A support material for solid-phase synthesis of oligonucleotides and peptides

Abstract

A cross-linked polysaccharide bead for use in a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis is provided. The bead has at least one nucleoside, nucleotide or amino acid attached thereto, or has an entity attached thereto which entity is to form the basis for attachment of a first unit of a sequence to be synthesized.

Description

[0001] A SUPPORT MATERIAL FOR SOLID-PHASE SYNTHESIS OF OLIGONUCLEOTIDES AND PEPTIDES

[0002] TECHNICAL FIELD

[0003] The present invention relates to a solid-phase support material for oligonucleotide sequence synthesis, peptide synthesis, or oligonucleotide-peptide conjugate sequence synthesis.

[0004] BACKGROUND ART

[0005] Oligonucleotides are short synthesized sequences of nucleotides or their modified versions or analogs that are used in a variety of applications, including basic research, diagnostics, and therapeutics.

[0006] Peptides are short synthesized sequences of amino acids or their modified versions or analogs. Synthesized peptides may have many applications and can be used as antigens to generate custom antibodies, used as standards in mass spectrometry to aid in quantitation and identification, and be used as peptide drugs in a wide range of therapeutic areas.

[0007] Developers of oligonucleotide-based or peptide-based drugs and molecular diagnostic kits have a clear need for regulatory-compliant and cost-effective production methods.

[0008] Solid-phase synthesis is a common method for synthesis of oligonucleotide or peptide sequences. In this approach, the oligonucleotide or peptide is synthesized on a solid support, such as a resin bead, using repetitive steps for each chain extension cycle. The process is automated and can be easily scaled up or down depending on the amount of oligonucleotide or peptide sequence needed. Common solid-phase materials used in oligonucleotide and peptide sequence synthesis include controlled pore glass (CPG) or polystyrene, onto which e.g. polyethylene glycol (PEG or POE) is grafted.

[0009] Oligonucleotide or peptide synthesis using a solid-phase support material is commonly carried out in a closed vessel, e.g. a column, why a pressure can build up in the vessel. Swelling of the solid-phase material is a common problem encountered in solid-phase synthesis, which may lead to backpressure problems, a decrease in the efficiency of the reaction, and problems with up-scaling. There is an increasing demand for oligonucleotides and peptides in drug discovery and gene therapy, as well as advances in synthesis technologies that enable the production of longer and more complex oligonucleotides. A cost-efficient synthesis requires a high-loading capacity without compromising yield or purity and should offer scalability. As a result, there is a growing need for high-quality, stable, solid support materials that can provide improved performance and efficiency, as well as greater flexibility and scalability.

[0010] SUMMARY OF THE INVENTION

[0011] It is an object of the present disclosure to provide an improved or at least an alternative solidphase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis.

[0012] The invention is defined by the appended independent claims. Non-limiting embodiments emerge from dependent claims, the appended drawings, and the following description.

[0013] In one aspect, there is provided a cross-linked polysaccharide bead having at least one nucleoside, nucleotide or amino acid attached thereto, or having an entity attached thereto which entity is to form the basis for attachment of a first unit of a sequence to be synthesized. The entity can be a linker to which the first nucleotide or nucleoside or amino acid can be attached, but which is not intended to form part of the final synthesized sequence. Alternatively, the entity may be or include an entity which is intended to form part of the synthesized sequence at the 3' or C-terminal end of said sequence.

[0014] The present bead is thus intended for use as a solid-phase support material for the synthesis of peptide or oligonucleotide sequences or oligonucleotide-peptide conjugate sequences.

[0015] According to another aspect, there is provided a cross-linked polysaccharide bead for use in a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis, wherein a) at least one nucleotide or nucleoside binding compound is attached to the cross-linked bead via a first moiety of the compound, the nucleotide or nucleoside binding compound having a second moiety with nucleotide or nucleoside 3'-terminal binding function, or b) at least one amino acid binding compound is attached to the cross-linked bead via a first moiety of the compound, the amino acid binding compound having a second moiety with amino acid C-terminal binding function, or c) at least one nucleoside, nucleotide or amino acid is attached to the cross-linked bead, or d) at least one nucleoside or nucleotide is attached to the cross-linked bead through binding to a nucleotide or nucleoside binding compound, wherein said nucleotide or nucleoside binding compound is attached to the cross-linked bead via a first moiety of the compound, the nucleotide or nucleoside binding compound having a second moiety with nucleotide or nucleoside 3'-terminal binding function to which the nucleoside or nucleotide is bound, or e) at least one amino acid is attached to the cross-linked bead through binding to an amino acid binding compound, wherein the amino acid binding compound is attached to the cross-linked bead via a first moiety of the compound, the amino acid binding compound having a second moiety with amino acid C-terminal binding function to which the amino acid is bound.

[0016] The oligonucleotides are here synthesized sequences of nucleotides and / or their modified versions and / or analogues. The oligonucleotide may e.g. be DNA, RNA, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO) or locked nucleic acid (LNA).

[0017] The peptides are here synthesized sequences of amino acids and / or their modified versions and / or analogs.

[0018] The term "nucleotide" or "nucleoside" as used herein refer to individual nucleotides / nucleosides or varieties, analogues or modifications of nucleotides / nucleosides, such as nucleotides / nucleosides having an alternative linking group, an analogous form of purine, an analogous form of pyrimidine, a sugar analogue etc.

[0019] Oligonucleotide-peptide conjugates (OPCs) are synthetic constructs that combine peptides and nucleic acids in a single chimeric molecule.

[0020] The term amino acid comprises also varieties, analogues or modifications of amino acids, such as non-naturally occurring amino acids, etc.

[0021] With a nucleotide or nucleoside binding compound is here meant a compound having a first moiety and a second moiety, wherein the compound is attached to the cross-linked bead via its first moiety. The second moiety has the ability to bind to, or react with, a nucleotide or nucleoside 3'-terminal.

[0022] With an amino acid binding compound is here meant a compound having a first moiety and a second moiety, wherein the compound is attached to the cross-linked bead via its first moiety. The second moiety has the ability to bind to an amino acid C-terminal.

[0023] In the abovementioned alternative a) of the cross-linked polysaccharide bead, the bead is provided without any nucleoside / nucleotide bound to the second moiety of the nucleotide or nucleoside binding compound. Such a bead may then be used for oligonucleotide sequence synthesis by subsequent coupling of a nucleotide or nucleoside to the second moiety of the nucleotide or nucleoside binding compound having nucleotide or nucleoside 3'-terminal binding function.

[0024] In one embodiment, alternative d) of the cross-linked polysaccharide bead described above, the bead is provided with a first nucleoside / nucleotide bound to the second moiety of the nucleotide or nucleoside binding compound.

[0025] In the abovementioned alternative b) of the cross-linked polysaccharide bead, the bead is provided without any amino acid bound to the second moiety of the amino acid binding compound. Such a bead may then be used for peptide sequence synthesis by subsequent coupling of an amino acid to the second moiety of the amino acid binding compound having amino acid C-terminal binding function.

[0026] In one embodiment, alternative e) of the cross-linked polysaccharide particle described above, the bead is provided with a first amino acid bound to the second moiety of the amino acid binding compound.

