 nucleotides in length). Two or more complementary polynucleotide or oligonucleotide sequences can be attached to the template polynucleotide and a polymerase can fill each gap between each complementary polynucleotide or oligonucleotide sequence. A ligase can then be used to ligate each adjacent sequence together.

[0152] The final single-stranded product can be separated from the template using methods described herein. Optionally, the template can have properties described herein that allow the template to be reused for subsequent reactions.

[0153] In one example, a method is provided for making gapmers, which are oligonucleotides, typically 10-30 nucleotides in length, that contain at least one modified nucleotide residue. Generally, gapmers contain a central region that contains unmodified nucleotide residues and two wing regions (5' and 3' wings) on either side of the central region, each of which contains at least one modified nucleotide residue. In one embodiment, a polymerase is used to make the central region of the gapmer. In one example, the 5' segment can be used as a primer for the polymerase to make the central region of the gapmer, and the 3' segment can be used as a stopper for the polymerase. A ligase can then be used to fuse the central segment with the 3' segment to create the gapmer product. In one embodiment, a polymerase is used to make a portion of the central region of the gapmer. In one embodiment, the 5' segment can be used as a primer for the polymerase to make the segments of the gapmer, and the 3' segment can be used as a stopper sequence for the polymerase. The 5' segment can include or consist of the wing region. The 5' segment may comprise a portion of the wing region. The 3' segment may comprise or consist of the wing region. The 3' segment may comprise a portion of the wing region. In this respect, the complexity of the method is significantly reduced by reducing the number of rounds of production. In an embodiment, the central region comprises or consists of deoxynucleotides linked by phosphorothioate linkages, i.e., there are no sugar modifications in the central region, but the backbone is a full phosphorothioate backbone, and a polymerase is used to generate the central region of the gapmer. In an embodiment, the 5' segment is a primer and the 3' segment is a stopper, each segment comprises or consists of 2'-MOE sugar modified nucleotides linked by phosphorothioate linkages.In one embodiment, the central region of the gapmer comprises or consists of deoxynucleotides linked by phosphorothioate linkages, and a polymerase is used to generate the central region of the gapmer, the 5' segment is a primer and the 3' segment is a stopper, and each segment comprises or consists of 2'-MOE sugar modified nucleotides linked by phosphorothioate linkages. In one embodiment, the gapmer has a fully phosphorothioate backbone, a 5' wing that is fully 2'MOE sugar modified, a 3' wing that is fully 2'MOE sugar modified, and a central region that is fully deoxyribose, and the gapmer is generated using the 5' segment corresponding to the 5' wing as a primer, the 3' segment corresponding to the 3' wing as a stopper, and a pool of polymerase and deoxynucleoside alpha-thiotriphosphates to generate the central region. In one embodiment, the gapmer is a 5-10-5 gapmer.

[0154] In traditional polynucleotide production methods, such as solid-phase synthesis, single nucleotides are added to single-stranded oligonucleotides in a template-independent manner, allowing the synthesis of oligonucleotides with defined sequences. Such techniques can be used to create complete oligonucleotide products by repeatedly adding single bases. However, unless each synthesis cycle is performed with 100% yield, sequence deletion errors will be incorporated into the final product. For example, if an oligonucleotide is extended by one nucleotide in a synthesis cycle with a 99% yield, the remaining 1% is available to react in a subsequent synthesis cycle, but the product formed will be one nucleotide shorter than the desired product. Since the error rate increases cumulatively as the number of cycles increases, in this example, a cycle yield of 99% may result in the formation of 20% one-base-shortened sequences in the case of the production of a 20-mer.

[0155] Attempts to overcome this by using only short, typically 5-8 nucleotide long, sequences synthesized by the addition of single nucleotides using ligases or transferases capable of adding single nucleotides to single stranded oligonucleotides have resulted in short sequences with higher purity than longer sequences because the short sequences are exposed to fewer cycles of error accumulation. These short sequences are constructed on complementary DNA templates and then joined together. The use of complementary templates in conjunction with ligase ensures that only short oligonucleotides with both the correct length and the correct sequence are constructed. However, such methods still require multiple rounds of synthesis to add each nucleotide to another to create each short sequence and then combine them. This is time consuming and complicated.

[0156] Thus, the methods of the present disclosure provide more efficient methods for generating single-stranded oligonucleotide and polynucleotide products with higher overall yields and greater overall sequence accuracy.

[0157] According to the present disclosure, by using a polymerase in combination with a ligase, single-stranded oligonucleotide and polynucleotide products can be generated in an efficient manner and without repeated rounds of chemical synthesis.

[0158] The sequential accumulation of errors is also avoided.First, by using at least two segments of product sequence and using polymerase to fill gaps, this allows multiple shorter sequences to constitute the final product, resulting in fewer rounds of synthesis compared to, for example, solid-phase synthesis, in which errors may be introduced.Polymerase also has high sequence accuracy, reducing the possibility of error incorporation.This further reduces the restriction on the scale-up of the method for commercial oligonucleotide and polynucleotide production.

[0159] Second, assembly of the final product on a complementary polynucleotide template, with subsequent ligation, ensures that segments of the correct length and sequence required for the final product are assembled in the correct order and chirality, allowing highly accurate, individualized final products to be generated.

[0160] In an exemplary embodiment, the method is fully enzymatic, allowing for fewer rounds of synthesis and increased efficiency.In fact, the polymerase and ligase steps can be performed simultaneously, and thus the final product can be produced in one round of the method.When the polymerase and ligase steps are performed simultaneously, this may be known as "one-pot".

[0161] In another exemplary embodiment, the method is further simplified and provides further efficiency by using a short primer sequence for polymerase that is complementary to the 3' end of the template (in combination with the template polynucleotide or oligonucleotide described herein). Polymerase then extends the primer sequence along the template. A stopper that is complementary to the 5' end of the template can be used to stop the polymerase. Stop sequences can be unnecessary if the correct amount of nucleoside triphosphate is added to the reaction. This reduces the number of rounds of nucleotide addition required and the number of ligations required, resulting in a more efficient method with higher overall yield and higher overall sequence accuracy.

[0162] In some embodiments, to reduce or prevent destruction of the segment polynucleotide or oligonucleotide sequence, the polymerase is modified as described herein to eliminate both 3' to 5' exonuclease activity and 5' to 3' exonuclease activity.

[0163] In some embodiments, the polynucleotide product is an mRNA, and the coding region comprises nucleotides that are unmodified except for replacement of uridine residues with pseudouridine or N1-methyl-pseudouridine. In some embodiments, a polymerase is used to generate part or all of the coding region of the mRNA. In some embodiments, the 5'UTR, the 3'UTR, or both the 5'UTR and the 3'UTR comprise one or more modified nucleotides. In some embodiments, phosphorothioate linkages at either cytidine or both cytidine and uridine are included in the 5'UTR. In some embodiments, the polyA tail comprises one or more modifications that are not susceptible to 3'-5' exonuclease. In some embodiments, the polyA tail comprises one or more phosphorothioate linkages. In some embodiments, the polyA tail comprises phosphorothioate linkages at its 3' end. In some embodiments, the polyA tail comprises at least six phosphorothioate linkages at its 3' end.

[0164] Polynucleotide or oligonucleotide products according to this disclosure may possess at least one modified sugar moiety, nucleobase modification, and / or backbone modification described herein.

[0165] In some embodiments, one or more segment polynucleotides or oligonucleotides can have at least one modified nucleotide residue. In some embodiments, all segment polynucleotides or oligonucleotides have at least one modified nucleotide residue. In other embodiments, one or more segment polynucleotides or oligonucleotides can have no modified nucleotide residue. In some embodiments, the polymerase can incorporate at least one modified nucleotide residue into the extended sequence. In other embodiments, the polymerase can incorporate an unmodified nucleotide residue into the extended sequence.

[0166] In some embodiments, one or more segment polynucleotides or oligonucleotides can have at least one modified nucleotide residue and the polymerase can incorporate at least one modified nucleotide residue into the extended sequence. In some embodiments, one or more segment polynucleotides or oligonucleotides can have at least one modified nucleotide residue and the polymerase can incorporate an unmodified nucleotide residue into the extended sequence. In some embodiments, one or more segment polynucleotides or oligonucleotides can have no modified nucleotide residue and the polymerase can incorporate at least one modified nucleotide residue into the extended sequence. In some embodiments, one or more segment polynucleotides or oligonucleotides can have no modified nucleotide residue and the polymerase can incorporate an unmodified nucleotide residue into the extended sequence.

[0167] In embodiments, the product may be a gapmer. In embodiments, the wing regions (optionally the 5' and / or 3' segment oligonucleotides) may include backbone and sugar modifications, and the central region may include a backbone modification but no sugar modification. In embodiments, the wing regions may include at least one sugar modification or may consist entirely of modified sugars. In embodiments, the 5' and 3' wings of the gapmer comprise or consist of 2'-MOE modified nucleotides. In embodiments, the central region of the gapmer comprises or consists of nucleotides that contain a hydrogen at the 2' position of the sugar moiety, i.e., DNA-like. In embodiments, the 5' and 3' wings of the gapmer consist of 2'MOE modified nucleotides and the central region of the gapmer consists of nucleotides that contain a hydrogen at the 2' position of the sugar moiety (i.e., deoxynucleotides). In some embodiments, the 5' and 3' wings of the gapmer are composed of 2'MOE modified nucleotides, the central region of the gapmer is composed of nucleotides containing hydrogen at the 2' position of the sugar moiety (i.e., deoxynucleotides), and all internucleotide linkages are phosphorothioate linkages. In some embodiments, the gapmer is selected from the group consisting of valifolsene, bepirovirsen, custilsene, daprsilane, donidarolsen, eprontersen, furenosilsene, inotersen, rademirsene, mipomersen, olezarsen, tominersen, urefnersen, boranesorsen, and zircnersen. In some embodiments, the gapmer is a 5-10-5 gapmer. In some embodiments, the gapmer is a 6-8-6 gapmer. In some embodiments, the gapmer is a 4-12-4 gapmer. In some embodiments, the gapmer is a 7-6-7 gapmer.

[0168] Methods are provided in which the resulting product is greater than 90% pure. In embodiments, the product may be greater than 95% pure. In embodiments, the product may be greater than 96% pure. In embodiments, the product may be greater than 97% pure. In embodiments, the product may be greater than 98% pure. In embodiments, the product may be greater than 99% pure. The purity of a polynucleotide or oligonucleotide can be determined using any suitable method, such as high performance liquid chromatography (HPLC) or mass spectrometry (MS), particularly liquid chromatography-MS (LC-MS), HPLC-MS, or capillary electrophoresis mass spectrometry (CEMS).

[0169] In certain embodiments, the single stranded polynucleotides or oligonucleotides generated include alicaforsen, Apc001PE, AS1411, valifolsen, bepilovirsen, BIIB080, BIIB094, BIIB101, BIIB105, BIIB115, BIIB121, BIIB132, casimersen, simdelirsen, CpG1018, CpG7909, custilsen, daprsiran, donidarolsen, drisapersen, eprontersen, eteplirsen, fesomersen, homiversen, furenlosilsen, golodirsen, imetelstat, inotersen, ION224, ION260, ION306, ION363, ION455, ION464, ION532, ION541, ION582, ION839, ION859, ION904, IONIS-AGT-L Rx , IONIS-FB-L Rx , IONIS-MAPT Rx, rademirsen, lexanersen, mipomersen, mongelsen, nusinersen, NOX-E36, olezarsen, pegaptanib, peracarsen, plexigeversen, lenadilsen, RG6048, rugonersen, sapavursen, sepofarsen, subodirsen, tofersen sodium, tominersen, urefnersen, urtebrusen, besleteplirsen, viltolarsen, voranesorsen, WVE-003, WVE-004, WVE-006, WVE-N531, and dirganersen.

[0170] In some embodiments, the polynucleotide or oligonucleotide produced is an antisense polynucleotide or oligonucleotide. In some embodiments, the antisense oligonucleotide is selected from the group consisting of alicaforsen, valifolsen, bepilovirsen, BIIB080, BIIB094, BIIB101, BIIB105, BIIB115, BIIB121, BIIB132, simdelirsen, casimersen, custilsen, daprsiran, donidarolsen, drisapersen, eprontersen, eteplirsen, fesomersen, homiversen, furenlosilsen, golodirsen, imetelstat, inotersen, ION224, ION260, ION306, ION363, ION455, ION464, ION532, ION541, ION582, ION839, ION859, ION904, IONIS-AGT-L Rx , IONIS-FB-L Rx , IONIS-MAPT Rx , rademirsen, lexanersen, mipomersen, mongelsen, nusinersen, olezarsen, peracarsen, plexigeversen, lenadilsen, RG6048, rugonersen, sapabursen, sepofarsen, subodirsen, tofersen sodium, tominersen, urefnersen, urtebrusen, besleteplirsen, viltolarsen, voranesorsen, WVE-003, WVE-004, WVE-N531, and dirganersen.

[0171] In some embodiments, the polynucleotide or oligonucleotide produced is an aptamer. In some embodiments, the aptamer is pegaptanib, Apc001PE, AS1411, or NOX-E36.

[0172] In some embodiments, the polynucleotide or oligonucleotide produced is an mRNA, such as Cas9. In one embodiment, the polynucleotide or oligonucleotide produced is an mRNA vaccine. In some embodiments, the mRNA encodes one or more immunogens. In further embodiments, these immunogens can be selected from respiratory syncytial virus (RSV) immunogens, Epstein-Barr virus glycoprotein immunogens, cytomegalovirus glycoprotein immunogens, coronavirus spike polypeptide immunogens, influenza virus immunogens, varicella zoster virus glycoprotein immunogens, human papillomavirus 16 (HPV16) E6 immunogens, HPV16 E7 immunogens, or flavivirus immunogens. In further embodiments, the immunogens can be selected from coronavirus spike proteins, influenza antigens, and RSV antigens, such as f protein or g protein.

[0173] In certain embodiments, the mRNA is selected from the group consisting of AZD8601, BNT111, BNT112, BNT113, BNT115, BNT116, BNT122, BNT131, BNT141, BNT142, BNT151, BNT152, BNT153, BNT161, BNT162b2, BNT163 BNT164, BNT165, LUNAR-CF, LUNAR-COV19, LUNAR-FLU, LUNAR-GSDIII, LUNAR-OTC, MEDI1191, mRNA-1273, mRNA-0184, mRNA-1010, mRNA-1020, mRNA-1030, mRNA-1011, mR NA-1012, mRNA-1045, mRNA-1073, mRNA-1189, mRNA-1195, mRNA-1215, mRNA-1230, mRNA-1273, mRNA-1273.211, mRNA-1273.213, mRNA-1273.214, mRNA-1273.222, mRNA- 1273.351, mRNA-1273.529, mRNA-1273.617, mRNA-1283, mRNA-1287, mRNA-1345, mRNA-1365, mRNA-1468, mRNA-1574, mRNA-1608, mRNA-1644, mRNA-1647, mRNA-1653, mRNA-1893, mRNA2752, mRNA-3139, mRNA-3283, mRNA-3351, mRNA-3705, mRNA-3745, mRNA-3927, mRNA-4157, mRNA-4359, mRNA-5671, mRNA-6981, and VX-522. In further embodiments, the mRNA is selected from the group consisting of mRNA-1045, mRNA-1230, mRNA-1345, mRNA-1365, EBV mRNA-1189, mRNA-1195, mRNA-1647, BNT162b2, LUNAR-COV19, mRNA-1073, mRNA-1273.213, mRNA-1273.214, mRNA-1273.222, mRNA-1273.351, mRNA-1273.529, mRNA-1273.617, mRNA-1283, mRNA-1287, mRNA-1345, BNT161, mRNA-1468, BNT113, and mRNA-1893.In further embodiments, the mRNA is selected from the group consisting of mRNA-1045, mRNA-1230, mRNA-1345, mRNA-1365, BNT162b2, LUNAR-COV19, mRNA-1073, mRNA-1273.213, mRNA-1273.214, mRNA-1273.222, mRNA-1273.351, mRNA-1273.529, mRNA-1273.617, mRNA-1283, mRNA-1287, mRNA-1345, BNT161, and LUNAR-FLU.

