Methods, systems, and kits for polynucleotide synthesis

Abstract

The present invention relates to novel methods for synthesizing polynucleotide molecules according to predefined nucleotide sequences. The present invention also relates to methods for assembling synthetic polynucleotides after synthesis, as well as systems and kits for performing the synthesis and / or assembly methods.

Description

Technical Field

[0001] The present invention relates to a new method for synthesizing polynucleotide molecules according to a predefined nucleotide sequence. The present invention also relates to methods for assembling synthetic polynucleotides after synthesis, and systems and kits for performing the synthesis and / or assembly methods. Background Art

[0002] There are two main methods for synthesizing and assembling polynucleotide molecules, especially DNA.

[0003] Phosphoramidite chemistry is a synthetic method for assembling monomers of chemically activated T, C, A, or G into oligonucleotides with a length of approximately 100 / 150 bases through a stepwise process. The chemical reaction steps are highly sensitive, and the conditions alternate between completely anhydrous (completely absent of water), aqueous oxidation, and acidic conditions (Roy and Caruthers, Molecules, 2013, 18, 14268 - 14284). If the reagents from the previous reaction step have not been completely removed, this will be disadvantageous to future synthesis steps. Therefore, this synthetic method is limited to producing polynucleotides with a length of about 100 nucleotides.

[0004] The polymerase synthesis method uses a polymerase to synthesize a complementary strand of a DNA template using T, C, A, and G triphosphates. The reaction conditions are aqueous and mild, and this method can be used to synthesize DNA polynucleotides with a length of thousands of bases. The main disadvantage of this method is that single-stranded and double-stranded DNA cannot be de novo synthesized by this method, which requires a DNA template from which to prepare copies. (Kosuri and Church, Nature Methods, 2014, 11, 499 - 507).

[0005] Therefore, previous methods cannot be used to de novo synthesize double-stranded DNA without the aid of a pre-existing template molecule for copying.

[0006] The inventors have developed new methods by which single-stranded and double-stranded polynucleotide molecules can be de novo synthesized in a stepwise manner without replicating a pre-existing template molecule. These methods also avoid the extreme conditions associated with phosphoramidite chemistry techniques and instead are carried out under mild aqueous conditions near neutral pH. Such methods also enable the de novo synthesis of single-stranded or double-stranded polynucleotide molecules with a potential 10-fold improvement compared to current synthesis methods for nucleotide lengths of >100 mers to complete genomes, providing a wide range of possible applications in synthetic biology. 8 Improvements provide a wide range of possible applications in synthetic biology. Summary of the Invention

[0007] The present invention provides an in vitro method for synthesizing a double-stranded polynucleotide having a predefined sequence, the method comprising performing synthesis cycles, wherein each cycle comprises cleaving the double-stranded polynucleotide and extending the cleaved double-stranded polynucleotide by ligating nucleotide pairs, wherein the end of the first strand of the cleaved double-stranded polynucleotide is extended by adding nucleotides of the predefined sequence, and the end of the second strand of the cleaved double-stranded polynucleotide hybridized to the first strand is extended by adding partner nucleotides, thereby forming nucleotide pairs with the ligating nucleotides of the first strand. Preferably, the method is used for synthesizing DNA.

[0008] In any method of the present invention described herein, the method can provide synthesis of single-stranded polynucleotide molecules, wherein after synthesizing a double-stranded polynucleotide molecule having a predefined sequence, one strand of the double-stranded polynucleotide molecule is removed or replicated and / or amplified to provide a single-stranded polynucleotide molecule.

[0009] In any method of the present invention described herein, the method can provide synthesis of double-stranded or single-stranded oligonucleotides. Accordingly, all references herein to the synthesis of double-stranded or single-stranded polynucleotides using any method of the present invention apply, mutatis mutandis, to the synthesis of double-stranded or single-stranded oligonucleotides.

[0010] In the method of the present invention described above, each cycle can comprise extending the first strand by adding nucleotides of the predefined sequence together with a reversibly blocking group attached thereto, and then extending the second strand, wherein the reversibly blocking group is removed before or after the second strand is extended. In any such method, in each cycle, the nucleotides can be incorporated into a cleavable scaffold polynucleotide.