[0027] In one embodiment, the nucleotide or nucleoside binding compound or the amino acid binding compound constitutes or comprises a linker or spacer group. Alternatively, or in addition, the nucleotide or nucleoside binding compound or the amino acid binding compound may constitute or comprise a label, a dye, an enzyme, a universal linker and / or a solubility modifier. Hence, the nucleotide or nucleoside binding compound or the amino acid binding compound may be a linker / spacer, be e.g. a label or dye etc., or be a label or dye etc. connected to a linker / spacer, binding the label or dye etc. to the cross-linked bead.

[0028] In one embodiment of the cross-linked polysaccharide bead (alternative c) mentioned above), at least one nucleoside, nucleotide or amino acid is directly attached to the cross-linked bead (without the use of a nucleotide or nucleoside binding compound or amino acid binding compound).

[0029] A first nucleotide / nucleoside or amino acid pre-bound to the cross-linked polysaccharide bead may have a capped free end.

[0030] The described functionalized cross-linked polysaccharide bead is prepared for use in a solidphase support material for oligonucleotide or peptide sequence synthesis.

[0031] In conventional oligonucleotide and peptide sequence synthesis, the solid-phase support material is typically formed of beads of polystyrene resin, controlled pore glass, silica gel or polyethylene glycol, to which beads a first amino acid is attached via its carboxyl group or to which a first nucleotide is attached with its 3'-end, or a nucleoside is attached with its 3'-end via a linker, such as a succinyl linker.

[0032] A common problem encountered in conventional solid-phase synthesis is swelling of the solidphase support material, which may lead to backpressure problems in the column, a decrease in the efficiency of the synthesizing reaction, and problems with scalability. Surprisingly, the inventors found that a solid-phase support material based on the present polysaccharide beads was associated with less backpressure problems.

[0033] The polysaccharide bead used here is cross-linked. Cross-linking may allow for use of higher flows through a column in which the solid-phase support material is packed. The beads are porous beads that contain pores of a defined size range.

[0034] The cross-linked polysaccharide bead may be an agarose bead. Polysaccharide beads, such as agarose beads, useful in the present invention can be produced e.g. as described in US6602990 Bl, US7396467B2 or US8309709 B2, which are incorporated herein. For example, in accordance with the teachings of US6602990 Bl, cross-linked polysaccharide beads, e.g., agarose beads, may be obtained by the following steps: a) preparing a solution or dispersion of the polysaccharide, b) adding a bifunctional cross-linking agent having one active site and one inactive site to the solution or dispersion from step a), c) reacting hydroxyl groups of the polysaccharide with the active site of the crosslinking agent, d) forming a polysaccharide gel, e) activating the inactive site of the cross-linking agent, f) reacting the activated site from step e) with hydroxyl groups of the polysaccharide gel, whereby cross-linking of the gel takes place.

[0035] In accordance with the above-described process, the cross-linking agent is introduced into the polysaccharide solution or dispersion before gel formation takes place. The cross-linking agent is a bifunctional agent with one active site and one inactive site. When added to the polysaccharide solution or dispersion the active site of the agent is allowed to react with the hydroxyl groups of the polysaccharide. Thereby the cross-linking agent is chemically bound to the polymer chains before the gel formation process is started. In this manner an internal cross-linking agent is introduced into the polysaccharide. By use of said process, it is possible to manufacture highly rigid gel particles.

[0036] Such agarose beads may comprise 0.5-20 wt.% agarose. The agarose beads may comprise 0.5- 20 wt.% agarose. The agarose content may be 0.5-20 wt.%, 1.5-20 wt.%, 2-20 wt.%, >2-20 wt.%, 2.5-20 wt.%, 3-20 wt.%, 3.5-20 wt.%, 4-20 wt.%, 4.5-20 wt.%, 5-20 wt.%, 5.5-20 wt.%, 6-20 wt.%, 6.5-20 wt.%, 7-20 wt.%, 7.5-20 wt.%, 8-20 wt.%, 8.5-20 wt.%, 9-20 wt.%, 9.5-20 wt.%, 10-20 wt.%, 11-20 wt.%, 12-20 wt.%, 13-20 wt.%, 14-20 wt.%, 15-20 wt.%, 16-20 wt.%, 17-20 wt.%, 18-20 wt.%, 19-20 wt.%, 3- 10 wt.%, 5-15 wt.%, or 10-20 wt.%. The higher the initial agarose concentration, the smaller the pore sizes that form after cross-linking.

[0037] The bead size D50v may be 5-200 pm, wherein D50v is the median particle size of the cumulative volume distribution. Smaller particles than 5 pm may result in high pack-pressure and column-nets, i.e. leakage of small beads through the column filter. The bead size D50v may be 5-200 pm, 10-200 pm, 20-200 pm, 40-200 pm, 60-200 pm, 80-200 pm, 100-200 pm, 120-200 pm, 140-200 pm, 160-200 pm, 180-200 pm, 5-180 pm, 5-160 pm, 5-140 pm, 5-120 pm, 5-100 pm, 5-80 pm, 5-60 pm, 5-40 pm, 5-20 pm, or 10-50 pm.

[0038] After formation, the resulting slurry of cross-linked polysaccharide beads can be dried. When forming the solid-phase support material, the polysaccharide beads may be in dried form, at least initially. Dried beads may be re-suspended in a suitable solvent, for example acetonitrile, before use to form a slurry again.

[0039] Using cross-linked polysaccharide beads, such as agarose cross-linked beads, as solid-phase material to which a first amino acid is attached via its carboxyl group, or to which a first nucleotide is attached with its 3'-end, or to which a first nucleoside is attached with its 3'-end via a linker, or to which a first nucleotide / nucleoside or amino acid is attached through binding to a nucleotide / nucleoside or amino acid binding compound attached to the bead, there was an improved chemical and physical stability against the reagents used in the synthesis, as compared to when using standard beads of polystyrene. The solid-phase material with crosslinked polysaccharide beads was also observed with pressure flow properties similar to or better / lower than for the polystyrene beads. Using this support material was observed to result in a lower reagent consumption and a higher productivity due to shorter cycle time required for the synthesis. The solid-phase material comprising cross-linked polysaccharide beads showed similar swelling behavior as polystyrene, but created lower back-pressure problems, thereby enabling large-scale synthesis (scalability from ~1 pmol scale up to industrial production). Synthesis yield and purity were similar to solid-phase support material of polystyrene. Compared to when using polystyrene, there was no need to leave a headspace, i.e. free / empty space after packing of the support material in the column, thereby distribution of reagents in the column was improved. When using traditional polystyrene- based support material a free / empty space is created for possibly swelling of the polystyrene support.

[0040] The nucleotide or nucleoside binding compound or the amino acid binding compound may be or comprise a label, a dye, an enzyme, a universal linker, a sterol, a fatty acid, a vitamin and / or a solubility modifier. Such compounds may be intended to form part of the final synthesized sequence in order to prove a desired function thereto. Some examples of binding compounds comprise one or more fluorophore, quencher, biotin, brancher, alkyne and glycoconjugate, such as GalNAc (N-acetylgalactosamine), sterol such as cholesterol, fatty acid(s) or derivatives thereof, vitamins such as folic acid. These compounds may be included in a phosphoramidiate.

[0041] A universal linker may have a conformationally rigid and chemically stable bridge head ring oxygen atom carrying a conventional 4, 4' -dimethoxytrityl (DMT) and succinyl groups locked in a syn orientation.