[0174] In some embodiments, the polynucleotide or oligonucleotide product is an adjuvant. In some embodiments, the adjuvant is a CpG oligonucleotide. In some embodiments, the adjuvant is CpG1018 or CpG7909.

[0175] In embodiments, the product is a therapeutic polynucleotide or oligonucleotide.

[0176] In some embodiments, the method can generate double-stranded polynucleotides or oligonucleotides, where two complementary single-stranded polynucleotides or oligonucleotides are generated by the methods described herein and then mixed under conditions that allow annealing, such conditions being readily apparent to those skilled in the art. In some embodiments, the product is an siRNA. In some embodiments, the siRNA is selected from the group consisting of ALN-AAT02, ALN-APP, ALN-TTRsc04, ALN-HBV02, ALN-HSD, ALN-KHK, ALN-PNP, ARO-AAT, ARO-ANG3, AOC 1001, AOC 1020, AOC 1044, ARO-APOC3, ARO-C3, ARO-COV, ARO-DUX4, ARO-ENaC2, ARO-MUC5AC, ARO-MMP7, ARO-PNPLA3, ARO-RAGE, versesiran, semdisiran, cosdosilan, dapursilan, DCR-AUD, DCR-CM3, DCR-CM4, DCR-COMP1, DCR-COMP2, DCR-LIV2, DCR-LLY11, DCR-LLY12, DCR-NOVO1, DCR-NOVO2, elebsilan, fazilsilan, fitusilan, givosiran, HZN-457, inclisiran, JNJ-3989, lumasiran, LY3561774, LY3819469, nedosiran oran, olpaciran, patisiran, levusiran, RG6346, RIM730, SLN124, SLN501, SLN-HAN-1, SLN-MNK-2, SLN-MNK-3, SLN-AZ-1, SLN-AZ-2, STP122G, STP125G, STP135G, STP136G, STP144G, STP145G, STP146G, STP152G, STP155G, STP237G, STP247G, STP251G, STP355, STP369, STP702, STP779, STP705, STP707, teprasiran, chibanisiran, butrisiran, zerulasiran, dilebesiran, and difcasiran. In certain embodiments, the product is a miRNA or a miRNA mimic. In certain embodiments, the miRNA mimic is remlarsen.

[0177] The invention disclosed herein takes advantage of the properties of oligonucleotide binding to provide an improved method for their preparation. By providing a template oligonucleotide with 100% complementarity to the target sequence and controlling the reaction conditions so that the product can be released and separated under specific conditions, a product with a high degree of purity can be obtained.

[0178] Product (or impurity): Denaturation of the template duplex and separation of the product (or impurity) from the template Releasing the product (or any impurities) from the template requires that the Watson-Crick base pairing between the template polynucleotide or oligonucleotide strand and the product (or impurity) be broken (i.e., denaturing the duplex). The product (or impurity) can then be separated from the template, which can be done as two separate steps or as one combined step.

[0179] If the method is carried out in a column reactor, the release and separation of the product (or impurities) can be carried out in one step. Changing the pH or salt concentration, or carrying out in a buffer containing chemicals that disrupt base pairing (e.g., formamide or urea), causes denaturation of the polynucleotide or oligonucleotide chain, and the product (or impurities) is eluted in the buffer.

[0180] If the method is carried out in another reaction vessel, the release and separation of the product (or impurities) can be carried out as a two-step process. First, the Watson-Crick base pairs are broken to denature the strands, and then the product (or impurities) are separated from the template, e.g., removed from the reaction vessel. If the release and separation of the product is carried out as a two-step process, the cleavage of the Watson-Crick base pairs can be achieved by changing the buffer conditions (pH, salt) or by introducing chemical disrupting agents (formamide, urea). Alternatively, increasing the temperature can also cause the dissociation of the two strands, i.e., denaturation. The product (or impurities) can then be separated (and, if desired, removed from the reaction vessel) via methods including separation based on molecular weight, separation based on charge, separation based on hydrophobicity, separation based on specific sequences, or a combination of these methods.

[0181] When the method is carried out in a continuous or semi-continuous flow reactor, the release and separation of the product (or impurities) can be carried out in either one step or two steps. For example, the release and separation of the product (or impurities) in one step can be effected by increasing the temperature to cause dissociation of the two strands and separating the released strands based on molecular weight in the same part of the reactor used to increase the temperature. The release and separation of the product (or impurities) in two steps can be effected by increasing the temperature to cause dissociation of the two strands in one part of the reactor and separating the released strands based on molecular weight in a different part of the reactor.

[0182] Specific release and separation of impurities from the template and retention of products on the template Impurities arise when incorrect nucleotides are incorporated into the oligonucleotide chain during chain extension or when the chain extension reaction is terminated prematurely. Impurities also arise when a reaction includes a step of ligating segment polynucleotides or oligonucleotides and one or more of the ligation steps do not occur.

[0183] The Watson-Crick base pairing property can be exploited to specifically release any impurities bound to the template prior to release of the product. Each double-stranded polynucleotide or oligonucleotide dissociates under specific conditions, the conditions for sequences that do not have 100% complementarity being different when compared to sequences that have 100% complementarity. Determining such conditions is within the skill of the art.

[0184] A common means of denaturing polynucleotides or oligonucleotides is to increase the temperature. The temperature at which half of the base pairs dissociate, i.e., 50% of the duplex becomes single-stranded, is called the melting temperature T m The most reliable and accurate means of determining the melting temperature is by experience. However, this is tedious and usually not necessary. Several formulas exist for the T m Melting temperature calculators can be used to calculate melting temperature values ​​(Nucleic Acids Research 1987, 15(13):5069-5083; PNAS 1986, 83(11):3746-3750; Biopolymers 1997, 44(3):217-239), and numerous melting temperature calculators are maintained by reagent suppliers and universities and can be found online. For a given polynucleotide or oligonucleotide sequence, variants that are all phosphorothioate linkages are known to melt at a lower temperature than variants that are all phosphodiester linkages. Increasing the number of phosphorothioate linkages in a polynucleotide or oligonucleotide increases the T value of the polynucleotide or oligonucleotide as its intended target. m tends to decrease

[0185] To specifically separate impurities from the reaction mixture, the melting temperature of the product:template duplex is first calculated. The reaction vessel is then heated to a first temperature, for example, a temperature below the melting temperature of the product:template duplex, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 degrees Celsius below the melting temperature. This heating step causes denaturation of polynucleotides or oligonucleotides that are not template-derived products, i.e., that are not 100% complementary to the template. These denatured polynucleotides or oligonucleotides can then be removed from the reaction vessel using one of the methods disclosed above, for example, separation based on molecular weight, charge, hydrophobicity, specific sequence, or a combination of these methods. The reaction vessel is then raised to a second, higher temperature, for example, above the calculated melting temperature, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 degrees Celsius above the melting temperature, to cause denaturation of the template-derived products. The products can then be separated (and removed from the reaction vessel) using one of the methods disclosed above, for example, separation based on molecular weight, separation based on charge, separation based on hydrophobicity, separation based on specific sequences, or a combination of these methods.

[0186] Similar methods can be used when the disrupting agent is an agent that causes changes in pH or salt concentration or is a chemical disrupting agent.The disrupting agent is increased in concentration to just before the concentration at which product can dissociate, causing the denaturation of non-template-derived polynucleotides or oligonucleotides.These impurities can then be removed from the reaction vessel using one of the methods disclosed above.The disrupting agent is then increased in concentration to exceed the concentration at which product dissociates from template.Then, product can then be removed from the reaction vessel using one of the methods disclosed above.

[0187] The product obtained from the methods as disclosed above has a high degree of purity without the need for further purification steps, for example, the product obtained is greater than 95% pure.

[0188] Mold characteristics In some embodiments, the template may have properties that allow it to be retained in the reaction vessel when the product is removed to prevent it from becoming an impurity in the product. In one embodiment of the present invention, this retention is achieved by coupling the template to a support material. This coupling results in a template-support complex that has a high molecular weight and can therefore be retained in the reaction vessel when the impurities and product are removed, for example, by filtration. The template can be coupled to a solid support material such as polymer beads, fibrous supports, membranes, streptavidin-coated beads, and cellulose. The template can also be coupled to a soluble support material such as polyethylene glycol, soluble organic polymers, DNA, proteins, dendrimers, polysaccharides, oligosaccharides, and carbohydrates.

[0189] Each support material can have a plurality of points to which a template can be attached, and each attachment point can have a plurality of templates attached thereto.

[0190] The template may itself have a high molecular weight without being attached to a support material, for example, it may be a molecule having multiple copies of the template separated, for example, by linkers, in the manner shown in FIG.

[0191] The ability to retain the template in the reaction vessel also allows the template to be reused for subsequent reactions by recovery or by use in continuous or semi-continuous flow methods, which is useful, for example, in oligonucleotide production, such as gapmer production, and therapeutic mRNA production.

[0192] The properties of the template may allow for separation of the template and the product, or the template-bound product and impurities. Separation based on molecular weight, charge, hydrophobicity, specific sequence, or a combination of these methods can be used.

[0193] When the template is attached to a solid support, separation of the template from the product or separation of impurities from the template-bound product is achieved by washing the solid support under appropriate conditions that can be easily understood by those skilled in the art. When the template is coupled to a soluble support or the template itself is composed of a repeated template sequence, separation of the template from the product or separation of the template-bound product from impurities can be achieved by molecular weight based separation, for example, by using techniques such as ultrafiltration or nanofiltration, where the filter material is selected so that larger molecules are retained by the filter and smaller molecules pass through. If a single separation step of impurities from the template-product complex or separation of the product from the template is not efficient enough, multiple sequential filtration steps can be used to increase the separation efficiency and thus generate a product that meets the desired purity.

[0194] It would be desirable to provide a method for the separation of such polynucleotides or oligonucleotides that is efficient and applicable to industrial production scale. "Therapeutic oligonucleotides: The state of the art in purification technologies," Sanghvi et al., Current Opinion in Drug Discovery (2004) Vol. 7, No. 8, reviews the methods used for oligonucleotide purification.

[0195] WO01 / 55160A1 discloses the purification of oligonucleotides by forming imine linkages with contaminants and then removing the imine-linked impurities using chromatography or other techniques. "Size Fractionation of DNA Fragments Ranging from 20 to 30000 Base Pairs by Liquid / Liquid chromatography" Muller et al., Eur. J. Biochem (1982) 128-238 discloses the use of a solid column of microcrystalline cellulose on which a PEG / dextran phase is deposited for the separation of nucleotide sequences. "Separation and identification of oligonucleotides by hydrophilic interaction chromatography." Easter et al., The Analyst (2010); 135(10) discloses the separation of oligonucleotides using a modified HPLC method using a solid silica support phase. "Fractionation of oligonucleotides of yeast soluble ribonucleic acids by countercurrent distribution," Doctor et al., Biochemistry (1965) 4(1) 49-54, discloses the use of dry solid columns packed with dry DEAE-cellulose. "Oligonucleotide composition of a yeast lysine transfer ribonucleic acid," Madison et al., Biochemistry, 1974, 13(3), discloses the use of solid phase chromatography for the separation of nucleotide sequences.

[0196] Liquid-liquid chromatography is a known separation technique. "Countercurrent Chromatography The Support-Free Liquid Stationary Phase" Billardello, B.; Berthod, A.; Wilson & Wilson's Comprehensive Analytical Chemistry 38; Berthod, A. ed.; Elsevier Science BV: Amsterdam (2002) pp. 177-200 provides a useful general description of liquid-liquid chromatography. Various liquid-liquid chromatography techniques are known. One such technique is liquid-liquid countercurrent chromatography (referred to herein as "CCC"). Another known technique is centrifugal partition chromatography (referred to herein as "CPC").

[0197] The methods disclosed above and described in WO2013 / 030263 can be used, for example, to separate product polynucleotides or oligonucleotides from templates and / or impurities.

[0198] Polymerase In embodiments, the polymerase used may be a DNA polymerase, an RNA polymerase, or a combination thereof. The polymerase may be a mutant polymerase. The polymerase may be an engineered polymerase. The polymerase may catalyze the attachment of deoxyribonucleotides to deoxyribonucleotides, deoxyribonucleotides to ribonucleotides, ribonucleotides to deoxyribonucleotides, and / or ribonucleotides to ribonucleotides. The polymerase may lack strand displacement activity. The polymerase may lack 5'→3' exonuclease activity. The lack of 5'→3' exonuclease activity may prevent the destruction of the stopper. The polymerase may have 3'→5' exonuclease activity. The 3'→5' exonuclease activity typically allows the polymerase to remove misincorporated nucleotides, thereby ensuring high fidelity synthesis. The 3'→5' exonuclease activity may be known as proofreading ability. The polymerase may lack 3'→5' exonuclease activity. Different polymerase properties may be combined as required, for example, the polymerase may lack strand displacement activity, and / or may lack 5'→3' exonuclease activity, and / or may have 3'→5' exonuclease activity. For example, the polymerase may lack strand displacement activity, and / or may lack 5'→3' exonuclease activity, and / or may lack 3'→5' exonuclease activity. The polymerase may include DNA and / or RNA polymerases. The polymerase may be a DNA-dependent DNA polymerase (i.e., a polymerase that synthesizes DNA using a DNA template). The polymerase may be a DNA-dependent RNA polymerase (i.e., a polymerase that synthesizes RNA using a DNA template). The DNA polymerase may bind deoxyribonucleotides and / or ribonucleotides. RNA polymerase can bind ribonucleotides and / or deoxyribonucleotides.The polymerase can be a primer extension polymerase. The DNA polymerase can be a primer extension polymerase. The RNA polymerase can be a primer extension polymerase. The DNA and / or RNA polymerase can be a wild type polymerase. The DNA and / or RNA polymerase can be a mutant DNA and / or RNA polymerase. The DNA and / or RNA polymerase can be an engineered DNA and / or RNA polymerase. The polymerase can be capable of binding modified nucleotides. The DNA and / or RNA polymerase can be capable of binding modified nucleotides. The polymerase can fill at least one sequence gap along the template strand. The polymerase can extend at least one segment polynucleotide using a pool of nucleoside triphosphates. The DNA polymerase typically requires a primer and a template. Exemplary polymerases include wild-type Escherichia phage T7 polymerase, wild-type Sulfolobus solfataricus polymerase, mutant Thermococcus species (strain 9oN-7) polymerase, wild-type Enterobacteria phage T4 polymerase, wild-type Thermus aquaticus polymerase, and wild-type Thermococcus kodakarens polymerase.