[0011] The present invention provides the method as described above and herein, wherein each cycle comprises:

[0012] (1) providing a scaffold polynucleotide;

[0013] (2) cleaving the scaffold polynucleotide at a cleavage site;

[0014] (3) adding the nucleotides of the predefined sequence to the cleaved scaffold polynucleotide by the action of a nucleotide transferase or polymerase, the nucleotides comprising a reversible terminator group that blocks further extension by the enzyme;

[0015] (4) removing the reversible terminator group from the nucleotides of the predefined sequence; and

[0016] (5) ligating a ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising partner nucleotides of the nucleotides of the predefined sequence, wherein after ligation, the nucleotides of the predefined sequence are paired with the partner nucleotides.

[0017] In methods involving a scaffold polynucleotide, a scaffold polynucleotide can be provided that includes a synthetic strand and a support strand hybridized thereto, where the synthetic strand includes a primer strand portion and an auxiliary strand portion. In any such method, the auxiliary strand portion can be removed from the scaffold polynucleotide prior to any one, more, or all cleavage steps.

[0018] In any such method involving a scaffold polynucleotide, the synthetic strand can be the first strand and the support strand can be the second strand.

[0019] In any such method, the support strand can be extended by a linking polynucleotide that is linked to the support strand, where in each synthesis cycle, the linking polynucleotide contains nucleotides that form nucleotide pairs with predetermined nucleotides incorporated into the first strand in that cycle.

[0020] The linking polynucleotide can be single-stranded or double-stranded. Preferably, the linking polynucleotide is double-stranded.

[0021] In methods where the linking polynucleotide is double-stranded, the linking polynucleotide can preferably include a support strand and an auxiliary strand. The auxiliary strand can be removed from the scaffold polynucleotide prior to the cleavage step in the next synthesis cycle, and in these methods, the auxiliary strand is removed after the linking step.

[0022] The present invention provides a method as described above, where step (1) includes providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, where the synthetic strand includes a primer strand portion and the support strand includes universal nucleotides; where step (2) includes cleaving the scaffold polynucleotide at a cleavage site defined by a sequence including the universal nucleotides in the support strand, where cleavage includes cleaving the support strand and removing the universal nucleotides from the scaffold polynucleotide; and where in step (5), the linking polynucleotide includes a support strand that includes a partner nucleotide and universal nucleotides defining a cleavage site for the next cycle, and where the linking polynucleotide is linked to the support strand of the cleaved scaffold polynucleotide to form nucleotide pairs.

[0023] The present invention provides a method as described above, which includes:

[0024] (1) providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, where the synthetic strand includes a primer strand portion and an auxiliary strand portion separated by a single-strand break, and the support strand includes universal nucleotides;

[0025] (2) Cleave the scaffold polynucleotide at a cleavage site defined by a sequence including the universal nucleotide in the support strand, wherein cleavage includes cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double-stranded scaffold polynucleotide, the cleaved double-stranded scaffold polynucleotide including a support strand and a synthetic strand including a primer strand portion;

[0026] (3) Extend the end of the primer strand portion of the synthetic strand of the cleaved double-stranded scaffold polynucleotide together with the first nucleotide of the predefined sequence by the action of a nucleotide transferase or polymerase, the first nucleotide including a reversible terminator group that blocks further extension by the enzyme;

[0027] (4) Remove the terminator group from the first nucleotide;

[0028] (5) Ligate a double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide including a support strand and a helper strand hybridized thereto and further including complementary ligation ends, the ligation ends including:

[0029] (i) In the support strand, a universal nucleotide and a partner nucleotide of the first nucleotide, wherein the partner nucleotide of the first nucleotide overhangs in the helper strand; and

[0030] (ii) A terminal nucleotide lacking a phosphate group in the helper strand;

[0031] wherein after ligating the support strand, the first nucleotide pairs with the partner nucleotide;

[0032] (6) Cleave the scaffold polynucleotide at a cleavage site defined by a sequence including the universal nucleotide in the support strand, wherein cleavage includes cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double-stranded scaffold polynucleotide, the cleaved double-stranded scaffold polynucleotide including a support strand and a synthetic strand including a primer strand portion;

[0033] (7) Extend the end of the primer strand portion of the synthetic strand of the cleaved double-stranded scaffold polynucleotide together with the next nucleotide of the predefined nucleotide sequence by the action of a nucleotide transferase or polymerase, the next nucleotide including a reversible terminator group that blocks further extension by the enzyme;