[0042] The at least one nucleoside, nucleotide or amino acid, may be attached to the bead via a linker. In embodiments, the nucleotide or nucleoside binding compound or the amino acid binding compound may represent said linker. In other embodiments, the nucleotide or nucleoside binding compound or amino acid binding compound may be attached to the bead via a linker, in which case the linker may optionally be regarded as forming part of the nucleotide or nucleoside binding compound or the amino acid binding compound. Hence in such embodiments, the nucleotide or nucleoside binding compound or the amino acid binding compound may comprise a linker.

[0043] The linker or spacer group may be a cleavable linker, such that the formed nucleotide or peptide sequence is cleavable from the solid-phase support material, and may be a succinyl linker, a polyethylene glycol linker, an aliphatic linker, a nitrobenzyl functionalised photolabile linker, a silyl linker, a disulfide linker, or a universal linker.

[0044] In addition to the linker / spacer group, the nucleotide or nucleoside binding compound or the amino acid binding compound may constitute or comprise a label, a dye, an enzyme, a universal linker and / or a solubility modifier (as discussed above).

[0045] The linker or spacer group may be a cleavable linker, such that the formed nucleotide or peptide sequence is cleavable from the solid-phase support material. The linker may be a linker as exemplified above.

[0046] The first building block or monomer may be attached to the solid support material through a covalent or non-covalent bond. A nucleotide may be attached directly to the solid support material through its 3' carbon end. A linker or spacer, such as succinate, may be used for covalent attachment of a nucleoside to the support material. The first nucleotide may be attached to the support without any cleavable linker.

[0047] The first amino acid may be non-covalently or covalently attached to the solid support via its C -terminus. For covalent coupling, the linker used may depend on the type of peptide being synthesized. The linker may for example be a benzyl alcohol-type linker.

[0048] The linker may be selected from a succinyl linker, a polyethylene glycol linker, an aliphatic linker, a nitrobenzyl functionalised photolabile linker, a silyl linker, and a disulfide linker.

[0049] The at least one nucleoside, nucleotide or amino acid attached to the cross-linked polysaccharide bead (optionally via a nucleotide or nucleoside binding compound or amino acid binding compound as described above, or via a linker) may be protected with a protection group.

[0050] Before a next amino acid or nucleotide can be added, the protection group is removed. Common amino acid deprotection reagents are known in the art and include trifluoroacetic acid (TFA) and hydrofluoric acid (HF). Fluorenylmethyloxycarbonyl (FMOC) is commonly used for protection of amino acids, which can be deprotected with amine solutions. The nucleotide or nucleoside protective group may be dimethoxy trityl and deprotection may be performed using chloroacetic acid in dichloromethane (DCM), or toluene.

[0051] According to another aspect there is provided a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis, comprising the cross-linked polysaccharide beads described above.

[0052] The solid-phase support material, prepared as described above, may be provided in dry form or in slurry form. When provided in dry form the material may be resuspended in a suitable solvent, for example acetonitrile, to form a slurry before or during use in oligonucleotide or peptide synthesis.

[0053] The solid-phase support material may have a first nucleotide, nucleoside or amino acid loading capacity of 50-1000 pmol / g. The loading capacity of the solid-support material for oligonucleotide or peptide synthesis refers to the amount of nucleoside / nucleotide / amino acid that can be attached to the solid support material. It is typically measured in pmol of the first nucleoside, nucleotide or amino acid attached to the support material per gram of resin / solid support material. Solid supports for conventional oligonucleotide synthesis are typically manufactured with a loading of 20- 350 pmol of nucleotide per gram of resin. The loading capacity can be measured be means of a trityl assay for oligonucleotide or FMOC assay for peptide. The loading capacity may be 50- 1000 pmol / g, 100-1000 pmol / g, 200-1000 pmol / g, 300-1000 pmol / g, 400-1000 pmol / g, 500- 1000 pmol / g, 600-1000 pmol / g, 700-1000 pmol / g, 800-1000 pmol / g, 900-1000 pmol / g, 50- 900 pmol / g, 50-800 pmol / g, 50-700 pmol / g, 50-600 pmol / g, 50-500 pmol / g, 50-400 pmol / g, or 50-300 pmol / g, 100-200 pmol / g, 200-800 pmol / g, or 300-700 pmol / g.

[0054] According to another aspect there is provided a flow-through column comprising the solidphase support material described above.

[0055] According to another aspect there is provided a method of producing beads for use in a solidphase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis. The method comprises: providing cross-linked polysaccharide beads, and i) coupling a nucleoside, nucleotide or amino acid attached thereto, or ii) coupling an entity attached thereto which entity is to form the basis for attachment of a first unit of a sequence to be synthesized, optionally followed by coupling of a nucleoside, nucleotide or amino acid to said entity.

[0056] In another aspect, a method of producing beads for use in a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis comprises providing cross-linked polysaccharide beads, and a) coupling, optionally via a linker, at least one nucleotide or nucleoside binding compound to a cross-linked bead via a first moiety of the compound, the nucleotide or nucleoside binding compound having a second moiety with nucleotide or nucleoside 3'-terminal binding function, and, optionally, binding a nucleoside or nucleotide to the nucleotide or nucleoside binding compound, or b) coupling, optionally via a linker, at least one amino acid binding compound to a cross-linked bead via a first moiety of the compound, the amino acid binding compound having a second moiety with amino acid C-terminal binding function, and optionally binding an amino acid to the amino acid binding compound, or c) coupling at least one linker to a cross-linked bead, and, optionally, binding a nucleoside or nucleotide, or an amino acid to the linker, or d) coupling at least one nucleoside, nucleotide or amino acid to a cross-linked bead, and e) optionally, adding a protection group to any free end of a nucleoside, nucleotide or amino acid.

[0057] According to a further aspect there is provided a method of synthesizing an oligonucleotide sequence, a peptide sequence, or an oligonucleotide-peptide conjugate sequence, the method comprising the following steps:

[0058] 1) providing a solid-phase support material, the solid-phase support material comprising cross-linked polysaccharide beads produced as above, wherein nucleosides, nucleotides or amino acids are coupled to the cross-linked beads, and provided with a protection group at any free end,

[0059] 2) de-protecting the protected nucleosides, nucleotides or amino acids,

[0060] 3) coupling a nucleotide or an amino acid to a free end of a nucleoside, nucleotide or amino acid attached to the solid-phase support material,

[0061] 4) capping free ends of nucleotides or amino acids coupled to the solid-phase support material, and

[0062] 5) repeating steps 2-4 until a desired oligonucleotide or peptide sequence is assembled.

[0063] Step 1) of the method, wherein a solid-phase support material is provided, may comprise providing beads wherein the nucleosides, nucleotides or amino acids are pre-attached to the cross-linked polysaccharide beads. Optionally, the free ends are already provided with a protection group at any free end, or protection groups are added in step 1. Alternatively, step 1) of the method may comprise a step of attaching nucleosides, nucleotides or amino acids to the cross-linked polysaccharide beads at discussed above, and further add protection groups to any free end. The formed oligonucleotides covalently attached to the beads may be extended by DNA polymerases, using nucleoside 5'-triphosphates (NTPs).

[0064] The flow rate used during the synthesis of the oligonucleotide or peptide sequence depends on the scale of the synthesis. There are advantages and disadvantages of using high flow rates in oligonucleotide / peptide synthesis on solid-phase support material. High flow rates can increase the speed of the synthesis process. However, high flow rates can also decrease the purity of the formed product.