[0199] Polymerases capable of binding unmodified nucleotides to other unmodified nucleotides, polymerases capable of binding unmodified nucleotides to modified nucleotides (i.e., modified 5' nucleotides to unmodified 3' nucleotides and / or unmodified 5' nucleotides to modified 3' nucleotides), and polymerases capable of binding modified nucleotides to other modified nucleotides are within the scope of the present disclosure. The polymerase can bind unmodified nucleotides to other unmodified nucleotides. Each unmodified nucleotide can then be modified. Examples of modifications of nucleotides are disclosed herein and include modifications selected from the group including modified sugar moieties, modifications of nucleobases, modifications of the backbone, substitution of one or more uracil residues, and combinations thereof.

[0200] Additionally, exemplary engineered DNA and RNA polymerases capable of incorporating modified nucleotides include those disclosed in "Engineering and application of polymerases for synthetic genetics", Houlihan et al., Current Opinion in Biotechnology 2017, 48;168-179.

[0201] In embodiments, DNA and / or RNA polymerases can be engineered to accept 2' sugar modifications, including polymerases with mutations in the polymerase thumb subdomain of Thermococcus gogonarius replicative DNA polymerase, optionally including the E664K and Y409G mutations. Such polymerases provide for the inclusion of pseudouridine, 5-methyl-C, 2'-fluoro, or 2-azido modified NTPs, or combinations thereof, primed from, for example, DNA, RNA, locked nucleic acid, or 2'-OMe RNA modified nucleotides.

[0202] In some embodiments, RNA polymerases engineered to accept 2' sugar modifications include T7 RNA polymerase, such as T7 RNA polymerases that contain the Y639F mutation, which can facilitate the inclusion of, for example, 2' fluoropyrimidines and 2' aminopyrimidines.

[0203] In some embodiments, a variant of the Stoffel fragment of Taq polymerase (SM19) that is engineered to accept 2' sugar modifications is used. For example, the introduction of a negatively charged amino acid at 614 and the mutation E615G achieves the inclusion of 2' sugar modifications. SM19 can be further evolved into polymerases SFM4-3 and SFM4-9. For example, SFM4-3 can transcribe a fully modified 2'-OMe 60 nucleotide sequence.

[0204] In some embodiments, a thermophilic RNA polymerase from the marine cyanophage Syn5 that has been engineered to accept 2' sugar modifications is used.

[0205] In some embodiments, a Tgo polymerase containing the mutations Y409G, I521L, F545L, and E664K is used, which is capable of synthesizing DNA and RNA with regioisomeric 2'-5' linkages by incorporation of 3' deoxy or 3'-OMe nucleotides.

[0206] Compartmentalized self-replication methods are useful for evolving polymerases to incorporate modified nucleotides. "Directed evolution of polymerase function by compartmentalized self-replication," Ghadessy et al., Proc. Natl. Acad. Sci. USA 2001, 98:4552-4557, describes an exemplary compartmentalized self-replication method.

[0207] Ligase In some embodiments, the ligase can be an ATP-dependent ligase. The ATP-dependent ligase ranges in size from 30 to less than 100 kDa. In some embodiments, the ligase can be an NAD-dependent ligase. NAD-dependent enzymes are highly homologous, monomeric proteins of 60 to 90 kDa, optionally 70 to 80 kDa. In some embodiments, the ligase can be a thermostable ligase. The thermostable ligase can be derived from a thermophilic bacterium.

[0208] In embodiments, the ligase can be a template-dependent ligase. In embodiments, ligation is performed on a template. In embodiments, ligating the segmented polynucleotides and / or extended segmented polynucleotides is performed on a template using a ligase to form a single-stranded polynucleotide product. In embodiments, the ligase can be a duplex-acting ligase. The duplex can be a duplex DNA. The duplex can be an RNA:DNA hybrid duplex. The duplex can be a duplex RNA. The ligase can be a DNA ligase. The ligase can be an RNA ligase.

[0209] In some embodiments, the ligase can catalyze the joining of two segment polynucleotides and / or extended segment polynucleotides. In some embodiments, the ligase can catalyze the joining of two segment oligonucleotides. The ligase can catalyze the joining of a segment polynucleotide or extended segment polynucleotide comprising naturally occurring nucleotides with a segment polynucleotide or extended segment polynucleotide comprising naturally occurring nucleotides. The ligase can catalyze the joining of a segment polynucleotide or extended segment polynucleotide comprising at least one modified nucleotide with a segment polynucleotide or extended segment polynucleotide comprising naturally occurring nucleotides. The segment polynucleotide or extended segment polynucleotide comprising at least one modified nucleotide may be located at the 3' end of the junction. The segment polynucleotide or extended segment polynucleotide comprising naturally occurring nucleotides may be located at the 5' end of the junction. The modified nucleotide may or may not be located at the junction, i.e., one or both of the linking nucleotides are modified nucleotides, or none of them are modified nucleotides. The ligase can catalyze the ligation of a segment polynucleotide or an extended segment polynucleotide that comprises at least one modified nucleotide with a segment polynucleotide or an extended segment polynucleotide that comprises at least one modified nucleotide. Those skilled in the art will understand that the arrangement of the at least one modified nucleotide within the segment polynucleotide or extended segment polynucleotide can be configured to enhance ligation efficiency.

[0210] The ligase may be a wild type ligase. The ligase may be a mutant ligase. The ligase may be an engineered ligase. The DNA and / or RNA ligase may be a wild type DNA and / or RNA ligase. The DNA and / or RNA ligase may be a mutant DNA and / or RNA ligase. The DNA and / or RNA ligase may be an engineered DNA and / or RNA ligase. Those skilled in the art will understand that a suitable ligase can be selected based on the segment polynucleotide and / or extended segment polynucleotide that require ligation and / or the number and / or type and / or position of modifications in said segment polynucleotide and / or extended segment polynucleotide.

[0211] Exemplary ligases include wild-type Enterobacteria phage T3 ligase and wild-type bacteriophage T4 DNA ligase.

[0212] In some embodiments, the ligase can be immobilized, for example, on a bead.

[0213] Pool of polynucleotides or polynucleotides The polynucleotide or oligonucleotide used to generate the "pool of polynucleotides" of the disclosed method may comprise at least two segments of the product sequence. The polynucleotide or oligonucleotide used to generate the pool of the disclosed method may comprise at least two different segments of the product sequence. The at least two segments of the product sequence may differ in sequence. Each of the at least two segments corresponds to a different region of the product sequence. The pool of polynucleotides or oligonucleotides may comprise at least one segment polynucleotide or oligonucleotide that comprises at least one modified nucleotide residue. The pool of polynucleotides or oligonucleotides may comprise at least two segment polynucleotides or oligonucleotides, each of which comprises at least one modified nucleotide residue.

[0214] Thus, the pool is a heterogeneous set of polynucleotides or oligonucleotides. At least two segment polynucleotides or oligonucleotides vary in sequence, may be shorter than the target sequence, and may not have the same sequence as the target sequence.

[0215] At least two segment polynucleotides or oligonucleotides can be produced by enzymatic synthesis, chemical synthesis, optionally solid-supported synthesis or liquid-phase synthesis, or a combination thereof. Enzymatic synthesis can be performed using single-stranded ligases, transferases, polymerases, or a combination thereof.

[0216] In other examples, one or more or all of the segment polynucleotides or oligonucleotides can be produced using chemical synthesis. In other examples, one or more or all of the segment polynucleotides or oligonucleotides can be produced using chemical synthesis in combination with enzymatic synthesis, such as the use of polymerase, single-stranded ligase or transferase, or a combination thereof. In other examples, one or more or all of the segment polynucleotides or oligonucleotides can be produced using chemical synthesis, single-stranded ligase and / or transferase in combination with the use of polymerase to produce one or more segment oligonucleotides. In other examples, one or more or all of the segment polynucleotides or oligonucleotides can be produced using single-stranded ligase, transferase, polymerase, or a combination thereof in a fully enzymatic synthesis method. In a simplified method, single-stranded ligase and polymerase can be used in a fully enzymatic method for the production of a pool of two or more or all of the segment polynucleotides or oligonucleotides.

[0217] In one example of oligonucleotide production, a pool of oligonucleotides can include a 5' primer segment oligonucleotide that contains at least one modified nucleotide residue, produced using chemical and / or enzymatic synthesis (e.g., using single-stranded ligase, transferase, and / or polymerase), and a different 3' segment oligonucleotide that contains at least one modified nucleotide residue, produced using chemical and / or enzymatic synthesis (e.g., using single-stranded ligase, transferase, and / or polymerase).

[0218] A segment polynucleotide or oligonucleotide may act as a primer. A segment polynucleotide or oligonucleotide may act as a stopper. A segment polynucleotide or oligonucleotide may act as both a primer and a stopper. A segment polynucleotide or oligonucleotide may act as both a primer and a stopper when there is a sequence gap on both sides of the segment polynucleotide or oligonucleotide.

[0219] In one embodiment, in any of the methods of the disclosure, a segmented oligonucleotide may be provided that is a 5' primer segment oligonucleotide comprising at least 3 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 5' primer segment oligonucleotide comprising 3-50 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 5' primer segment oligonucleotide comprising 3-40 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 5' primer segment oligonucleotide comprising 3-30 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 5' primer segment oligonucleotide comprising 3-25 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 5' primer segment oligonucleotide comprising 3-20 nucleotides. In some embodiments, the 5' primer segment oligonucleotide comprises at least 5 nucleotides. In some embodiments, the 5' primer segment oligonucleotide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, optionally one or more of the nucleotides are modified nucleotides. In some embodiments, the modified nucleotide in the 5' primer segment oligonucleotide is pseudouridine, N1-methylpseudouridine, 5-Me, 2'-F, 2'OMe, or 2'MOE.

[0220] In one embodiment, a segmented oligonucleotide may be provided that is a 3' stopper segment oligonucleotide comprising at least 3 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 3' stopper segment oligonucleotide comprising 3 to 50 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 3' stopper segment oligonucleotide comprising 3 to 40 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 3' stopper segment oligonucleotide comprising 3 to 30 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 3' stopper segment oligonucleotide comprising 5 to 30 nucleotides. In one embodiment, a segmented oligonucleotide may be provided that is a 3' stopper segment oligonucleotide comprising 3 to 20 nucleotides. In some embodiments, the 3' stopper segment oligonucleotide comprises at least 5 nucleotides. In some embodiments, the 3' stopper segment oligonucleotide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, optionally one or more of the nucleotides are modified nucleotides. In some embodiments, the modified nucleotide in the 3' stopper segment oligonucleotide is pseudouridine, N1-methylpseudouridine, 5-Me, 2'-F, 2'OMe, or 2'MOE.

[0221] In one embodiment, a segment oligonucleotide that is a 5' primer segment oligonucleotide comprising 3-50 nucleotides may be provided together with a segment oligonucleotide that is a 3' stopper segment oligonucleotide comprising 3-50 nucleotides. In one embodiment, a segment oligonucleotide that is a 5' primer segment oligonucleotide comprising 3-25 nucleotides may be provided together with a segment oligonucleotide that is a 3' stopper segment oligonucleotide comprising 3-30 nucleotides. In one embodiment, a segment oligonucleotide that is a 5' primer segment oligonucleotide comprising 5-25 nucleotides may be provided together with a segment oligonucleotide that is a 3' stopper segment oligonucleotide comprising 5-30 nucleotides. In one embodiment, a segment oligonucleotide that is a 5' primer segment oligonucleotide comprising 5-20 nucleotides may be provided together with a segment oligonucleotide that is a 3' stopper segment oligonucleotide comprising 5-30 nucleotides.

[0222] Enzymatic generation of segmented polynucleotides or oligonucleotides 1) Polymerase The polymerase can catalyze the linkage of the 3' hydroxyl group of the terminal nucleotide of a short oligonucleotide (primer) to the 5' phosphate of a nucleotide that is added in a template-dependent manner. The nucleotide that is added (i.e., the nucleoside triphosphate in the pool of nucleotide triphosphates) can be unmodified, i.e., naturally occurring, or modified as described herein. Separate templates and primers can be used, or a self-priming template can be used. The polynucleotide or oligonucleotide can be subsequently modified or further modified.

[0223] 2) Single-stranded ligases, such as RNA ligases Single-stranded ligases catalyze, for example, the ATP-driven addition of 3',5' nucleotide bisphosphates, 3',5' nucleotide thiophosphates (e.g., 3',5' bisthiophosphates or 3'-phosphate-5'-thiophosphates or 3'-thiophosphate-5'-phosphates), or 3',5' nucleotide dithiophosphates (e.g., 3',5' bisdithiophosphates or 3'-phosphate-5'-dithiophosphates or 3'-dithiophosphate-5'-phosphates) to the 3'-OH of short oligonucleotides (primers) in a template-independent manner. Those skilled in the art will appreciate that, although no additional phosphate (or thiophosphate) moieties are required, diphosphates (or dithiophosphates) or triphosphates (or other oligophosphates in which one or more oxygen atoms are replaced by sulfur) at the 3' position of the sugar moiety may also be used. Equivalently modified dinucleotides, trinucleotides, or tetranucleotides may be used in place of the individual nucleotides described above. Oligonucleotide primers are usually a minimum of three nucleotides in length. The resulting polynucleotide or oligonucleotide from this addition reaction is one nucleotide longer than the starting polynucleotide or oligonucleotide (or 2, 3, or 4 nucleotides longer than the starting polynucleotide or oligonucleotide, respectively, if di-, tri-, or tetra-nucleotides are used). The introduced nucleotide may be unmodified, i.e., naturally occurring, or modified as described herein. Here, the new 3' position is phosphorylated. To add the subsequent nucleotide, the 3' phosphate of the growing polynucleotide or oligonucleotide is removed by hydrolysis to generate a 3' OH. This hydrolysis is typically carried out using a phosphatase enzyme.

[0224] For example, single-stranded ligase can be used in a method of making segmented polynucleotides or oligonucleotides, where the method using single-stranded ligase includes a two-step reaction, i.e., 3' extension for segment synthesis, which includes addition and deprotection. An exemplary addition step involves ATP-dependent ligation of nucleotide-3',5'-bis(thio)phosphate to the 3'OH of a single-stranded nucleic acid primer, followed by deprotection of the 3' phosphate in the single-stranded polynucleotide or oligonucleotide by phosphatase. In another example, single-stranded ligase can be used in a method of making segmented polynucleotides or oligonucleotides, where the method includes an exemplary 3' extension (addition and deprotection) to make a segment sequence, followed by strand cleavage using a site-specific nuclease (e.g., endonuclease V, which cleaves the second phosphodiester bond one base after inosine, i.e., 3' to inosine) to release the segment.