[0034] (8) Remove the terminator group from the next nucleotide; and

[0035] (9) Ligate a double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide including a support strand and a helper strand hybridized thereto and further including complementary ligation ends, the ligation ends including:

[0036] (i) In the support strand, a universal nucleotide and a conjugate nucleotide of the next nucleotide, wherein the conjugate nucleotide of the next nucleotide overhangs in the auxiliary strand; and

[0037] (ii) A terminal nucleotide lacking a phosphate group in the auxiliary strand;

[0038] Wherein after connecting the support strand, the next nucleotide pairs with the conjugate nucleotide;

[0039] (10) Repeat steps 6 to 9 multiple times to provide a double-stranded polynucleotide having a predefined nucleotide sequence.

[0040] Any such method as described above can be carried out according to the method of synthesis method version 1 of the present invention, wherein:

[0041] a) Before and during the cleavage step of the first cycle (step 2), the universal nucleotide occupies position n in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, in which cycle, after adding it to the end of the primer strand portion, the position in the synthesis strand will be occupied by the first nucleotide of the predefined sequence, wherein the nucleotide at position n in the support strand is opposite to and pairs with the terminal nucleotide of the auxiliary strand;

[0042] b) In the cleavage step of the first cycle (step 2), the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where position n-1 is the next nucleotide position in the support strand relative to position n in the direction of the distal side of the auxiliary strand / proximal side of the primer strand portion;

[0043] c) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the conjugate nucleotide for the first nucleotide of the predefined sequence is the terminal nucleotide of the support strand and occupies position n, wherein the universal nucleotide occupies position n+1 in the support strand and pairs with the terminal nucleotide of the auxiliary strand, wherein in step 5, after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide, position n is the nucleotide position opposite to the first nucleotide of the predefined sequence.

[0044] d) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles:

[0045] i. The universal nucleotide occupies position n in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in said cycle, after being added to the end of the primer strand portion, the position in the synthesis strand will be occupied by the next nucleotide of the predefined sequence; and

[0046] ii. The support strand of the scaffold polynucleotide is cleaved between positions n and n - 1, where n - 1 is the next nucleotide position in the support strand relative to position n in the proximal direction of the distal / primer strand portion of the auxiliary strand; and

[0047] e) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in said cycle is the terminal nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n + 1 in the support strand and pairs with the terminal nucleotide of the auxiliary strand; where position n is the nucleotide position when ligating the ligation polynucleotide to the cleaved scaffold polynucleotide, and said position n will be opposite to the next nucleotide of the predefined sequence bound in said cycle (step 7).

[0048] Alternatively, any such method as described above may be performed according to the method of synthesis method version 2 specified in the present invention, wherein:

[0049] a) Before and during the cleavage step of the first cycle (step 2), the universal nucleotide occupies position n + 1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in said cycle, after being added to the end of the primer strand portion, the position in the synthesis strand will be occupied by the first nucleotide of the predefined sequence, where the nucleotide at position n in the support strand is opposite to and pairs with the terminal nucleotide of the auxiliary strand, and where position n + 1 is the next nucleotide position in the support strand relative to the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0050] b) In the cleavage step of the first cycle (step 2), the support strand of the scaffold polynucleotide is cleaved between positions n and n - 1, where n - 1 is the next nucleotide position in the support strand relative to position n in the proximal direction of the distal / primer strand portion of the auxiliary strand;

[0051] c) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence is the terminal nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; wherein in step 5, after ligating the ligation polynucleotide into the cleaved scaffold polynucleotide, position n is the nucleotide position opposite to the first nucleotide of the predefined sequence, and relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand, position n+2 is the second position in the support strand;

[0052] d) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles:

[0053] i. The universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in said cycle, after adding it to the end of the primer strand portion, the position in the synthesis strand will be occupied by the next nucleotide of the predefined sequence; and where relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand, n+1 is the next nucleotide position in the support strand; and

[0054] ii. The support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where relative to position n in the direction proximal to the distal / primer strand portion of the auxiliary strand, n-1 is the next nucleotide position in the support strand; and