[0065] The coupling of a nucleotide or an amino acid to a free end of a nucleoside, nucleotide or amino acid attached to the solid-phase support material in step 3) may be performed enzymatically. In such enzymatic synthesis, nucleotides or oligonucleotide initiators (>3 nucleotides) or amino acids may be provided attached to the support material with protection groups at their free ends. The oligonucleotide initiators may be pre-produced and attached to the solid support. Alternatively, the oligonucleotide initiators may first be chemically synthesized using the method described above, before enzymatic synthesizing is used. When enzymatically synthesizing an oligonucleotide a DNA polymerase and nucleoside 5'- triphosphates (NTPs) may be used. When enzymatically synthesizing a peptide, proteolytic enzymes capable of hydrolyzing peptide bonds may be used.

[0066] The use of solid support in enzymatic synthesis of oligonucleotides / peptides has several advantages over traditional solution-phase enzymatic synthesis, including improved reaction efficiency, reduced reagent consumption, and simplified product purification. In the enzymatic synthesis of oligonucleotides and peptides a solid support assembly of the growing chain is carried out by the enzymes performing chemical reactions. These reactions can be performed either with assistance or without assistance of template sequence and initiation point assembled on the solid support.

[0067] When the polysaccharide beads are agarose beads, which are hydrophilic, this is preferable for enzymatic synthesis.

[0068] The enzymatic synthesis reaction is carried out under hydrated and mild conditions, coupled with the specificity of the enzyme. The enzymatic synthesis can reduce the formation of byproducts and the need for purification as compared to chemical synthesis. The method of synthesizing an oligonucleotide or peptide sequence may further comprise a step 6) of cleaving the formed oligonucleotide or peptide sequence from the solid-phase support material. The cleaving may be an enzymatic or chemical cleaving from the solid-phase support material.

[0069] In the methods described above, the solid-phase support material may be provided in a flow- through column.

[0070] According to a sixth aspect there is provided a kit of parts for a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotidepeptide conjugate sequence synthesis comprising: cross-linked polysaccharide beads as described above, and optionally, nucleotides, nucleosides or amino acids for coupling to the beads. The nucleotides, nucleosides or amino acids may be used for forming of a oligonucleotide, peptide or oligonucleotide / peptide conjugate.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Fig. 1 shows schematically a method of synthesizing an oligonucleotide sequence, a peptide sequence or an oligonucleotide-peptide conjugate sequence on a solid-phase support material.

[0073] Fig. 2 shows an example of synthesis of an oligonucleotide sequence on a solid-phase support material.

[0074] Fig. 3 shows the trityl area for synthesis of a DNA sequence with 13 nucleotides on a solidphase support material comprising cross-linked agarose beads (dots) and cross-linked polystyrene beads (squares), respectively.

[0075] Fig. 4 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides using different solid-phase support materials (support material comprising cross-linked agarose beads with different agarose content (diamonds, dots and squares) and reference support material comprising polystyrene beads (triangles)).

[0076] Fig. 5 shows the detritylation time required for synthesis of a DNA sequence with 84 nucleotides on different solid-phase support materials (support material comprising cross- linked agarose beads (squares) and reference support material comprising polystyrene beads (dots and diamonds)).

[0077] Fig. 6 shows the deprotection time required for synthesis of a DNA sequence with 21 nucleotides on different solid-phase support materials (support material comprising crosslinked agarose beads (squares, diamonds and triangles) and reference support material comprising polystyrene beads (dots)).

[0078] Figs. 7a and 7b show the volume of solvent needed for synthesis of a DNA sequence with 21 nucleotides using a solid-phase support material comprising polystyrene beads and crosslinked agarose beads, respectively.

[0079] Figs. 8a and 8b show the time needed for synthesis of a DNA sequence with 21 nucleotides using a solid-phase support material comprising polystyrene beads and cross-linked agarose beads, respectively.

[0080] Fig. 9 shows the distribution of reagents, amidite and tetrazole-based activator during recirculation, a solid-phase support material comprising polystyrene beads (dotted line) and a support material comprising cross-linked agarose beads (solid line).

[0081] Figs. lOa-lOg schematically illustrate different embodiments of a solid-phase support material comprising a cross-linked polysaccharide bead.

[0082] Fig. 11 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solidphase support material comprising cross-linked agarose beads where thiolation (dots) and oxidation (squares) is performed, respectively.

[0083] Fig. 12 shows the trityl area for synthesis of a DNA (dots) and RNA (squares) sequence with 84 nucleotides on a solid-phase support material comprising cross-linked agarose beads, respectively.

[0084] Fig. 13 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solidphase support material comprising cross-linked agarose beads where lower equivalence of amidite (dots) and higher equivalence of amidite (squares), respectively. Fig. 14 shows the duration for detritylation for synthesis of a purine-rich sequence with 30 nucleotides on a solid-phase support material comprising cross-linked agarose beads (squares) and cross-linked polystyrene beads (dots), respectively.

[0085] Fig. 15 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solidphase support material comprising cross-linked agarose beads where base loaded support (dots) and UnyLinker loaded support (squares) were used, respectively.

[0086] Fig. 16 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solidphase support material comprising cross-linked agarose beads where UnyLinker loaded support was verified with different amidite equivalence as 3 eq (dots), 2 eq (squares) and 1.5 eq (triangles), respectively.

[0087] Fig. 17 shows the duration required for detritylation with synthesis of a DNA sequence with 21 nucleotides on a solid-phase support material comprising cross-linked agarose beads where bed height of 2 cm (dots), 8 cm (squares).

[0088] DETAILED DESCRIPTION

[0089] Below is described in more detail the use of a solid-phase support material for synthesis of oligonucleotide sequences, peptide sequences and oligonucleotide-peptide conjugate sequences. The solid-phase support material comprises cross-linked polysaccharide beads, here exemplified by cross-linked agarose beads. The solid-phase oligonucleotide or peptide sequence synthesis is performed in a flow-through column reactor using a pump-driven system. Prior to synthesis, the column reactor is filled with the solid-phase support material with or without the first nucleotide, nucleoside or amino acid of the sequence to be synthesized already attached to the beads. If the beads filled into the column reactor are without pre-attached first nucleotides, nucleosides or amino acids, once packed in the column, there is a step of attaching the first nucleotides, nucleosides or amino acids to the beads. The first nucleosides, nucleotides or amino acids are the anchors for the growing the oligonucleotides or peptides. Sequence synthesis is then carried out in a number of sequential steps. In Fig. 1 is schematically illustrated a method of synthesizing an oligonucleotide or peptide sequence, the method comprising the following steps: 1) providing 100 a solid-phase support material, the solid-phase support material comprising cross-linked polysaccharide beads with nucleosides, nucleotides or amino acids attached thereto, which nucleosides, nucleotides or amino acids are provided with protection groups at their free ends; 2) deprotecting 101 the protected nucleotides, nucleosides or amino acids; 3) coupling 102 a nucleotide or an amino acid to a free end of a nucleotide, nucleoside or amino acid attached to the solid-phase support material; 4) capping 103 free ends of nucleotides or amino acids attached to the solid-phase support material; 5) repeating 104 steps 2-4 until a desired nucleotide or peptide sequence is assembled. The method may also comprise a step 6) of cleaving 105 the formed nucleotide or amino acid sequence from the solid-phase support material. Instead of chemical synthesis, the method can be an enzymatic method with an enzyme present in step 3) for enzymatic formation of oligonucleotide or peptide.