[0225] 3) Transferase Terminal deoxynucleotidyl transferase (TdT) enzyme catalyzes the addition of 3'-protected nucleotide triphosphates, for example protected by 3'-O-azidomethyl, 3'-aminoxy or 3'-O-allyl groups, to the 3'OH of short oligonucleotides (primers) in a template-independent manner. The oligonucleotide primers are usually a minimum of three nucleotides in length. The introduced nucleotides can be unmodified, i.e. naturally occurring, or modified as described herein.

[0226] Suitable methods are described, for example, in EP2796552, US8808989, WO16128731A1, and WO16139477A1.

[0227] The primer oligonucleotides used in the above-described methods for generating segment polynucleotides or oligonucleotides are (1) If desired, it can be maintained as part of a segmented polynucleotide or oligonucleotide, or (2) Can be cleaved from the product polynucleotide or oligonucleotide to allow for separation of the desired product and the possibility of reuse to make additional segment polynucleotides or oligonucleotides. Cleavage of the primer from the segment polynucleotide or oligonucleotide can be performed using sequence-specific nucleases and appropriate design of the primer and segment so that cleavage is efficient and precise.

[0228] The present invention is exemplified by the following clauses: 1. A method for generating a single stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleotides and a polymerase and extending at least one segment polynucleotide using the pool of nucleotides and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; and g) changing conditions to denature the annealed template and the single-stranded polynucleotide products and separate the single-stranded polynucleotide products. A method comprising:

[0229] 2. The method of claim 1, wherein at least one segment polynucleotide comprises at least one modified nucleotide residue.

[0230] 3. The method of clause 1 or 2, wherein at least one segment polynucleotide comprises a 5' phosphate, a 5' phosphorothioate, a 5' phosphorodithioate, or a 5' methylphosphonate.

[0231] 4. The method of any one of clauses 1 to 3, wherein the pool of nucleotides in step (d) comprises (i) naturally occurring nucleotides, (ii) at least one modified nucleotide, or (iii) a plurality of modified nucleotides.

[0232] 5. The method of any one of clauses 1 to 4, wherein at least one modified nucleotide comprises a sugar moiety modification, a nucleobase modification, and / or a backbone modification.

[0233] 6. The method of any one of clauses 1 to 5, wherein at least one modified nucleotide comprises a modification at the 2' position of the sugar moiety, a bicyclic sugar or a 4'-CH(CH3)-O-2' group, or a combination thereof.

[0234] 7. The method of any one of clauses 1-6, wherein at least one modified nucleotide comprises 2'-F, 2'-OMe, 2'-MOE, or 2'-amino.

[0235] 8. The method of any one of clauses 1-7, wherein at least one modified nucleotide comprises a modified cytosine, 5-methylcytosine, 5-methylpyrimidine, 7-deazaguanosine, or an abasic nucleotide.

[0236] 9. The method of any one of clauses 1-8, wherein at least one modified nucleotide comprises a phosphorothioate, phosphoramidate, phosphorodiamidate, or phosphorodithioate.

[0237] 10. The method of any one of clauses 1-9, wherein at least one modified nucleotide comprises 1-methyl-pseudouridine, 5-methoxy-uracil, 1-ethyl-pseudouracil, pseudouracil, 1-methylpseudouracil, 5-methyl-cytidine, 5-methyl-cytosine, N6-methyladenosine, or 7-methylguanosine.

[0238] 11. The method of any one of clauses 1 to 10, wherein at least one modified nucleotide comprises 1-methyl-pseudouridine.

[0239] 12. The method of any one of clauses 1 to 4, wherein at least one modified nucleotide comprises a PMO, an LNA, a c-Et, a PNA, a BNA, or an L-ribonucleic acid.

[0240] 13. The method of any one of clauses 1-12, wherein the ligase in step (e) ligates the 3' and / or 5' end of each segment polynucleotide or extended segment polynucleotide to each adjacent segment polynucleotide or adjacent extended segment polynucleotide to form a product polynucleotide chain.

[0241] 14. The method of any one of clauses 1-13, wherein the ligase in step (e) is capable of ligating RNA to RNA, DNA to DNA, RNA to DNA, and / or DNA to RNA.

[0242] 15. The method of any one of clauses 1 to 14, wherein the ligase in step (e) is an RNA and / or DNA ligase.

[0243] 16. The method of any one of clauses 1-15, wherein the polymerase in step (d) lacks strand displacement activity.

[0244] 17. The method of any one of clauses 1-16, wherein the polymerase in step (d) lacks 5' to 3' exonuclease activity.

[0245] 18. The method of any one of clauses 1-17, wherein the polymerase in step (d) has 3' to 5' exonuclease activity or lacks 3' to 5' exonuclease activity.

[0246] 19. The method of any one of clauses 1 to 18, wherein the polymerase is a DNA polymerase or an RNA polymerase.

[0247] 20. The method of any one of clauses 1-19, wherein the DNA or RNA polymerase is an engineered or mutated DNA or RNA polymerase.

[0248] 21. The method of any one of clauses 1 to 20, wherein two or more of steps a) to g) are performed simultaneously, and optionally steps d) and e) are performed simultaneously.

[0249] 22. The method of any one of clauses 1 to 21, wherein two or more of steps a) to g) are performed sequentially, and optionally steps d) and e) are performed sequentially.

[0250] 23. The method of any one of clauses 1-22, wherein the template has a property that allows it to be separated from the single-stranded polynucleotide product.

[0251] 24. The method of claim 23, wherein the property that enables the template to be separated from the single-stranded polynucleotide product is that the template is attached to a support material.

[0252] 25. The method of claim 24, wherein the support material is a soluble support material, and optionally the support material is selected from the group consisting of polyethylene glycol, soluble organic polymers, DNA, proteins, dendrimers, polysaccharides, oligosaccharides, and carbohydrates.

[0253] 26. The method of claim 24, wherein the support material is an insoluble support material, and optionally the support material is selected from the group consisting of glass beads, polymer beads, fibrous supports, membranes, streptavidin-coated beads, cellulose, and reaction walls, and optionally the reaction walls are part of the reaction vessel.

[0254] 27. The method of any one of clauses 24-26, wherein multiple replicate copies of the template are successively attached to the support material via a single attachment point.

[0255] 28. The method of any one of clauses 23 to 27, wherein the property that enables the template to be separated from the product is the molecular weight of the template.

[0256] 29. The method of any one of clauses 1-28, wherein the method further comprises the step h) of reusing the mold.

[0257] 30. The method of claim 29, wherein the method further comprises the step i) of repeating steps a) to g) or steps a) to h) with the reused template.

[0258] 31. The method of any one of clauses 1 to 30, wherein the method is semi-continuous or continuous.

[0259] 32. The method of any one of clauses 1-31, wherein the template polynucleotide consists of a sequence complementary to the single-stranded polynucleotide product.

[0260] 33. The method of any one of clauses 1-32, wherein the single stranded polynucleotide product is 3-30 nucleotides in length, optionally 10-30 nucleotides in length, 3-15 nucleotides in length, 15-20 nucleotides in length, 20-25 nucleotides in length, or 20-30 nucleotides in length.

[0261] 34. The single-stranded polynucleotide product is 20 nucleotides in length and at least two segment polynucleotides are (i) a 5' segment that is 7 nucleotides in length and a 3' segment that is 7 nucleotides in length; (ii) a 5' segment that is 6 nucleotides in length and a 3' segment that is 6 nucleotides in length; (iii) a 5' segment that is 5 nucleotides in length and a 3' segment that is 5 nucleotides in length; (iv) a 5' segment that is 4 nucleotides in length and a 3' segment that is 4 nucleotides in length; or (v) a 5' segment that is 3 nucleotides long and a 3' segment that is 3 nucleotides long 34. The method according to any one of clauses 1 to 33, comprising:

[0262] 35. The method of any one of clauses 1-34, wherein the single stranded polynucleotide product is a gapmer.

[0263] 36. The method of any one of clauses 1-32, wherein the single stranded polynucleotide product is 30-20,000 nucleotides in length, optionally 30-10,000 nucleotides in length, 30-5,000 nucleotides in length, 30-1,000 nucleotides in length, 30-500 nucleotides in length, 30-400 nucleotides in length, 30-300 nucleotides in length, 30-200 nucleotides in length, 30-100 nucleotides in length, 30-50 nucleotides in length, or 30-40 nucleotides in length.

[0264] 37. The method of any one of clauses 1-36, wherein the single-stranded polynucleotide product is a DNA polynucleotide product, an RNA polynucleotide product, or a DNA and RNA hybrid polynucleotide product.

[0265] 38. The method of clause 37, wherein the RNA polynucleotide product is mRNA.

[0266] 39. The method of any one of clauses 1-38, wherein the product is produced on a gram, kilogram, or larger scale and / or the method is carried out in a reaction volume of at least 1 L.

[0267] 40. The method of any one of clauses 1-39, wherein the resulting single-stranded polynucleotide product is at least 80% pure, optionally, the single-stranded polynucleotide or oligonucleotide product is at least 90% pure, optionally, the single-stranded polynucleotide or oligonucleotide product is at least 95% pure, optionally, the single-stranded polynucleotide or oligonucleotide product is at least 98% pure.

[0268] 41. A method for producing a double-stranded polynucleotide product, wherein two complementary single-stranded polynucleotides produced by the method of any one of clauses 1 to 40 are mixed under conditions that allow annealing.

[0269] 42. A method for generating a double-stranded polynucleotide product having at least one modified nucleotide residue, comprising: a) providing a template polynucleotide comprising a sequence complementary to the single-stranded polynucleotide product; b) providing a pool of polynucleotides comprising at least two segment polynucleotides; c) contacting the template polynucleotide of step (a) with the pool of polynucleotides of step (b) under conditions that allow annealing of at least two segment polynucleotides to the template polynucleotide, whereby at least one sequence gap is formed between the at least two segment polynucleotides; d) providing a pool of nucleotides and a polymerase and extending at least one segment polynucleotide using the pool of nucleotides and the polymerase to fill at least one sequence gap; e) ligating the segment polynucleotides and / or extended segment polynucleotides using a ligase to form single stranded polynucleotide products; f) optionally changing conditions to denature the annealed template and any impurities and separate the impurities; g) changing conditions to denature the annealed template and the single-stranded polynucleotide products and separate the single-stranded polynucleotide products; and h) using the single stranded polynucleotide product as a template in step a) and repeating steps a) to e) to generate a double stranded polynucleotide product. A method comprising:

[0270] 43. The method of clause 42, wherein the double-stranded polynucleotide product is purified.

[0271] 44. The method of any one of clauses 41-43, wherein the double-stranded polynucleotide product is a siRNA.

[0272] 45. The method of any one of clauses 1 to 44, wherein the polynucleotide product is a therapeutic polynucleotide product.

[0273] 46. ​​The method of clause 37 or 38, wherein the RNA polynucleotide product comprises a sequence encoding one or more immunogens.

[0274] 47. The method of clause 46, wherein the immunogen is selected from a respiratory syncytial virus (RSV) immunogen, an Epstein-Barr virus glycoprotein immunogen, a cytomegalovirus glycoprotein immunogen, a coronavirus spike polypeptide immunogen, an influenza virus immunogen, a varicella zoster virus glycoprotein immunogen, a human papillomavirus 16 (HPV16) E6 immunogen, an HPV16 E7 immunogen, or a flavivirus immunogen.

[0275] 48. The method according to clause 46 or 47, wherein the immunogen is selected from a coronavirus spike protein, an influenza antigen, and a RSV antigen, such as the f protein or g protein. EXAMPLES

[0276] Abbreviation HPLC High Performance Liquid Chromatography LCMS Liquid Chromatography Mass Spectrometry SEC Size Exclusion Chromatography TEAA Triethylammonium Acetate PO phosphodiester PS phosphorothioate * Phosphorothioate / 3Phos / 3' phosphate group / 5Phos / 5' phosphate group / Me-dC / 5-methylcytosine / 5Biosg / 5'Biotin EDTA Ethylenediaminetetraacetic acid dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP deoxythymidine triphosphate dATPαS 2'-deoxyadenosine-5'-(α-thio)-triphosphate dCTPαS 2'-deoxycytidine-5'-(α-thio)-triphosphate dGTPαS 2'-deoxyguanosine-5'-(α-thio)-triphosphate dTTPαS 2'-deoxythymidine-5'-(α-thio)-triphosphate ATP Adenosine Triphosphate MgCl2 Magnesium chloride Tris 2-amino-2-(hydroxymethyl)propane-1,3-diol TBuAA Tributylammonium acetate 2'MOE 2'-O-Methoxy-ethyl 2'OMe 2'-O-methyl 2'F 2'Fluoro LNA Locked Nucleic Acid CTP Cytidine triphosphate GTP Guanosine triphosphate UTP Uridine triphosphate mA 2'-O-methyladenosine mC 2'-O-methylcytidine mG 2'-O-methylguanosine mU 2'-O-methyluridine rA 2'-hydroxyadenosine rC 2'-hydroxyl cytidine rG 2'-hydroxylguanosine rU 2'-hydroxyadenosine fA 2' Fluoroadenosine fC 2' Fluorocytidine fG 2' Fluoroguanosine fU 2' Fluorouridine eA 2'-O-Methoxy-ethyladenosine eC 2'-O-Methoxy-ethyl 5-methylcytosine eG 2'-O-Methoxy-ethylguanosine eT 2'-O-methoxy-ethylthymidine ΨTP Pseudouridine-5'-triphosphate m1ΨTP N1-methylpseudouridine-5'-triphosphate Ψ Pseudouridine m1Ψ N1-Methylpseudouridine ATPαS Adenosine-5'-(α-thio)-triphosphate CTPαS Cytidine-5'-(α-thio)-triphosphate GTPαS Guanosine-5'-(α-thio)-triphosphate UTPαS Uridine-(α-thio)-triphosphate

[0277] Example 1: Synthesis of a 5 bp 2'H oligonucleotide on a 42 bp 2'H oligonucleotide template by gap-filling between a 10 bp 2'H oligonucleotide primer and a 27 bp 2'H oligonucleotide 3' block. Objective: To demonstrate gap filling between unmodified DNA oligonucleotide segments using unmodified deoxyribonucleoside triphosphates and show the general applicability of this technique.

[0278] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 39-43) were obtained from commercial sources and used directly.

[0279] The reaction was set up according to Table 1. The oligonucleotides contained in the reaction were annealed by heating to 95°C and cooling to 15°C at 0.1°C / s. The polymerase was then added to start the reaction. The reaction was incubated at 25°C for 2 hours. The reaction was then quenched by the addition of 50 μL of 100 mM EDTA, and the reaction was then buffer exchanged to 10 mM EDTA using SEC. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:11. Figure 3a shows a chromatogram of the reaction starting material. Figure 3b shows a chromatogram of the product-forming reaction.

[0280] [Table 1]

[0281] Reaction results:

[0282] [Table 2]

[0283] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0284] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0285] Conclusion: A genetically diverse set of primer extension polymerases are shown to perform base filling between two adjacent oligonucleotides. The above examples show high (>80% by area) product formation and low (<20% by area) incomplete reactions or by-product formation.