[0090] In Fig. 2 is shown an example of synthesis of an oligonucleotide sequence on a solid-phase support material. In a first step, de-block (detritylation), a 5'-dimethoxytrityl protecting group is removed from the support-bound nucleotide using e.g. chloroacetic acid in DCM or toluene. In the second step, coupling, a phosphoramidite monomer (A, C, G, U, or T) mixed with an activator, such as 5-(benzylmercapto)-lH-tetrazole (BMT), is added and a phosphite triester internucleotide bond is formed. In a third step, capping and oxidation, a 5'-cap is added to the growing chain of nucleotides. The cap is usually an acetyl group that protects the 5'-hydroxyl group from further reaction during the synthesis. In the oxidation the phosphite triester is converted to phospo triester. This step is carried out using an oxidizing agent such as iodine or tert-butyl hydroperoxide (TBHP). Next there is a new step of de-blocking. The formed oligonucleotide may be deprotected and purified or the described synthesis steps repeated one or more cycles.

[0091] Figs. lOa-lOg schematically illustrate different embodiments of the solid-phase support material comprising a cross-linked polysaccharide bead. In Fig. 10a and Fig. 10c a first nucleotide / nucleoside or amino acid 4 is coupled to a nucleotide or nucleoside binding compound or amino acid binding compound 3. The nucleotide / nucleoside binding compound 3 has a first moiety and a second moiety, wherein the second moiety has nucleotide or nucleoside 3'-terminal binding function to which the nucleotide / nucleoside 4 is coupled. The first moiety of the compound 3 is attached to the bead 1 via a linker / spacer 2, Fig. 10a, or is directly attached to the bead, Fig. 10c. The amino acid binding compound 3 has a first moiety and a second moiety with amino acid C-terminal binding function to which the first amino acid 4 is coupled, Fig. 10a and Fig. 10c. The first moiety of the amino acid binding compound 3 is attached to the bead 1 via a linker / spacer 2, Fig. 10a, or is directly attached to the bead 1, Fig. 10c. In Fig. 10 d) is shown a first nucleotide / nucleoside or amino acid 4 directly attached to the bead 1 and in Fig. 10b is shown a nucleoside / nucleotide or amino acid 4 attached to the bead 1 via a linker / spacer 2. In Figs. lOe and lOf is shown a bead 1 functionalised with a nucleotide or nucleoside binding compound or amino acid binding compound 3, either directly attached to the bead 1, Fig. lOe, or coupled to the bead 1 via a linker / spacer, Fig. lOf. In Fig. 10g is illustrated a cross-linked polysaccharide bead with a linker / spacer 2 attached thereto. Below are described experiments using the solid-phase support material of Fig. 10b.

[0092] EXPERIMENTAL

[0093] Derivatization of crosslinked agarose support material with T succinate nucleoside

[0094] Support material, i.e. cross-linked agarose beads, produced as described in US6602990 Bl, of three different agarose concentrations were used: cross-linked agarose resin A / B / C of increasing agarose concentration (A<B<C) within the range 0.5-20 %. The resins having a respective bead size D50v of ~ 60 pm; ~ 53 pm and ~ 40 pm, respectively, and compared to reference support materials of polystyrene beads (PS5G, PS40S Cytiva®, where PS40S has a higher crosslinking degree than PS5G). The agarose support material was washed with 5*2GV (5 times with the double gel volume (GV) of the support material) of acetone followed by 10*2GV of acetonitrile. Further, gel was sucked dry on vacuum and 1GV of dry acetonitrile, nucleoside succinate to be loaded (3.2-3.5 mmol), followed by diisopropylethylamine (DIPEA;1.5-3.5 mmol / same succinate scale) and O-(Benzotriazol-l-yl)-N, N, N', N'- tetramethyluronium hexafluorophosphate (HBTU; 3.2-3.5 mmol) were added to the gel slurry. The reaction was allowed to proceed on a shaker overnight at room temperature. Gel was washed by 2*2GV acetonitrile and unreacted amine groups were capped by using the equimolar volume of cap A (20% N-methylimidazole in acetonitrile) and cap B (Bl: 40% acetic anhydride in acetonitrile, B2: 60% lutidine in acetonitrile). After 3 h on a shaker, the agarose support material was filtered and washed with 2*2GV of acetonitrile followed by 5*2GV of ethanol. First nucleotide can also be assembled on the solid support material using automated pump driven system. The agarose support material was dried on a vacuum and the loading was measured by performing a trityl assay. Deprotection was performed using 0.1 M p- toluene sulfonic acid in acetonitrile. The property being measured is the degree of nucleotide substitution, expressed in pmol / g.

[0095] Oligonucleotide synthesis

[0096] For oligonucleotides (DNA, RNA, phosphodiester or phosphorothioate), the nucleotide sequence was synthesized on the AKTA oligosynt instrument (Cytiva®). All oligos were synthesized using a standard cycle of acid-catalyzed detritylation (3%, v / v, dichloroacetic acid in toluene), coupling (5-(benzylmercapto)-lH-tetrazole (BMT) as activating agent, 0.3 M in acetonitrile), capping (Cap A, 20%, v / v, N-methylimidazole / acetonitrile and an equal volume of Bl (40%, v / v, acetic anhydride in acetonitrile) and B2 (60%, v / v lutidine in acetonitrile) as Cap B were mixed in situ for capping), and iodine-based oxidation (0.05 M iodine in pyridine with 10% v / v water) using cross-linked agarose support on automated solid-phase synthesizer and P-cyanoethyl phosphoramidite monomers. The phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.150 M and used in the presence of molecular sieves (0.3 nm rods of 1.6 mm). The coupling time (recycle) was 3-10 min with 1.5-5 eq. of amidite, the support was treated with diethylamine (DEA) before deprotection with ammonium hydroxide for 16-20 h at 55°C. In Table 1 below, the sequences and molecular weights of three different oligonucleotides synthesized, SEQ ID NO: 1 (13 nucleotides), SEQ ID NO: 2 (21 nucleotides) and SEQ ID NO: 3 (84 nucleotides) are shown.

[0097] Table 1. Oligonucleotide sequences synthesized

[0098] Detritylation

[0099] The first reaction, detritylation, removed a 5'-dimethoxytrityl protecting group from the support-bound nucleoside. The release of the protecting group generated a bright red-orange color and its relative quantities could be measured on-line by UV-VIS. Deprotection was performed using 3%, v / v, dichloroacetic acid in toluene.

[0100] Wash After detritylation, the support was washed with acetonitrile to remove the detritylation reagent.

[0101] Synthesis of DNA phosphodiester oligonucleotides

[0102] The second reaction involved coupling the appropriate phosphoramidite monomer (A, C, G, U or T) mixed with an activator. In this reaction, a phosphite triester internucleotide bond was formed in very high yields, usually over 99 %. For DNA, coupling was most often performed in a single pass of reagents through the column. With slower coupling kinetics, e.g. for RNA, a longer reaction time is required. This is achieved by re-circulating coupling reagents over the column.

[0103] Wash

[0104] After coupling, the solid-phase support material was washed with acetonitrile.

[0105] Oxidation / thiolation

[0106] The newly formed phosphite triester internucleotide bond was then converted to the corresponding phosphorothioate or phosphodiester with a thiolating or oxidation reagent respectively.

[0107] Wash

[0108] After oxidation / thiolation, the solid-phase support material was washed with acetonitrile.