[0286] Example 2: One-pot synthesis of 5 bp 2'H oligonucleotides on a 42 bp 2'H oligonucleotide template by gap-filling between a 10 bp 2'H oligonucleotide primer and a 27 bp 2'H oligonucleotide 3' block and ligation to the synthetic 42 bp product. Objective: To demonstrate gap filling and ligation using unmodified DNA oligonucleotide segments and unmodified deoxyribonucleoside triphosphates to show the general applicability of this technique.

[0287] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from a commercial supplier. Primer extension polymerases (SEQ ID NOs: 39-43) were obtained from a commercial supplier and used directly. Double-stranded ligase (SEQ ID NO: 45) was obtained from a commercial supplier and used directly.

[0288] The reactions were set up according to Table 3. The oligonucleotides contained in the reactions were annealed by heating to 95°C and cooling to 15°C at 0.1°C / s. The polymerase and ligase were then added to initiate the reaction. The reactions were incubated at 25°C for 2 hours. The reactions were then quenched by the addition of 50 μL of 100 mM EDTA, and the reaction's buffer was then exchanged to 10 mM EDTA using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:38.

[0289] [Table 3]

[0290] Reaction results:

[0291] [Table 4]

[0292] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0293] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0294] Conclusion: A genetically diverse set of primer extension polymerases have been shown to perform base filling between two adjacent oligonucleotides, and ligases can perform tandem reactions resulting in full-length oligonucleotides. Examples above with high product formation (>95% by area) and low (<5% by area) incomplete reactions or by-product formation.

[0295] Example 3: Synthesis of 6 bp 2'H oligonucleotides on a 42 bp 2'H oligonucleotide template by gap-filling between a 10 bp 2'OH oligonucleotide primer and a 26 bp 2'H oligonucleotide 3' block Objective: To demonstrate the use of unmodified deoxyribonucleoside triphosphates to fill the gap between an RNA oligonucleotide segment (primer) and an unmodified DNA oligonucleotide segment (stopper / block) and show the application of this technology to sugar groups (2'OH) commonly found in oligonucleotide therapeutics.

[0296] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 40, 41 and 42) were obtained from commercial sources and used directly.

[0297] The reactions were set up according to Table 5. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to initiate the reaction. The reactions were incubated at 25°C for 2 hours. The reactions were then quenched by the addition of 25μL of 100mM EDTA, and the reaction's buffer was then exchanged to 10mM EDTA using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:12.

[0298] [Table 5]

[0299] Reaction results:

[0300] [Table 6]

[0301] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0302] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0303] Conclusion: Proof of concept that primer extension polymerases can be used to perform base filling between two adjacent oligonucleotides extending from a 2'-OH sugar modified 5' primer. The above exemplary polymerase with over 24% product formation by area.

[0304] Example 4: Synthesis of 15 bp 2'H oligonucleotides on a 42 bp 2'H oligonucleotide template by gap-filling between a 10 bp 2'H oligonucleotide primer and a 17 bp 2'H 5-methylcytosine base oligonucleotide 3' block. Objective: To demonstrate gap filling between one modified and one unmodified oligonucleotide segment using unmodified deoxyribonucleoside triphosphates and show the application of this technology to base modifications commonly found in oligonucleotide therapeutics.

[0305] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 40, 42 and 44) ​​were obtained from commercial sources and used directly.

[0306] The reaction was set up according to Table 7. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reaction was incubated at 25°C for 2 hours. The reaction was then quenched by the addition of 25μL of 100mM EDTA, and the reaction's buffer was then exchanged to 10mM EDTA using SEC. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:21.

[0307] [Table 7]

[0308] Reaction results:

[0309] [Table 8]

[0310] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0311] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0312] Conclusion: Proof of concept that primer extension polymerases can be used to perform base filling between two adjacent oligonucleotides where at least a base modification is contained in the adjacent oligonucleotide. The above exemplary polymerase with over 79% product formation by area.

[0313] Example 5: Synthesis of 6 bp 2'H fully PS modified oligonucleotides by gap filling between a 26 bp 2'H oligonucleotide 3' block of a 10 bp 2'H oligonucleotide primer on a 42 bp 2'H oligonucleotide template Objective: To demonstrate gap filling between unmodified oligonucleotide segments with modified deoxyribonucleoside triphosphates and show the application of this technology to backbone modifications commonly found in oligonucleotide therapeutics.

[0314] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 40, 41 and 42) were obtained from commercial sources and used directly.

[0315] The reactions were set up according to Table 9. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 25°C for 2 hours. The reactions were then quenched by the addition of 5 μL of 500 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:12.

[0316] [Table 9]

[0317] Reaction results:

[0318] [Table 10]

[0319] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0320] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0321] Conclusion: Proof of concept that primer extension polymerases can be used to perform backbone-modified base filling between two adjacent oligonucleotides. Filling of the gap by synthesizing a fully phosphorothioate-modified backbone. Exemplary polymerase above with product formation of over 33% by area.

[0322] Example 6: Synthesis of 6 bp 2'H deoxyguanosine PS modified oligonucleotides by gap filling between a 26 bp 2'H oligonucleotide 3' block of a 10 bp 2'H oligonucleotide primer on a 42 bp 2'H oligonucleotide template Objective: To demonstrate gap filling between unmodified oligonucleotide segments using modified deoxyguanosine triphosphate, but not unmodified deoxyadenosine triphosphate, unmodified deoxycytidine triphosphate, or unmodified deoxythymidine triphosphate, and to show the application of this technology to modular backbone modifications commonly found in oligonucleotide therapeutics.

[0323] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 40, 41 and 42) were obtained from commercial sources and used directly.

[0324] The reactions were set up according to Table 11. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 25°C for 2 hours. The reactions were then quenched by the addition of 5 μL of 500 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:12.

[0325] [Table 11]

[0326] Reaction results:

[0327] [Table 12]

[0328] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0329] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0330] Conclusion: Proof of concept that primer extension polymerases can be used to perform specific backbone-modified base filling between two adjacent oligonucleotides. Filling of the gap by synthesizing a phosphorothioate-modified backbone linkage between the desired bases. Exemplary polymerase above with over 89% product formation by area.

[0331] Example 7: Synthesis of 6 bp 2'H deoxythymidine PS modified oligonucleotides by gap filling between a 26 bp 2'H oligonucleotide 3' block of a 10 bp 2'H oligonucleotide primer on a 42 bp 2'H oligonucleotide template Objective: To demonstrate gap filling between unmodified oligonucleotide segments using modified deoxythymidine triphosphate, but not unmodified deoxyadenosine triphosphate, unmodified deoxycytidine triphosphate, or unmodified deoxyguanosine triphosphate, and to show the application of this technology to modular backbone modifications commonly found in oligonucleotide therapeutics.

[0332] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 40, 41 and 42) were obtained from commercial sources and used directly.

[0333] The reactions were set up according to Table 13. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 25°C for 2 hours. The reactions were then quenched by the addition of 5 μL of 500 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:12.

[0334] [Table 13]

[0335] Reaction results:

[0336] [Table 14]

[0337] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0338] Buffer A: 100mM TEAA, pH7 Buffer B: 100 mM TEAA in 25% acetonitrile / 75% HO, pH 7

[0339] Conclusion: Proof of concept that primer extension polymerases can be used to perform specific backbone-modified base filling between two adjacent oligonucleotides. Filling of the gap by synthesizing a phosphorothioate-modified backbone linkage between the desired bases. Exemplary polymerase above with over 89% product formation by area.

[0340] Example 8: One-pot synthesis of 15 bp 2'H oligonucleotides by gap-filling between a 10 bp 2'H oligonucleotide primer and a 17 bp 2'H 5-methylcytosine base oligonucleotide 3' block on a 42 bp 2'H oligonucleotide template and ligation to the synthetic 42 bp product. Objective: To demonstrate gap filling and ligation with one modified and one unmodified oligonucleotide segment and unmodified deoxyribonucleoside triphosphates, and to show the application of this technology to base modifications commonly found in oligonucleotide therapeutics.

[0341] Oligonucleotides (SEQ ID NOs: 1-38) were obtained from a commercial source. Primer extension polymerases (SEQ ID NOs: 40, 42, and 44) ​​were obtained from a commercial source and used directly. Double-stranded ligase (SEQ ID NO: 45) was obtained from a commercial source and used directly.

[0342] The reactions were set up according to Table 15. The oligonucleotides contained in the reactions were annealed by heating to 95°C and cooling to 15°C at 0.1°C / s. The polymerase and ligase were then added to start the reaction. The reactions were incubated at 25°C for 2 hours. The reactions were then quenched by the addition of 25μL of 100mM EDTA, and the reaction's buffer was then exchanged to 10mM EDTA using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:38.

[0343] [Table 15]

[0344] Reaction results:

[0345] [Table 16]

[0346] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 35-71% buffer B was run over 18 min, then increased to 95% buffer B over 7 min.

[0347] Conclusion: Proof of concept that a primer extension polymerase can be used to perform base filling between two adjacent oligonucleotides where at least one base modification is contained in one of the adjacent oligonucleotides. Also, proof of concept that a ligase can perform a tandem ligation reaction resulting in a full-length oligonucleotide from a synthesized fragment. Exemplary polymerase and ligase above with over 81% product formation by area.

[0348] Example 9: One-pot synthesis of 6 bp 2'F modified oligonucleotides by gap-filling between a 10 bp 2'H oligonucleotide primer and a 26 bp 2'H oligonucleotide 3' block 2'H on a 45 bp 2'H oligonucleotide template and ligation to the synthetic 42 bp product. Objective: To demonstrate gap filling between unmodified oligonucleotide segments using unmodified deoxythymidine triphosphate, but not modified deoxyadenosine triphosphate, modified deoxycytidine triphosphate, or modified deoxyguanosine triphosphate, and to show the application of this technology to sugar modifications commonly found in oligonucleotide therapeutics.

[0349] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 67, 69, 73, 77 and 87) were obtained from commercial sources and used directly. Double-stranded ligase (SEQ ID NO: 45) was obtained from commercial sources and used directly.

[0350] The reactions were set up according to Table 17. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase and ligase were then added to start the reaction. The reactions were incubated at 25°C for 4 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:38.

[0351] [Table 17]

[0352] Reaction results:

[0353] [Table 18]

[0354] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0355] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0356] Conclusions: Proof of concept that a primer extension polymerase can be used to perform specific sugar modified base filling between two adjacent oligonucleotides. Filling of the gap by incorporating a 2'-fluoro modification at the desired base. Also, proof of concept that a ligase can perform a tandem ligation reaction resulting in a full length oligonucleotide from a synthesized fragment. Exemplary polymerase and ligase above with over 90.1% product formation by area.

[0357] Example 10: One-pot synthesis of 6 bp 2'H deoxycytidine Me-dC modified oligonucleotides by gap-filling between a 26 bp 2'H oligonucleotide 3' block 2'H of a 10 bp 2'H oligonucleotide primer on a 45 bp 2'H oligonucleotide template and ligation to the synthetic 42 bp product. Objective: To demonstrate gap filling between unmodified oligonucleotide segments using modified deoxycytidine triphosphate, but not unmodified deoxyadenosine triphosphate, unmodified deoxyguanosine triphosphate, or unmodified deoxythymidine triphosphate, and to show the application of this technology to modular base modifications commonly found in oligonucleotide therapeutics.

[0358] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 42, 44 and 86) were obtained from commercial sources and used directly. Double-stranded ligase (SEQ ID NO: 45) was obtained from commercial sources and used directly.

[0359] The reactions were set up according to Table 19. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase and ligase were then added to start the reaction. The reactions were incubated at 25°C for 4 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:38.

[0360] [Table 19]

[0361] Reaction results:

[0362] [Table 20]

[0363] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0364] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0365] Conclusion: Proof of concept that a primer extension polymerase can be used to perform specific modified base filling between two adjacent oligonucleotides. Filling of the gap by incorporating a 5-methylcytosine modification at the desired base. Also, proof of concept that a ligase can perform a tandem ligation reaction resulting in a full-length oligonucleotide from a synthesized fragment. Exemplary polymerase and ligase above with over 98% product formation by area.

[0366] Example 11: Synthesis of 6 bp 2'F fully modified oligonucleotides by gap filling between a 26 bp 2'H oligonucleotide 3' block of a 10 bp 2'F modified oligonucleotide primer on a 45 bp 2'H oligonucleotide template Objective: To demonstrate gap filling between modified and unmodified oligonucleotide segments with modified deoxyribonucleoside triphosphates and show the application of this technology to sugar modifications commonly found in oligonucleotide therapeutics.

[0367] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 80, 84 and 89) were obtained from commercial sources and used directly.

[0368] The reactions were set up according to Table 21. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 25°C for 4 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:12.

[0369] [Table 21]

[0370] Reaction results:

[0371] [Table 22]

[0372] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0373] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0374] Conclusion: Proof of concept that primer extension polymerases can be used to perform base filling between two adjacent oligonucleotides extending from a 2'-fluoro modified oligonucleotide primer. Filling of the gap by incorporating a 2'-fluoro sugar modification at the desired base position. Exemplary polymerase above with product formation of over 14% by area.

[0375] Example 12: Synthesis of 8 bp 2'OH oligonucleotides on a 53 bp 2'H oligonucleotide template by gap-filling between a 16 bp 2'OH oligonucleotide primer and a 26 bp 2'H oligonucleotide 3' block Objective: To demonstrate the use of nucleoside triphosphates to fill the gap between an RNA oligonucleotide segment (primer) and an unmodified DNA oligonucleotide segment (block) and to show the application of this technology to sugar groups (2'OH) commonly found in oligonucleotide therapeutics.

[0376] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerase (SEQ ID NO: 95) was obtained from a commercial source and used directly.

[0377] The reactions were set up according to Table 23. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 55°C for 20 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:20.

[0378] [Table 23]

[0379] Reaction results:

[0380] [Table 24]

[0381] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0382] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0383] Conclusion: Proof of concept that a primer extension polymerase can be used to perform gap filling between two adjacent oligonucleotides extending from an RNA 5' primer. The polymerase yielded 27% product formation by area.

[0384] Example 13: Synthesis of 8 bp 2'OH oligonucleotides on a 53 bp 2'H oligonucleotide template by gap-filling between a 26 bp 2'H oligonucleotide 3' block of a 16 bp 2'OMe modified oligonucleotide primer Objective: To demonstrate the use of nucleoside triphosphates to fill gaps between modified oligonucleotide segments (primers) and unmodified DNA oligonucleotide segments (blocks) and to show the application of this technology to sugar groups (2'OMe) commonly found in oligonucleotide therapeutics.

[0385] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerase (SEQ ID NO: 60) was obtained from a commercial source and used directly.

[0386] The reactions were set up according to Table 25. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 55°C for 20 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:20.

[0387] [Table 25]

[0388] Reaction results:

[0389] [Table 26]

[0390] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0391] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0392] Conclusion: Proof of concept that a primer extension polymerase can be used to perform gap filling between two adjacent oligonucleotides extending from a sugar-modified 5' primer. The polymerase with 32% product formation by area.