[0109] Capping

[0110] The final step in the synthesis cycle was capping the unreacted 5'-hydroxyl groups with acetic anhydride.

[0111] Wash

[0112] After capping, the solid-phase support material was thoroughly washed with acetonitrile before beginning the next cycle. The synthesis steps above can be repeated until the full- length oligonucleotide has been synthesized.

[0113] Deprotection of oligonucleotides

[0114] After the synthesis of the oligonucleotide sequence, cleavage from the solid support and deprotection of protecting groups were carried out by treating the resin with 25% aq. NH3 for 12-16 hours at 55 °C. Further, the supernatant solution was collected, and the support was washed (2*1GV (gel volume)) with water and 50% EtOH in water.

[0115] OD analysis

[0116] The fractions were collected, and the concentration was measured at 260 nm in a UV-VIS spectrophotometer.

[0117] UPLC-IEX-analysis

[0118] The purity of oligonucleotides was analyzed on IEX-UPLC with tris and sodium perchlorate as running buffers.

[0119] Parameters used

[0120] Flow rate: 1.0 mL / min, Temperature: 50 degrees, Gradient and run time: 0-55% B in 20 min, 55-100 % B in 4 min then stop method (zero flow and 0 % A and B), Detector: UV-260 nm.

[0121] The sample used was 1 mL (10 OD / mL) and the injection volume was 2 uL.

[0122] Results

[0123] Loading capacity

[0124] Table 2 below summarizes the loading capacity of the three agarose solid-phase support materials used, A, B, C / C1 / C2, and the reference material of polystyrene, P5SG, as measured with a trityl assay. The trityl assay is well-known in the art and is performed using UV spectrometry (here 411 nm) by separating (deprotecting) the protective group dimethoxy trityl from the nucleoside coupled to the support. Deprotection was performed using 0.1 M p- toluene sulfonic acid in acetonitrile. The property being measured is the degree of nucleoside substitution, expressed in pmol / g.

[0125] Table 2. Support material used and loading observed after succinate couplings. The results show that the agarose support material with cross-linked agarose beads can be used as solid-phase support material for oligonucleotide synthesis. Loading of the first nucleotide on the resin using optimized succinate coupling resulted in different activation levels depending on the particle size of the agarose resin. The particle size of the resin can affect the available surface area for coupling, as well as the diffusion rate of reagents to and from the resin. The observed trend for loading capacity was A<B<C, which may be associated with the particle size of these resins (A~ 60 pm; B ~ 53 pm and C ~ 40 pm).

[0126] It may be necessary to adjust the coupling conditions, such as the concentration of reagents, reaction time, or temperature, for each particle size of resin to achieve optimal activation levels and nucleotide loading as observed in Cl and C2.

[0127] Trityl area

[0128] The trityl group is a chromophore that is used to determine coupling efficiency in oligonucleotide synthesis. The synthesis cycle begins with the removal of the acid-labile DMT ether from the 5'-hydroxyl of the 3'-terminal nucleotide. This is usually accomplished by using dichloroacetic acid (DCA) in toluene or dichloromethane. The resulting trityl cation chromophore can be quantitated to determine coupling efficiency.

[0129] The trityl area for synthesis of a DNA sequence with 13 nucleotides on a solid-phase support material comprising cross-linked agarose beads, material B (dots), and cross-linked polystyrene material (PS5G, squares), respectively, is shown in Fig. 3 (nucleotides on the X-axis and trityl area (mAU) on the Y-axis). The graph shows that the detritylation profile for a 13- nucleotide long oligo synthesis is similar in nature (trend) for an agarose-based support and a polystyrene support. Absolute numbers for trityl area are dependent on the scale of the synthesis and time required for the deblock step as it is assessed with the volume / time of reagent used for this step. Lower values for agarose-based supports can be attributed to the quicker deblock times compared to polystyrene-based supports.

[0130] In Fig. 4 is shown the trityl area for synthesis of a DNA sequence with 21 nucleotides using different solid-phase support substrates: agarose support material A (diamonds), agarose support material B (dots), agarose support material C (squares) and cross-linked polystyrene material (PS5G, triangles). The graph shows that the detritylation profile for a 21-nucleotide long oligo synthesis is similar in nature (trend) for agarose-based supports and polystyrene supports. Absolute numbers for the trityl area are dependent on the scale of the synthesis and the time required for the deblock step as it is assessed with the volume / time of reagent used in this step. Lower values observed for all the agarose-based supports can be attributed to the quicker deblock times compared to the polystyrene-based supports.

[0131] Detritylation

[0132] In Fig. 5 is shown the detritylation time required for synthesis of a DNA sequence with 84 nucleotides (base number vs trityl time (minutes)) using different solid-phase support substrates: agarose support material A (squares) and reference materials P5SG (dots) and PS40S (diamonds).

[0133] The processing time for agarose support material A was observed to be lower than for the reference solid-phase support material as the detritylation profiles were better. Less detritylation means less risk of depurination of the sequence and that will result in higher quality oligonucleotide sequences, in particular for longer lengths of the sequence where many deblock steps are performed (based on the length of sequence).

[0134] Deprotection time

[0135] Fig. 6 shows the deprotection time required for synthesis of a DNA sequence with 21 nucleotides vs cycle number on agarose support material C (squares) (scale: 400 pmol), agarose support material C (diamonds), (scale: 309 pmol), agarose support material B (triangles), (scale: 309 pmol) and PS5G (dots), (scale: 229 pmol). The graph shows that the time required for the deprotection (deblock step) is quicker on agarose support material (~6-7 minutes) compared to polystyrene support (~9-10 minutes). It is worth mentioning that higher scale means required more volume of reagents to perform the deblocking step, however, the time required for the agarose support was observed to be similar for different scales of synthesis which are up to 60 % higher than the usual scale for polystyrene supports in similar dimensions of columns used.

[0136] Yield

[0137] Yields are discussed in terms of optical density (OD) observed (at 260 nm) for crude oligonucleotides after cleavage from the solid support and deprotection of protecting groups. Higher OD / mL means a higher amount of oligonucleotide produced from the same column volume (CV), provided similar scales are used. Higher scales can result in a higher amount of OD / CV, which results in higher productivity and lower reage nts / solve nt consumption for oligonucleotide synthesis.

[0138] For agarose-based supports, total OD observed was higher than for the polystyrene support (e.g agarose-based synthesis of a 21-nucleotide long DNA resulted in ~44K total OD, whereas polystyrene based supports is known to have ~31K total OD when used in columns of the same dimensions). This can be attributed to the possibility of higher scales with agarose-based supports (packing higher amount results in a higher scale of synthesis).

[0139] Yields are also assessed for the purity of oligonucleotide observed when analysed on HPLC. Agarose-based supports resulted in a higher purity of oligo as compared to the polystyrene supports. In particular, when an 84 nucleotide-long DNA sequence was synthesized the full- length oligo product (FLP) was observed to be 46 % using agarose-based supports whereas with the polystyrene support a purity for full-length product was up to 30 %.

[0140] Reagent consumption

[0141] Figs. 7a and 7b show the volume of solvent needed for synthesis of a DNA sequence with 21 nucleotides using a solid-phase support material comprising polystyrene beads and crosslinked agarose beads, respectively. Use of the agarose support resulted in the usage of ~38 mL of deblock solution, whereas use of the polystyrene support was observed to require ~88 mL of deblock solution. This corresponds to the usage of a 50 % lower volume of deblock solution for each deblock step for 21 nucleotides DNA when synthesis is performed using agarose supports.