[0393] Example 14: Synthesis of a 54 bp 2'OH oligonucleotide by gap filling between a 26 bp 2'H oligonucleotide 3' block of a 16 bp 2'OH oligonucleotide primer on a 100 bp 2'H oligonucleotide template Objective: To demonstrate longer gap filling between an RNA oligonucleotide segment (primer) and an unmodified DNA oligonucleotide segment (block) using a 100 bp 2'H oligonucleotide template and nucleoside triphosphates to show the general applicability of the technology.

[0394] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 56, 60, 84, 89 and 93) were obtained from commercial sources and used directly.

[0395] The reactions were set up according to Table 27. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 55°C for 20 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:55.

[0396] [Table 27]

[0397] Reaction results:

[0398] [Table 28]

[0399] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 60-85% buffer B was run over 15 min, followed by a decrease to 60% buffer B over 5 min.

[0400] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0401] Conclusion: Proof of concept that primer extension polymerases can be used to perform longer base filling between two adjacent oligonucleotides extending from an RNA 5' primer. Exemplary polymerase above with over 57% product formation by area.

[0402] Example 15: Synthesis of a 54 bp 2'OH oligonucleotide on a 100 bp 2'H oligonucleotide template by gap filling between a 26 bp 2'H oligonucleotide 3' block of a 16 bp 2'OMe oligonucleotide primer Objective: To demonstrate longer gap filling between modified oligonucleotide segments (primers) and unmodified DNA oligonucleotide segments (blocks) using a 100 bp 2'H oligonucleotide template and nucleoside triphosphates to show the general applicability of the technology.

[0403] Oligonucleotides (SEQ ID NOs: 1-38 and 46-54) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 60, 83, 84 and 89) were obtained from commercial sources and used directly.

[0404] The reactions were set up according to Table 12. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 55°C for 20 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:55.

[0405] [Table 29]

[0406] Reaction results:

[0407] [Table 30]

[0408] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 60-85% buffer B was run over 15 min, followed by a decrease to 60% buffer B over 5 min.

[0409] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0410] Conclusion: Proof of concept that primer extension polymerases can be used to perform longer base filling between two adjacent oligonucleotides extending from a sugar-modified 5' primer. Exemplary polymerase above with over 33% product formation by area.

[0411] Example 16: One-pot synthesis of 6 bp 2'H fully PS modified oligonucleotides by gap filling between a 9 bp 2'MOE oligonucleotide primer and a 7 bp 2'MOE oligonucleotide 3' block 2 on a 22 bp 2'H oligonucleotide template and ligation to the synthetic 22 bp product Objective: To demonstrate gap filling between 2'MOE modified oligonucleotide segments with PS modified deoxyribonucleotides and show the application of this technology to backbone and sugar modifications commonly found in gapmer oligonucleotide therapeutics.

[0412] Oligonucleotides (SEQ ID NO: 96-98) were obtained from a commercial supplier. Primer extension polymerase (SEQ ID NO: 41) was obtained from a commercial supplier and used directly. Double-stranded ligase (SEQ ID NO: 45) was obtained from a commercial supplier and used directly.

[0413] The reactions were set up according to Table 31. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase and ligase were then added to start the reaction. The reactions were incubated at 25°C for 4 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:99.

[0414] [Table 31]

[0415] Reaction results:

[0416] [Table 32]

[0417] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0418] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0419] Conclusions: Proof of concept that a primer extension polymerase can be used to perform specific backbone modified base filling between two adjacent 2'MOE oligonucleotides. Filling of the gap by incorporating PS modifications between the desired bases. Also, proof of concept that a ligase can perform a tandem ligation reaction resulting in a full length oligonucleotide from a synthesized fragment. Exemplary polymerase and ligase with over 13.2% product formation by area.

[0420] Example 17: One-pot synthesis of 6 bp 2'H oligonucleotides by gap-filling between a 9 bp 2'MOE oligonucleotide primer and a 7 bp 2'MOE oligonucleotide 3' block 2'H on a 22 bp 2'H oligonucleotide template and ligation to the synthetic 22 bp product. Objective: To demonstrate gap filling between 2'MOE modified oligonucleotide segments with deoxyribonucleoside triphosphates and show the application of this technology to sugar modifications commonly found in gapmer oligonucleotide therapeutics.

[0421] Oligonucleotides (SEQ ID NOs: 96-98) were obtained from a commercial supplier. Primer extension polymerases (SEQ ID NOs: 40-42) were obtained from a commercial supplier and used directly. Double-stranded ligase (SEQ ID NO: 45) was obtained from a commercial supplier and used directly.

[0422] The reactions were set up according to Table 33. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase and ligase were then added to start the reaction. The reactions were incubated at 25°C for 4 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:99.

[0423] [Table 33]

[0424] Reaction results:

[0425] [Table 34]

[0426] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0427] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0428] Conclusion: Proof of concept that a primer extension polymerase can be used to perform specific base filling between two adjacent 2'MOE oligonucleotides. Also, a ligase can perform a tandem ligation reaction resulting in a full-length oligonucleotide from a synthesized fragment. Exemplary polymerase and ligase above with over 64% product formation by area.

[0429] Example 18: Synthesis of 8 bp 2'OMe oligonucleotides on a 53 bp 2'H oligonucleotide template by gap-filling between a 26 bp 2'H oligonucleotide 3' block of a 16 bp 2'OMe oligonucleotide primer Objective: To demonstrate gap filling with 2'OMe sugar-modified nucleoside triphosphates between 2'OMe-modified oligonucleotide segments (primers) and unmodified DNA oligonucleotide segments (blocks) using a 53 bp 2'H oligonucleotide template, demonstrating the general applicability of this technology.

[0430] Oligonucleotides (SEQ ID NOs: 4, 47 and 52) were obtained from commercial sources. Primer extension polymerases (SEQ ID NOs: 62 and 87) were obtained from commercial sources and used directly.

[0431] The reactions were set up according to Table 35. The oligonucleotides contained in the reactions were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. The polymerase was then added to start the reaction. The reactions were incubated at 55°C for 20 hours. The reactions were then quenched by the addition of 60 μL of 100 mM EDTA, and the reaction's buffer was then exchanged into water using SEC. The reactions were then analyzed by HPLC for the presence of SEQ ID NO:20.

[0432] [Table 35]

[0433] Reaction results:

[0434] [Table 36]

[0435] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 60-85% buffer B was run over 15 min, followed by a decrease to 60% buffer B over 5 min.

[0436] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0437] Conclusion: Proof of concept that primer extension polymerases can be used to perform base filling between two adjacent oligonucleotides extending from a 2'OMe sugar-modified 5' primer with 2'OMe sugar-modified nucleoside triphosphates. Exemplary polymerases above with greater than 15% product formation by area.

[0438] Example 19: Synthesis of an 850 bp 2'OH oligonucleotide on an 895 bp 2'H oligonucleotide template by gap filling between a 16 bp 2'OH oligonucleotide primer and a 26 bp 2'OH oligonucleotide 3' block and ligation to generate an 892 bp product. Objective: To demonstrate gap filling between an RNA oligonucleotide segment (primer) and an RNA oligonucleotide segment (block) using nucleoside triphosphates and show the application of this technology to sugar groups (2'OH) commonly found in longer oligonucleotide therapeutics.

[0439] Oligonucleotides (SEQ ID NO:51, 76 and 101) were obtained from a commercial source. Primer extension polymerase (SEQ ID NO:95) was obtained from a commercial source and used directly. Double stranded ligase (SEQ ID NO:45) was obtained from a commercial source and used directly.

[0440] The reaction was set up according to Table 37. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. RNAse inhibitor was then added. Subsequently, polymerase and ligase were then added to start the reaction. The reaction was incubated at 25°C for 16 hours. 1 uL of DNase1 was then added and the reaction was further incubated at 25°C for 2 hours. The reaction was then quenched by adding 4 μL of 10 mM EDTA and the reaction was then analyzed by gel electrophoresis for the presence of SEQ ID NO:102. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:101.

[0441] [Table 37]

[0442] Reaction results:

[0443] [Table 38]

[0444] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0445] Buffer A: 5 mM TbuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TbuAA in 20% HO / 80% acetonitrile, pH 7

[0446] Conclusion: Proof of concept that primer extension polymerase can be used to perform gap filling between two adjacent oligonucleotides extending from an RNA 5' primer. Polymerase and tandem ligation yielded product formation of 18.6% by area.

[0447] Example 20: Synthesis of an 850 bp 2'OH oligonucleotide on an 895 bp 2'H oligonucleotide template by gap filling between a 16 bp 2'Ome oligonucleotide primer and a 26 bp 2'OH oligonucleotide 3' block and ligation to generate an 892 bp product. Objective: To demonstrate gap filling between a 2'Ome-containing oligonucleotide segment (primer) and an RNA oligonucleotide segment (block) using nucleoside triphosphates and show the application of this technology to sugar groups (2'OH) commonly found in longer oligonucleotide therapeutics.

[0448] Oligonucleotides (SEQ ID NO:53, 76 and 101) were obtained from a commercial source. Primer extension polymerase (SEQ ID NO:95) was obtained from a commercial source and used directly. Double stranded ligase (SEQ ID NO:45) was obtained from a commercial source and used directly.

[0449] The reaction was set up according to Table 39. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. RNAse inhibitor was then added. Subsequently, polymerase and ligase were then added to start the reaction. The reaction was incubated at 25°C for 16 hours. 1 uL of Dnase1 was then added and the reaction was further incubated at 25°C for 2 hours. The reaction was then quenched by adding 4 μL of 10 mM EDTA and the reaction was then analyzed by gel electrophoresis for the presence of SEQ ID NO:103. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:101.

[0450] [Table 39]

[0451] Reaction results:

[0452] [Table 40]

[0453] HPLC analysis was performed using a Waters Xbridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0454] Buffer A: 5 mM TbuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TbuAA in 20% HO / 80% acetonitrile, pH 7

[0455] Conclusion: Proof of concept that primer extension polymerase can be used to perform gap filling between two adjacent oligonucleotides extending from a 2'Ome-containing 5' primer. Polymerase and tandem ligation resulted in product formation of 36.52% by area.

[0456] Example 21: Synthesis of an 850 bp 2'OH oligonucleotide on an 895 bp 2'H oligonucleotide template by gap filling between a 16 bp 2'OH PS oligonucleotide primer and a 26 bp 2'OH oligonucleotide 3' block and ligation to generate an 892 bp product. Objective: To demonstrate gap filling between an RNA phosphorothioate-containing oligonucleotide segment (primer) and an RNA oligonucleotide segment (block) using nucleoside triphosphates and show the application of this technology to sugar groups (2'OH) commonly found in longer oligonucleotide therapeutics.

[0457] Oligonucleotides (SEQ ID NO: 76, 101 and 104) were obtained from a commercial source. Primer extension polymerase (SEQ ID NO: 95) was obtained from a commercial source and used directly. Double stranded ligase (SEQ ID NO: 45) was obtained from a commercial source and used directly.

[0458] The reaction was set up according to Table 41. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. RNAse inhibitor was then added. Subsequently, polymerase and ligase were then added to start the reaction. The reaction was incubated at 25°C for 16 hours. 1 uL of Dnase1 was then added and the reaction was further incubated at 25°C for 2 hours. The reaction was then quenched by adding 4 μL of 10 mM EDTA and the reaction was then analyzed by gel electrophoresis for the presence of SEQ ID NO:105. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:101.

[0459] [Table 41]

[0460] Reaction results:

[0461] [Table 42]

[0462] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0463] Buffer A: 5 mM TbuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TbuAA in 20% HO / 80% acetonitrile, pH 7

[0464] Conclusion: Proof of concept that primer extension polymerase can be used to perform gap filling between two adjacent oligonucleotides extending from a 2'OH phosphorothioate-containing 5' primer. The polymerase and tandem ligation yielded product formation of 10.78% by area.

[0465] Example 22: Synthesis of an 850 bp 2'OH oligonucleotide containing pseudouridine by gap filling between a 16 bp 2'OH oligonucleotide primer and a 26 bp 2'OH oligonucleotide 3' block on an 895 bp 2'H oligonucleotide template and ligation to generate an 892 bp product. Objective: To demonstrate gap filling between an RNA oligonucleotide segment (primer) and an RNA oligonucleotide segment (block) using pseudouridine nucleoside triphosphate and nucleoside triphosphates, and to show the application of this technology to base modifications (Ψ) commonly found in oligonucleotide therapeutics.

[0466] Oligonucleotides (SEQ ID NO:51, 76 and 101) were obtained from a commercial source. Primer extension polymerase (SEQ ID NO:95) was obtained from a commercial source and used directly. Double stranded ligase (SEQ ID NO:45) was obtained from a commercial source and used directly.

[0467] The reaction was set up according to Table 43. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. RNAse inhibitor was then added. Subsequently, polymerase and ligase were then added to start the reaction. The reaction was incubated at 25°C for 16 hours. 1 uL of Dnase1 was then added and the reaction was further incubated at 25°C for 2 hours. The reaction was then quenched by adding 4 μL of 10 mM EDTA and the reaction was then analyzed by gel electrophoresis for the presence of SEQ ID NO:106. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:101.

[0468] [Table 43]

[0469] Reaction results:

[0470] [Table 44]

[0471] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0472] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0473] Conclusion: Proof of concept that primer extension polymerase can be used to perform gap filling to form pseudouridine-containing sequences. Polymerase and tandem ligation resulted in product formation of 24.4% by area.

[0474] Example 23: Synthesis of an 850 bp 2'OH oligonucleotide containing N1-methylpseudouridine by gap filling between a 16 bp 2'OH oligonucleotide primer and a 26 bp 2'OH oligonucleotide 3' block on an 895 bp 2'H oligonucleotide template and ligation to generate an 892 bp product. Objective: To demonstrate gap filling between an RNA oligonucleotide segment (primer) and an RNA oligonucleotide segment (block) using N1-methylpseudouridine nucleoside triphosphate and nucleoside triphosphates, and to show the application of this technology to base modifications (m1Ψ) commonly found in oligonucleotide therapeutics.

[0475] Oligonucleotides (SEQ ID NO:51, 76 and 101) were obtained from a commercial source. Primer extension polymerase (SEQ ID NO:95) was obtained from a commercial source and used directly. Double stranded ligase (SEQ ID NO:45) was obtained from a commercial source and used directly.

[0476] The reaction was set up according to Table 45. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. RNAse inhibitor was then added. Subsequently, polymerase and ligase were then added to start the reaction. The reaction was incubated at 25°C for 16 hours. 1 uL of DNase1 was then added and the reaction was further incubated at 25°C for 2 hours. The reaction was then quenched by adding 4 μL of 10 mM EDTA and the reaction was then analyzed by gel electrophoresis for the presence of SEQ ID NO:107. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:101.

[0477] [Table 45]

[0478] Reaction results:

[0479] [Table 46]

[0480] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0481] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0482] Conclusion: Proof of concept that primer extension polymerase can be used to perform gap filling to form N1-methylpseudouridine-containing sequences. Polymerase and tandem ligation resulted in product formation of 21.64% by area.

[0483] Example 24: Synthesis of 850 bp 2'OH PS modified oligonucleotides by gap filling between a 16 bp 2'OH oligonucleotide primer and a 26 bp 2'OH oligonucleotide 3' block on an 895 bp 2'H oligonucleotide template and ligation to generate a 892 bp product. Objective: To demonstrate gap filling between an RNA oligonucleotide segment (primer) and an RNA oligonucleotide segment (block) using modified nucleoside triphosphates and show the application of this technology to backbone modifications commonly found in oligonucleotide therapeutics.

[0484] Oligonucleotides (SEQ ID NO:51, 76 and 101) were obtained from a commercial source. Primer extension polymerase (SEQ ID NO:95) was obtained from a commercial source and used directly. Double stranded ligase (SEQ ID NO:45) was obtained from a commercial source and used directly.

[0485] The reaction was set up according to Table 45. The oligonucleotides contained in the reaction were annealed by heating to 95°C at 0.1°C / s and cooling to 15°C. RNAse inhibitor was then added. Subsequently, polymerase and ligase were then added to start the reaction. The reaction was incubated at 25°C for 16 hours. 1 uL of DNase1 was then added and the reaction was further incubated at 25°C for 2 hours. The reaction was then quenched by adding 4 μL of 10 mM EDTA and the reaction was then analyzed by gel electrophoresis for the presence of SEQ ID NO:108. The reaction was then analyzed by HPLC for the presence of SEQ ID NO:101.

[0486] [Table 47]

[0487] Reaction results:

[0488] [Table 48]

[0489] HPLC analysis was performed using a Waters XBridge Peptide BEH C18 column (300 Å, 3.5 μm, 2.1 mm×150 mm) pumped at 0.5 ml / min with absorbance monitored at 260 nm. The column was maintained at 50° C. 5 μl of sample was injected and a gradient of 40-95% buffer B was run over 25 min, followed by a decrease to 40% buffer B over 4 min.

[0490] Buffer A: 5 mM TBuAA in 90% HO / 10% acetonitrile, pH 7 Buffer B: 5 mM TBuAA in 20% HO / 80% acetonitrile, pH 7

[0491] Conclusion: Proof of concept that primer extension polymerase can be used to perform gap filling to form phosphorothioate modified sequences. Polymerase and tandem ligation resulted in product formation of 21.64% by area.

[0492] Overall conclusion The inventors have shown that it is possible to synthesize polynucleotides or polynucleotides (including oligonucleotides) or oligonucleotides with a variety of therapeutically relevant modifications by constructing polynucleotide or oligonucleotide segments on complementary templates, extending the segments using a polymerase to fill in gaps, ligating the segments together, and separating the product polynucleotides or oligonucleotides from both impurities and their complementary templates in an efficient method that is scalable and suitable for large-scale production of therapeutic polynucleotides and oligonucleotides.

[0493] When using the inherent properties of nucleic acids, such as DNA or RNA, to specifically recognize and bind complementary sequences with an affinity that reflects both the accuracy and length of the complementary sequence, we can produce highly pure polynucleotides and oligonucleotides without the need for chromatography, which improves both the efficiency and scalability of the production method.By recovering the template in an unchanged state during the separation process, we can reuse the template for further rounds of synthesis, thus avoiding the economic consequences of having to produce one equivalent of template for each equivalent of product oligonucleotide formed.In some embodiments, oligonucleotides can also be synthesized in solution, which avoids the constraints on scale-up imposed by solid-phase methods.

[0494] Finally, while wild-type polymerases are effective, modifications of the polymerase (leading to mutant or engineered polymerases) may be utilized to increase efficiency, template recovery, or to incorporate modified nucleotides. Similarly, while wild-type ligases are effective, with appropriate mutation and evolution of the ligase, ligation efficiency can be increased, and appropriately modified ligases are effective catalysts for synthesizing oligonucleotides containing multiple modifications.

[0495] Sequence Listing

[0496] [Table 49] JPEG2025511938000050.jpg249154JPEG2025511938000051.jpg248151JPEG2025511938000052.jpg249155JPEG2025511938 000053.jpg249155JPEG2025511938000054.jpg250153JPEG2025511938000055.jpg249154JPEG2025511938000056.jpg36153

[0497] SEQ ID NO:1 / 5Biosg / GCTAATGGCTTTGGTGCGAAGCAGACTGAGGCACCGAGGAGT SEQ ID NO:2 ACTCCTCGGT SEQ ID NO:3 / 5phos / AGTCTGCTTCGCACCAAAGCCATTAGC SEQ ID NO:4 / 5phos / GTCTGCTTCGCACCAAAGCCATTAGC SEQ ID NO:5 / 5Phos / GCACCAAAGC / Me-dC / ATTAGC SEQ ID NO:6 / 5Phos / GC / Me-dC / ATTAGC SEQ ID NO:7 ACTCCTCGGTG SEQ ID NO:8 ACTCCTCGGTGC SEQ ID NO:9 ACTCCTCGGTGCC SEQ ID NO:10 ACTCCTCGGTGCCT SEQ ID NO:11 ACTCCTCGGTGCCTC SEQ ID NO:12 ACTCCTCGGTGCCTCA SEQ ID NO:13 ACTCCTCGGTGCCTCAG SEQ ID NO:14 ACTCCTCGGTGCCTCAGT SEQ ID NO:15 ACTCCTCGGTGCCTCAGTC SEQ ID NO:16 ACTCCTCGGTGCCTCAGTCT SEQ ID NO:17 ACTCCTCGGTGCCTCAGTCTG SEQ ID NO:18 ACTCCTCGGTGCCTCAGTCTGC SEQ ID NO:19 ACTCCTCGGTGCCTCAGTCTGCT SEQ ID NO:20 ACTCCTCGGTGCCTCAGTCTGCTT SEQ ID NO:21 ACTCCTCGGTGCCTCAGTCTGCTTC SEQ ID NO:22 ACTCCTCGGTGCCTCAGTCTGCTTCG SEQ ID NO:23 ACTCCTCGGTGCCTCAGTCTGCTTCGC SEQ ID NO:24 ACTCCTCGGTGCCTCAGTCTGCTTCGCA SEQ ID NO:25 ACTCCTCGGTGCCTCAGTCTGCTTCGCAC SEQ ID NO:26 ACTCCTCGGTGCCTCAGTCTGCTTCGCACC SEQ ID NO:27 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCA SEQ ID NO:28 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAA SEQ ID NO:29 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAA SEQ ID NO:30 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAG SEQ ID NO:31 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGC SEQ ID NO:32 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCC SEQ ID NO:33 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCCA SEQ ID NO:34 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCCAT SEQ ID NO:35 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCCATT SEQ ID NO:36 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCCATTA SEQ ID NO:37 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCCATTAG SEQ ID NO:38 ACTCCTCGGTGCCTCAGTCTGCTTCGCACCAAAGCCATTAGC SEQ ID NO:39 MIVSDIEANALLESVTKFHCGVIYDYSTAEYVSYRPSDFGAYLDALEAEVARGGLIVFHNGHKYDVPALTKLAKLQLNREFHLPRENCIDTLVLSRLIHSNLKDTDMGLLRSGKLPGKRFGSHALEAWGYRLGEMKGEYKDDFKRMLEEQGEEYVDGMEWWNFNEEMMDYNVQDVV VTKALLEKLLSDKHYFPPEIDFTDVGYTTFWSESLEAVDIEHRAAAWLLAKQERNGFPFDTKAIEELYVELAARRSELLRKLTETFGSWYQPKGGTEMFCHPRTGKPLPKYPRIKTPKVGGIFKKPKNKAQREGREPCELDTREYVAGAPYTPVEHVVFNPSSRDHIQKKLQEAGWV PTKYTDKGAPVVDDEVLEGVRVDDPEKQAAIDLIKEYLMIQKRIGQSAEGDKAWLRYVAEDGKIHGSVNPNGAVTGRATHAFPNLAQIPGVRSPYGEQCRAAFGAEHHLDGITGKPWVQAGIDASGLELRCLAHFMARFDNGEYAHEILNGDIHTKNQIAAELPTRDNAKTFIYGF LYGAGDEKIGQIVGAGKERGKELKKKFLENTPAIAALRESIQQTLVESSQWVAGEQQVKWKRRWIKGLDGRKVHVRSPHAALNTLLQSAGALICKLWIIKTEEMLVEKGLKHGWDGDFAYMAWVHDEIQVGCRTEEIAQVVIETAQEAMRWVGDHWNFRCLLDTEGKMGPNWAICH SEQ ID NO:40 MIVLFVDFDYFYAQVEEVLNPSLKGKPVVVCVFSGRFEDSGAVATANYEARKFGVKAGIPIVEAKKILPNAVYLPMRKEVYQQVSSRIMNLLREYSEKIEIASIDEAYLDISDKVRDYREAYNLGLEIKNKILEKEKITVTVGISKNKVFAKIAADMAKPNGIKVIDDEEVKRLIR ELDIADVPGIGNITAEKLKKLGINKLVDTLSIEFDKLKGMIGEAKAKYLISLARDEYNEPIRTRVRKSIGRIVTMKRNSRNLEEIKPYLFRAIEESYYKLDKRIPKAIHVVAVTEDLDIVSRGRTFPHGISKETAYSESVKLLQKILEEDERKIRRIGVRFSKFIEAIGLDKFFDT SEQ ID NO:41 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:42 MKEFYISIETVGNNIVERYIDENGKERTREVEYLPTMFRHCKEESKYKDIYGKNCAPQKFPSMKDARDWMKRMEDIGLEALGMNDFKLAYISDTYGSEIVYDRKFVRVANCD IEVTGDKFPDPMKAEYEIDAITHYDSIDDRFYVFDLLNSMYGSVSKWDAKLAAKLDCEGGDEVPQEILDRVIYMPFDNERDMLMEYINLWEQKRPAIFTGWNIEGFDVPYIM NRVKMILGERSMKRFSPIGRVKSKLIQNMYGSKEIYSIDGVSILDYLDLYKKFAFTNLPSFSLESVAQHETKKGKLPYDGPINKLRETNHQRYISYNIIDVESVQAIDKIRG FIDLVLSMSYYAKMPFSGVMSPIKTWDAIIFNSLKGEHKVIPQQGSHVKQSFPGAFVFEPKPIARRYIMSFDLTSLYPSIIRQVNISPETIRGQFKVHPIHEYIAGTAPKPSD EYSCSPNGWMYDKHQEGIIPKEIAKVFFQRKDWKKKMFAEEMNAEAIKKIIMKGAGSCSTKPEVERYVKFSDDFLNELSNYTESVLNSLIEECEKAATLANTNQLNRKILIN SLYGALGNIHFRYYDLRNATAITIFGQVGIQWIARKINEYLNKVCGTNDEDFIAAGDTDSVYVCVDKVIEKVGLDRFKEQNDLVEFMNQFGKKKMEPMIDVAYRELCDYMNN REHLMHMDREAISCPPLGSKGVGGFWKAKKRYALNVYDMEDKRFAEPHLKIMGMETQQSSTPKAVQEALEESIRRILQEGEESVQEYYKNFEKEYRQLDYKVIAEVKTANDI AKYDDKGWPGFKCPFHIRGVLTYRRAVSGLGVAPILDGNKVMVLPLREGNPFGDKCIAWPSGTELPKEIRSDVLSWIDHSTLFQKSFVKPLAGMCESAGMDYEEKASLDFLFG SEQ ID NO:43 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:44 MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKR FLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGG YVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYE GFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT SEQ ID NO:45 MNIFNTNPFKAVSFVESAVKKALETSGYLIADCKYDGVRGNIVVDNVAEAAWLSRVSKFIPALEHLNGFDKRWQQLLNDDRCIFPDGFMLDGELMVKGVDFNTGSGLLRTKWVKRDNMGFHLTNVPTKLTPKGREVIDGKFEFHLDPKRLSVRLYAVMPIHIAESGEDYDVQN LLMPYHVEAMRSLLVEYFPEIEWLIAETYEVYDMDSLTELYEEKRAEGHEGLIVKDPQGIYKRGKKSGWWKLKPECEADGIIQGVNWGTEGLANEGKVIGFSVLLETGRLVDANNISRALMDEFTSNVKAHGEDFYNGWACQVNYMEATPDGSLRHPSFEKFRGTEDNPQEKM SEQ ID NO:46 / 5Biosg / GCTAATGGCTTTGGTGCGAAGCAGACTGAGGCACCGAGGAGTTTT SEQ ID NO:47 CTAATGGCTTTGGTGCGAAGCAGACCTGATGACTGAGGCACCGAGGAGTTTTT SEQ ID NO:48 CTAATGGCTTTGGTGCGAAGCAGACCTCTTCACCTTTGCTCACCATTTGTAGTCCATCGGATATATCTCCTTCGGATCCTGAGGCACCGAGGAGTTTTT SEQ ID NO:49 ACTCCfUfCfGfGfU SEQ ID NO:50 ACTCCTCGGTGCCTCA SEQ ID NO:51 rArCrUrCrCrUrCrGrGrUrGrCrCrUrCrA SEQ ID NO:52 mAmCmUmCmCmUmCmGmGmUmGmCmCmUmCmA SEQ ID NO:53 mAmCmUmCmCmUmCmGmGmUmGmCmCrUrCrA SEQ ID NO:54 GTCTGCTTCGCACCAAAGCCATTAGC SEQ ID NO:55 ACTCCTCGGTGCCTCAGGATCCGAAGGAGATATATCCGATGGACTACAAATGGTGAGCAAAGGTGAAGAG SEQ ID NO:56 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:57 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCGEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIGILANSFYGYYGYAKARWYCKECAESVTAWGRQYLETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTVGLEIVRVDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHRYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:58 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDVPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFATKKKYAVIDEEDKITTRGLKMVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEQLVIYQPITKQLHDYRARGPHVSVAKRLAARGIKIRPGTVISYIVPKGSGRIGDRAIPFDEFDPAKHKYDAGYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:59 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFAYLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAALEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVNPMMRRAAKTINFGVLYGMSAHRLSQVLAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRNAAERRAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:60 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDQPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLGPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHKQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:61 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLRATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:62 MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKR FLRVVKEKDPDVLITYDGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGG YVKEPERGLWENIVYLDFRSLGPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRLIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYE GFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRNWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHKQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT SEQ ID NO:63 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCGEYDVAPQVGHKFCKDFPGFIPSLLGPLLEERQKVKKKMKATIDPIEKKLLDYRQRLIGILANSFYGYYGYAKARWYCKECAESVTAWGRQYLETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTVGLEIVRVDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHRYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:64 MILDTDYITKDGKPIIRIFKKENGEFKIELDPHFQPYIYALLKDDSAIEEIKAIKGERHGKTVRVLDAVKVRKKFLGREVEVWKLIFEHPQDVPAMRGKIREHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETFYHEGDEFGKGEIIMISYADEEEARVITWKNIDLPYVDVVSNEREMIKR FVQVVKEKDPDVIITYNGDNFDLPYLIKRAEKLGVRLVLGRDKEHPEPKIQRMGDSFAVEIKGRIHFDLFPVVRRTINLPTYTLEAVYEAVLGKTKSKLGAEEIAAIWETEESMKKLAQYSMEDARATYELGKEFFPMEAELAKLIGQSVWDVSRSSTGNLVEWYLLRVAYARNELAPNKPDEEEYKRRLRTTY LGGYVKEPEKGLWENIIYLDFRSLYPSIIVTHNVSPDTLEKEGCKNYDVAPIVGYRFCKDFPGFIPSILGDLIAMRQDIKKKMKSTIDPIEKKMLDYRQRAIKLLANSYYGYMGYPKARWYSKECAESVTAWGRHYIEMTIREIEEKFGFKVLYADTDGFYATIPGEKPELIKKKAKEFLNYINSKLPGLLEL EYEGFYLRGFFVTKKRYAVIDEEGRITTRGLEVVRRDWSEIAKETQAKVLEAILKEGSVEKAVEVVRDVVEKIAKYRVPLEKLVIHEQITRDLKDYKAIGPHVAIAKRLAARGIKVKPGTIISYIVLKGSGKISDRVILLTEYDPRKHKYDPDYYIENQVLPAVLRILEAFGYRKEDLRYQSSKQTGLDAWLKR SEQ ID NO:65 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRKRAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:66 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:67 MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKR FLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGG YVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRRIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYE GFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHIQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT SEQ ID NO:68 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:69 MILDTDYITKDGKPIIRIFKKENGEFKIELDPHFQPYIYALLKDDSAIEEIKAIKGERHGKTVRVLDAVKVRKKFLGREVEVWKLIFEHPQDVPAMRGKIREHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKLLAFAIATFYHEGDEFGKGEIIMISYADEEEARVITWKNIDLPYVDVVSNEREMIKR FVQVVKEKDPDVIITYNGDNFDLPYLIKRAEKLGVRLVLGRDKEHPEPKIQRMGDSFAVEIKGRIHFDLFPVVRRTINLPTYTLEAVYEAVLGKTKSKLGAEEIAAIWETEESMKKLAQYSMEDARATYELGKEFFPMEAELAKLIGQSVWDVSRSSTGNLVEWYLLRVAYARNELAPNKPDEEEYKRRLRTTY LGGYVKEPEKGLWENIIYLDFRSLVPSIIVTHNVSPDTLEKEGCKNYDVAPIVGYRFCKDFPGFIPSILGDLIAMRQDIKKKMKSTIDPIEKKMLDYRQRLIKLLANSYYGYMGYPKARWYSKECAESVTAWGRHYIEMTIREIEEKFGFKVLYADTDGFYATIPGEKPELIKKKAKEFLNYINSKLPGLLEL EYEGFYLRGFFVTKKRYAVIDEEGRITTRGLEVVRRDWSEIAKETQAKVLEAILKEGSVEKAVEVVRDVVEKIAKYRVPLEKLVIHEQITRDLKDYKAIGPHVAIAKRLAARGIKVKPGTIISYIVLKGSGKISDRVILLTEYDPRKHKYDPDYYIENQVLPAVLRILEAFGYRKEDLRYQSSKQTGLDAWLKR SEQ ID NO:70 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHVEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHAIVEKILQYRELTKLKSTYIDPLPELIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKV SEQ ID NO:71 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRRIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:72 MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDQPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKR FLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGG YVKEPERGLWENIVYLDFRSLGPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRLIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYE GFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHKQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT SEQ ID NO:73 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFATKKKYAVIDEEDKITTRGLKMVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEQLVIYQPITKQLHDYRARGPHVSVAKRLAARGIKIRPGTVISYIVPKGSGRIGDRAIPFDEFDPAKHKYDAGYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:74 MILDTDYITKDGKPIIRIFKKENGEFKIELDPHFQPYIYALLKDDSAIEEIKAIKGERHGKTVRVLDAVKVRKKFLGREVEVWKLIFEHPQDVPAMRGKIREHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKLLAFAIATFYHEGDEFGKGEIIMISYADEEEARVITWKNIDLPYVDVVSNEREMIKR FVQVVKEKDPDVIITYNGDNFDLPYLIKRAEKLGVRLVLGRDKEHPEPKIQRMGDSFAVEIKGRIHFDLFPVVRRTINLPTYTLEAVYEAVLGKTKSKLGAEEIAAIWETEESMKKLAQYSMEDARATYELGKEFFPMEAELAKLIGQSVWDVSRSSTGNLVEWYLLRVAYARNELAPNKPDEEEYKRRLRTTY LGGYVKEPEKGLWENIIYLDFRSLVPSIIVTHNVSPDTLEKEGCKNYDVAPIVGYRFCKDFPGFIPSILGDLIAMRQDIKKKMKSTIDPIEKKMLDYRQRAIKLLANSYYGYMGYPKARWYSKECAESVTAWGRHYIEMTIREIEEKFGFKVLYADTDGFYATIPGEKPELIKKKAKEFLNYINSKLPGLLEL EYEGFYLRGFFVTKKRYAVIDEEGRITTRGLEVVRRDWSEIAKETQAKVLEAILKEGSVEKAVEVVRDVVEKIAKYRVPLEKLVIHEQITRDLKDYKAIGPHVAIAKRLAARGIKVKPGTIISYIVLKGSGKISDRVILLTEYDPRKHKYDPDYYIENQVLPAVLRILEAFGYRKEDLRYQSSKQTGLDAWLKR SEQ ID NO:75 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEVGWLLVVLDYSQMGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:76 CTAATGGCTTTGGTGCGAAGCAGACTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTGTTAGCAGCCGGATCTCTCGAGTTACTTATACAGTTCATCCATACCACCGGTACTATGGCGACCTTCTGCACGTTCATACTGTTCAACAATGGTATAATCCTCGTTGTGGCTGGTGATATCCAGTTGATATTAACATTATATGCACCAG GCAGCTGAACCGGCTTTTTGGCTTTGTAGGTGGTTTTTAACTTCTGCATCATAATGACCGCCATCTTTCAGCTTCAGACGCTGTTTAATTTCACCTTTCAGTGCGCCATCTTCCCGGATACATACGTTCGCTGCTTGCTTCCCAACCCATGGTCTTTTTCTGCATAACCGGACCATCACTCGGAAAATTCGTGCCACGCAGTTTAACTTTATAGATAAATTCACCA TCCTGCAGGCTGCTATCCTTGTGTAACGGTAACAACACCACCATCTTCAAAATTCATCACACGTTCCCATTTAAAACCTTCGGAAAGCTCAGTTCAGATAATCCGGGATATCTGCCGGATGTTTAACATAGGCTTTGCTACCATACATAAACTGCGGACTCAGAATATCCCCATGCAAACGGCAGCGGACCCACTTTGGTAACTTTCAGTTTTCGCGTCTGGGT GCCTTCATACGGACGACCTTCGCCTTCACCTTCAAATTCAAATTCGTGGCCATTAACGCTACCTTCCATATGACTTTGAAGCGCATGAATTCTTTGATGAGGCCATATTATCCTCTTCACCTTTGCCATGCTAGCCATATGGCTGCCGCGCGGCACCAGGCCGCTGTGTGATGATGATGATGGCTGCTGCCCATTGAGGCACCGAGGAGTTTTT sequence number77 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFAIATLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRRIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHIQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:78 MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKR FLRVVKEKDPDVLITYDGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGG YVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRLIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYE GFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHKQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT SEQ ID NO:79 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYQVPPQQLAIYQPITRALQDYKAKGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGKIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:80 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRTFIAEEGRQLVALDYSQTGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:81 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSQYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:82 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKR FLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTG GFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQ KAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYE GFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS SEQ ID NO:83 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPTQHLVIHQQITRALNDYKAIGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:84 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:85 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLSRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLDAVYEVVFGKPKEKVYAEEIAQAWKSGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYIKPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKCEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:86 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDVPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:87 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDVPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRRIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYIQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:88 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVNPMMRRAAKTINFGVLYGMSAHRLSQKLAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRNAAERRAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:89 MALGEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQVLAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:90 MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPIEVWKLYFNHPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVVTWKKIDLPYVDVVSTEKEMIKR FLRVVREKDPDVLITYNGDNFDFAYLKKRCEELGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYKRNELAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEEKLLDYRQRAIKFLANSFYGYYGYAKARWYCKECAESVTAWGREYIEMVIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEG FYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEFAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK SEQ ID NO:91 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCGEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYLETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHRYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:92 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDVPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYQVPPQQLAIYQPITRALQDYKAKGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGKIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:93 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRRRAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:94 MALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERL LWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAALEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLH TRFNQTATATGRLSSSSDPNLQSIPVRTPLGQRIRRAFIAEEGWLLVALDYSQEGLRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVNPLMRRAAKTINFGVLYGMSAHRLSQKLAIPYEEAQAFIERY FQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVRQAAERRAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE SEQ ID NO:95 MILDTDYITEDGKPVIRIFKKENGEFKIDYDRNFEPYIYALLKDDSAIEDVKKITAERHGTTVRVVRAEKVKKKFLGRPIEVWKLYFTHPQDQPAIRDKIKEHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFAIATLYHEGEEFAEGPILMISYADEEGARVITWKNIDLPYVDVVSTEKEMIKR FLKVVKEKDPDVLITYNGDNFDFAYLKKRSEKLGVKFILGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGQPKEKVYAEEIAQAWETGEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAG GYVKEPERGLWENIVYLDFRSLGPSIIITHNVSPDTLNREGCEEYDVAPQVGHKFCKDFPGFIPSLLGDLLEERQKVKKKMKATIDPIEKKLLDYRQRLIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLDYINAKLPGLLELEY EGFYKRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIYKQITRDLKDYKATGPHVAVAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLGAWLKPKT SEQ ID NO:96 / 5Biosg / GCACTTCGCTTCACCTCTCTGCTTT SEQ ID NO:97 eGeCeAeGeAeGeAGG SEQ ID NO:98 / 5phos / GAeAeGeTeGeC SEQ ID NO:99 eGeCeAeGeAeGeAGGTGAAGCGAeAeGeTeGeC SEQ ID NO:100 eGeCeAeGeAeGeAGGTGAAGC SEQ ID NO:101 / 5Phos / rGrUrCrUrGrCrUrUrCrGrCrArCrCrArArArGrCrCmAmUmUmAmGmC SEQ ID NO:102 SEQ ID NO:103 SEQ ID NO:104 rArCrUrCrCrUrCrGrGrUrGrCrCrUrCrA SEQ ID NO:105 SEQ ID NO:106 SEQ ID NO:107 SEQ ID NO:108