[0142] There was less reagent consumption during oligonucleotide synthesis using agarose-based substrates as compared to when using polystyrene substrates.

[0143] Overall consumption of deblock reagent was observed to be 25% less compared to the polystyrene support material. This might be attributed to the nature of the agarose-based supports where it is efficient to perform the deblock step with deblock reagent in a toluene solvent compared to the polystyrene support. Properties of agarose-based support material might allow higher flow rates and fewer pressure possibilities, which allow faster kinetics for cleavage and washing of cleaved protecting groups from the agarose supports.

[0144] Synthesis time

[0145] Figs. 8a and 8b show the time needed for the synthesis of a DNA sequence with 21 nucleotides using a solid-phase support material comprising polystyrene beads and cross-linked agarose beads, respectively. The graph shows that the time required for synthesis using agarose support was ~25 minutes less compared to when using the polystyrene support. The faster synthesis time can be due to less time required for each deblock step, quicker washing steps and possibly less back pressure developed related to swelling of the supports in contact with different reagents and solvents used during the synthesis cycles.

[0146] Distribution of reagents, amidite and activator

[0147] Fig. 9 shows the distribution of reagents, amidite, and tetrazole-based activators during recirculation (i.e. recirculation of reagents over the column through the pump and back in the column for a certain time or a number of times), comparing a solid-phase support material comprising polystyrene beads (PS5G, dotted line) and a support material comprising crosslinked agarose beads (solid line). This was measured using conductance reading for the recirculation step. As observed in the figure the distribution of tetrazole activated amidite, which is charged, resulted in a more homogenous distribution (based on conductance) over the column for each recirculation step compared to polystyrene support where conductance was saturated.

[0148] Packing density

[0149] The packing density in columns for the agarose material was about 159 mg / mL compared to 111 mg / mL for the polystyrene material, PS5G, which resulted in possibilities of higher scales (up to 60% higher) synthesis. Higher scales with the same column dimensions means lower consumption of reagents and solvents used in synthesis. In particular, lower wash volumes were required for higher-loaded support compared to low-loaded supports.

[0150] Thiolation

[0151] In Fig. 11 is shown the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solid-phase support material comprising cross-linked agarose beads where thiolation (dots) and oxidation (squares) is performed, respectively. The total trityl area and nature (trend) of detritylation represent similar synthesis for both the oxidation and thiolations in the synthesis cycle. Yield for thiolation was observed similar (143 OD / pmol and 82 % full length product) to oxidation (136 OD / pmol and 80 % full length product) and LCMS confirmed no S-0 impurity in the product.

[0152] RNA synthesis

[0153] Fig. 12 shows the trityl area for synthesis of a DNA (dots) and RNA (squares) sequence with 84 nucleotides on a solid-phase support material comprising cross-linked agarose beads, respectively. The total trityl area and nature (trend) of detritylation represent similar synthesis for both the DNA and RNA monomers in the synthesis cycle. Agarose support had similar performance for synthesis on both the DNA and RNA long oligonucleotides.

[0154] Amidite equivalence

[0155] Fig. 13 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solidphase support material comprising cross-linked agarose beads where lower equivalence (1.8 ) of amidite (dots) and higher equivalence (3.0) of amidite (squares), respectively. Both the 3 and 1.8 equivalence resulted into similar synthesis and similar yields and purity (~130 OD / pmol and ~82 % full length product). Similarly, Fig. 16 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solid-phase support material comprising cross-linked agarose beads where UnyLinker loaded support was verified with different amidite equivalence as 3 eq (dots) , 2 eq (squares) and 1.5 eq (triangles), respectively. Agarose support with UnyLinker resulted into superior quality and yields for 21 nucleotide long DNA even with 1.5 equivalence of amidite which suggested better synthesis performance for agarose based support compared to the polystyrene based support.

[0156] Purine rich DNA sequence

[0157] Fig. 14 shows the duration for detritylation for synthesis of a purine rich sequence with 30 nucleotides on a solid-phase support material comprising cross-linked agarose beads (dots) and cross-linked polystyrene beads (squares), respectively. Synthesis with agarose base d support resulted lower volumes for detritylation as also observed for 84 nucleotide long DNA synthesis. This is important aspect in terms of challenging / complex DNA oligonucleotide synthesis such as purine rich oligos where longer exposure of detritylation reagent can result into depurinations and impurity formation. Agarose based support can reduce such impurity formation.

[0158] UnyLinker chemistry

[0159] Fig. 15 shows the trityl area for synthesis of a DNA sequence with 21 nucleotides on a solidphase support material comprising cross-linked agarose beads where nucleobase succinate loaded support (dots) and UnyLinker succinate loaded support (squares) were used, respectively. Both base loaded and UnyLinker loaded succinates on agarose showed similar synthesis and results or UnyLinker showed even better purity and required lower equivalence of amidite monomer.

[0160] Bed heights

[0161] Fig. 17 shows the duration required for detritylation with synthesis of a DNA sequence with 21 nucleotides on a solid-phase support material comprising cross-linked agarose beads where bed height of 2 cm (dots) , 8 cm (squares). Result suggests the improved bed height does not impact the synthesis and the time required for detritylation (trend) is similar for both the bed heights. This was also confirmed with the yield and purity of synthesis where quality was similar for higher bed heights to the smaller bed heights. This is advantageous as the synthesis scale can be improved with new support as higher bed heights as possible compared to the polystyrene base support where only 2 cm bed height was possible in stainless steel column.

[0162] Peptide synthesis

[0163] The major steps in solid-phase peptide synthesis include:

[0164] Activation of the resin

[0165] The first step is to activate the resin by treating it with a suitable activating agent. This is done to ensure that the amino acids are attached to the resin.

[0166] Coupling

[0167] The next step is coupling, where the activated resin is reacted with an amino acid derivative that has been protected with a suitable protecting group. Some of the most common coupling reagents used in solid-phase peptide synthesis include HATU, HBTU, PyBOP, and TBTU. Deprotection

[0168] After coupling, the protecting group is removed from the amino acid derivative. Common deprotection reagents include TFA, HF and amine derivatives.

[0169] Washing and drying

[0170] The peptide resin is washed and dried after each coupling and deprotection step.

[0171] Cleavage

[0172] The final step is cleavage, where the peptide is cleaved from the resin using a suitable cleavage agent such as TFA.

[0173] Enzymatic synthesis of oligonucleotide or peptide sequences

[0174] In the enzymatic synthesis of oligonucleotides or peptides a solid support assembly of the growing oligonucleotide or peptide chain is carried out by enzymes performing chemical reactions. These reactions can be performed either with assistance or without assistance of template sequence and initiation point assembled on the solid support.

[0175] The major steps include:

[0176] Activation of the resin

[0177] The first step is to activate the resin by treating it with a suitable activating agent.

[0178] Coupling

[0179] The next step is coupling of a 3'-protected nucleotide / a 3'-protected nucleotide initiator strand or coupling of an amino acid derivative that has been protected with a suitable protecting group to the solid support. The oligonucleotide initiator (> 3nt) may be attached to the support or may be formed on the support as described above for chemical synthesis of oligonucleotides.

[0180] Deprotection

[0181] After coupling, the protecting group is removed.

[0182] Coupling

[0183] The next nucleotide is coupled to the 3'-OH group of the uncapped nucleotide or the next amino acid is coupled to the uncapped amino acid end in the presence of an enzyme. Once the enzymes or substrates are immobilized on the solid support, the enzymatic reaction can take place. The reaction conditions such as temperature, pH, and buffer concentration can be optimized to improve the reaction efficiency.

[0184] When enzymatically synthesizing an oligonucleotide a DNA polymerase such as nucleoside 5'- triphosphates (NTPs) may be used. When enzymatically synthesizing a peptide an enzyme such as a-chymotrypsin, papain, subtilisin or trypsin may be used.

[0185] Capping

[0186] Any unreacted end groups are capped.

[0187] The steps are repeated until the desired oligonucleotide sequence is synthesized. Cleavage

[0188] The final step is cleavage.

[0189] For the purposes of enzymatic release of the oligonucleotide, uracil may be placed at the 3' end of the initiator for a point of cleavage from the solid support using e.g. uracil DNA glycosylase. Alternatively, the formed oligonucleotide may be cleaved chemically as discussed above. The peptide may be cleaved as discussed above.

Claims

CLAIMS1. A cross-linked polysaccharide bead for use in a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis, wherein the bead has at least one nucleoside, nucleotide or amino acid attached thereto, or wherein the bead has an entity attached thereto which entity is to form the basis for attachment of a first unit of a sequence to be synthesized.

2. A cross-linked polysaccharide bead according to claim 1, wherein a) at least one nucleotide or nucleoside binding compound is attached to the cross-linked bead via a first moiety of the compound, the nucleotide or nucleoside binding compound having a second moiety with nucleotide or nucleoside 3'-terminal binding function, or b) at least one amino acid binding compound is attached to the cross-linked bead via a first moiety of the compound, the amino acid binding compound having a second moiety with amino acid C-terminal binding function, or c) at least one nucleoside, nucleotide or amino acid is attached to the cross-linked bead, or d) at least one nucleoside or nucleotide is attached to the cross-linked bead through binding to a nucleotide or nucleoside binding compound, wherein said nucleotide or nucleoside binding compound is attached to the cross-linked bead via a first moiety of the compound, the nucleotide or nucleoside binding compound having a second moiety with nucleotide or nucleoside 3'-terminal binding function to which said nucleoside or nucleotide is bound, or e) at least one amino acid is attached to the cross-linked bead through binding to an amino acid binding compound, wherein said amino acid binding compound is attached to the crosslinked bead via a first moiety of the compound, the amino acid binding compound having a second moiety with amino acid C-terminal binding function to which the amino acid is bound.

3. The cross-linked polysaccharide bead of claim 1, wherein said entity is a nucleotide or nucleoside binding compound or an amino acid binding compound as defined in claim 2.

4. The cross-linked polysaccharide bead of claim 2 or 3, wherein the nucleotide or nucleoside binding compound or the amino acid binding compound is or comprises a label, a dye, an enzyme, a universal linker, a sterol, a fatty acid, a vitamin, and / or a solubility modifier.

5. The cross-linked polysaccharide bead of any one of claims 1 to 4, wherein the at least one nucleoside, nucleotide, or amino acid attached to the polysaccharide bead is protected with a protection group.1006. The cross-linked polysaccharide bead of any one of claims 1-5, wherein said entity is a linker.

7. The cross-linked polysaccharide bead of any of claims 2-5, wherein the nucleotide or105 nucleoside binding compound or the amino acid binding compound is or comprises a linker.

8. The cross-linked polysaccharide bead of any one of claims 2-5, wherein the at least one nucleoside, nucleotide, amino acid, nucleotide or nucleoside binding compound or amino acid binding compound is attached to the bead via a linker. no9. The cross-linked polysaccharide bead of any one of claims 6-8, wherein the linker is selected from a succinyl linker, a polyethylene glycol linker, an aliphatic linker, a nitrobenzyl functionalised photolabile linker, a silyl linker, and a disulfide linker.115 10. The cross-linked polysaccharide bead of any one of claims 1-9, wherein the polysaccharide bead is an agarose bead.

11. A solid-phase support material for oligonucleotide and / or peptide sequence synthesis, comprising cross-linked polysaccharide beads of any one of claims 1-10.12012. The solid-phase support material of claim 11 having a loading capacity of 50-1000 pmol / g.

13. A flow-through column comprising a solid-phase support material of claim 11 or 12.

14. A method of producing beads for use in a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis, the method comprising:- providing cross-linked polysaccharide beads, and i) coupling a nucleoside, nucleotide or amino acid attached to said cross-linked polysaccharide beads, or ii) coupling an entity attached to said cross-linked polysaccharide beads, optionally followed by coupling of a nucleoside, nucleotide or amino acid to said entity, wherein said entity is to form the basis for attachment of a first unit of a sequence to be synthesized.

15. The method of claim 14, comprising a) coupling, optionally via a linker, at least one nucleotide or nucleoside binding compound to said cross-linked polysaccharide beads via a first moiety of the compound, the nucleotide or nucleoside binding compound having a second moiety with nucleotide or nucleoside 3'- terminal binding function, and, optionally, binding a nucleoside or nucleotide to the nucleotide or nucleoside binding compound, or b) coupling optionally via a linker, at least one amino acid binding compound to said crosslinked polysaccharide beads via a first moiety of the compound, the amino acid binding compound having a second moiety with amino acid C-terminal binding function, and optionally binding an amino acid to the amino acid binding compound, or c) coupling at least one linker to said cross-linked polysaccharide beads, and, optionally, binding a nucleoside or nucleotide, or an amino acid to the linker, or d) coupling at least one nucleoside, nucleotide or amino acid to said cross-linked polysaccharide beads, and e) optionally, adding a protection group to any free end of a nucleoside, nucleotide or amino acid.

16. The method of claim 14 or 15, wherein the polysaccharide beads are agarose beads.

17. A method of synthesizing an oligonucleotide sequence, a peptide sequence, or an oligonucleotide-peptide conjugate sequence, the method comprising the following steps:1) providing (100) a solid-phase support material comprising cross-linked polysaccharide beads produced according to any one of claims 14-16, wherein nucleosides, nucleotides or amino acids are coupled to the cross-linked beads, and provided with a protection group at any free end,2) de-protecting (101) the protected nucleosides, nucleotides or amino acids,3) coupling (102) a nucleotide or an amino acid to a free end of a nucleoside, nucleotide or amino acid coupled to the solid-phase support material,4) capping (103) free ends of nucleotides or amino acids coupled to the solidphase support material, and5) repeating (104) steps 2-4 until a desired oligonucleotide or peptide sequence is assembled.

18. The method of claim 17, wherein coupling a nucleotide or an amino acid to a free end of a nucleoside, nucleotide or amino acid coupled to the solid-phase support material in step 3) is performed enzymatically.

19. The method of claim 17 or 18, further comprising a step 6) of cleaving (105) the formed oligonucleotide or peptide sequence from the solid-phase support material.

20. The method of any one of claims 17-19, wherein the solid-phase support material, is provided in a flow-through column.

21. A kit of parts for a solid-phase support material for oligonucleotide sequence synthesis, peptide sequence synthesis, or oligonucleotide-peptide conjugate sequence synthesis, comprising:- cross-linked polysaccharide beads of any one of claims 1-10, and- optionally, nucleosides, nucleotides or amino acids for coupling to the beads.

Images