Claims

1. A method for producing a single-stranded polynucleotide product having at least one modified nucleotide residue, a) A step of producing a template polynucleotide annealed by at least two segment polynucleotides, wherein a template polynucleotide containing a sequence complementary to a single-stranded polynucleotide product and a pool of at least two segment polynucleotides are brought into contact under conditions that enable annealing of at least two segment polynucleotides to the template polynucleotide, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides. b) A step of extending at least one of the annealed segment polynucleotides using a pool of nucleoside triphosphates and polymerase to fill at least one sequence gap and produce at least one extended segment polynucleotide, c) Ligating one or more segmented polynucleotides and / or one or more extended segmented polynucleotides using a ligase to form a single-stranded polynucleotide product bound to a template polynucleotide in a double-stranded form, d) A step of modifying the conditions to denature a double helix containing a single-stranded polynucleotide product and a template polynucleotide, thereby producing a single-stranded polynucleotide product. Methods that include...

2. The method according to claim 1, further comprising step d) changing conditions to denature a double helix containing an impurity polynucleotide and a template polynucleotide, and changing conditions to separate one or more arbitrary impurity polynucleotides before denature a double helix containing a single-stranded polynucleotide product and a template polynucleotide.

3. The method according to claim 1 or 2, further comprising the step of separating a single-stranded polynucleotide product.

4. The method according to claim 1 or 2, wherein at least one segment polynucleotide comprises at least one modified nucleotide residue.

5. The method according to claim 1 or 2, wherein at least one segment polynucleotide comprises 5'-phosphate, 5'-thiophosphate, 5'-dithiophosphate, or 5'-methylphosphate, and optionally, the segment polynucleotide at the 3' end of the sequence gap comprises 5'-phosphate, 5'-phosphorothioate, 5'-phosphorodithioate, or 5'-methylphosphate.

6. The method according to claim 1 or 2, wherein the pool of nucleoside triphosphates comprises (i) naturally occurring nucleoside triphosphates, (ii) modified nucleoside triphosphates, or (iii) naturally occurring nucleoside triphosphates and modified nucleoside triphosphates.

7. The method according to claim 1 or 2, wherein at least one modified nucleotide comprises modification of a sugar moiety, modification of a nucleic acid base, and / or modification of the backbone, and optionally at least one modified nucleotide is N1-methyl-pseuduridine.

8. The method according to claim 1 or 2, wherein the ligase ligates the 3' and / or 5' ends of a segmented polynucleotide or an elongated segmented polynucleotide to an adjacent segmented polynucleotide or an adjacent elongated segmented polynucleotide to form a single-stranded polynucleotide product.

9. The method according to claim 1 or 2, wherein the ligase in step (c) is capable of ligating two oligonucleotides together, and one or both of the linked nucleotides to be joined are modified nucleotides.

10. The method according to claim 1 or 2, wherein the template has properties that allow it to be separated from a single-stranded polynucleotide product.

11. The method according to claim 1 or 2, further comprising the step of reusing a template polynucleotide.

12. The method according to claim 1 or 2, wherein the template polynucleotide is a recycled template polynucleotide.

13. The method according to claim 1 or 2, wherein the method is semi-continuous or continuous.

14. A method for producing a double-stranded polynucleotide product, comprising the step of annealing two complementary single-stranded polynucleotide products, A method wherein at least one of two complementary single-stranded polynucleotide products is prepared by the method of claim 1 or 2, and optionally both of the two complementary single-stranded polynucleotide products are prepared by the method of claim 1 or 2.

15. A method for producing a double-stranded polynucleotide product having at least one modified nucleotide residue, a) A step of producing a template polynucleotide annealed by at least two segment polynucleotides, wherein a template polynucleotide containing a sequence complementary to a single-stranded polynucleotide product and a pool of at least two segment polynucleotides are brought into contact under conditions that enable annealing of at least two segment polynucleotides to the template polynucleotide, wherein at least one sequence gap is formed between the at least two annealed segment polynucleotides. b) A step of extending at least one of the annealed segment polynucleotides using a pool of nucleoside triphosphates and polymerase to fill at least one sequence gap and produce at least one extended segment polynucleotide, c) Ligating one or more segmented polynucleotides and / or one or more extended segmented polynucleotides using a ligase to form a single-stranded polynucleotide product bound to a template polynucleotide in a double-stranded form. d) A step of modifying the conditions to denature a double helix containing a single-stranded polynucleotide product and a template polynucleotide, thereby producing a single-stranded polynucleotide product, and, e) Using a single-stranded polynucleotide product as a template polynucleotide in step a), and repeating steps a) to c) to produce a double-stranded polynucleotide product. Methods that include...

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