[0055] e) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in said cycle is the terminal nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; where position n is the nucleotide position when ligating the ligation polynucleotide into the cleaved scaffold polynucleotide, and position n will be opposite to the next nucleotide of the predefined sequence bound in said cycle (step 7), and relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand, position n+2 is the second position in the support strand;

[0056] Alternatively, any such method as described above may be performed according to the method of synthesis method version 3 specified in the present invention, wherein:

[0057] a) Before and during the cleavage step of the first cycle (step 2), the universal nucleotide occupies position n in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in this cycle, after adding it to the end of the primer strand portion, the position in the synthesis strand will be occupied by the first nucleotide of the predefined sequence, where the nucleotide at position n in the support strand is opposite to and pairs with the terminal nucleotide of the auxiliary strand;

[0058] b) In the cleavage step of the first cycle (step 2), the support strand of the scaffold polynucleotide is cleaved between positions n - 1 and n - 2, where positions n - 1 and n - 2 are the next nucleotide position and the consecutive nucleotide position in the support strand, respectively, relative to position n in the direction of the distal side of the auxiliary strand / proximal side of the primer strand portion;

[0059] c) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the penultimate nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n + 1 in the support strand and pairs with the terminal nucleotide of the auxiliary strand; where in step 5, after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide, position n is the nucleotide position opposite to the first nucleotide of the predefined sequence, and where position n + 1 is the next nucleotide position in the support strand relative to position n in the direction of the proximal side of the auxiliary strand / distal side of the primer strand portion;

[0060] d) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles:

[0061] i. The universal nucleotide occupies position n in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in this cycle, after adding it to the end of the primer strand portion, the position in the synthesis strand will be occupied by the next nucleotide of the predefined sequence; and

[0062] ii. The support strand of the scaffold polynucleotide is cleaved between positions n - 1 and n - 2, where positions n - 1 and n - 2 are the next nucleotide position and the consecutive nucleotide position in the support strand, respectively, relative to position n in the direction of the distal side of the auxiliary strand / proximal side of the primer strand portion; and

[0063] e) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in the cycle is the penultimate nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+1 in the support strand and pairs with the terminal nucleotide of the auxiliary strand; where position n is the nucleotide position when the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide, and this position n will be opposite to the next nucleotide of the predefined sequence bound in the cycle (step 7), and where position n+1 is the next nucleotide position in the support strand relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0064] Alternatively, any such method as described above can be carried out according to the method of synthesis method version 4 specified in the present invention, wherein:

[0065] a) Before and during the cleavage step of the first cycle (step 2), the universal nucleotide occupies position n+2 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in this cycle, after it is added to the end of the primer strand portion, this position in the synthesis strand will be occupied by the first nucleotide of the predefined sequence, where the nucleotide at position n in the support strand is opposite to and pairs with the terminal nucleotide of the auxiliary strand, and where position n+2 is the second nucleotide position in the support strand relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0066] b) In the cleavage step of the first cycle (step 2), the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where position n-1 is the next nucleotide position in the support strand relative to position n in the direction proximal to the distal / primer strand portion of the auxiliary strand;

[0067] c) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the terminal nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n+3 in the support strand and pairs with the nucleotide that is two positions removed from the terminal nucleotide of the auxiliary strand in the distal direction of the primer strand portion; where in step 5, after the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide, position n is the nucleotide position opposite to the first nucleotide of the predefined sequence, and position n+3 is the third position in the support strand relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0068] d) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles:

[0069] i. The universal nucleotide occupies position n+2 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in said cycle, after it is added to the end of the primer strand portion, the position in the synthesis strand will be occupied by the next nucleotide of the predefined sequence; and where n+2 is the second nucleotide position in the support strand relative to position n in the direction proximal to the primer strand portion of the auxiliary strand; and

[0070] ii. The support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where n-1 is the next nucleotide position in the support strand relative to position n in the direction distal to the primer strand portion of the auxiliary strand; and

[0071] e) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in said cycle is the terminal nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+3 in the support strand and pairs with the nucleotide that is two positions removed from the terminal nucleotide of the auxiliary strand in the distal direction of the primer strand portion; where position n is the nucleotide position when the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide, and said position n will be opposite to the next nucleotide of the predefined sequence in said cycle (step 7), and relative to position n in the direction proximal to the primer strand portion of the auxiliary strand, position n+3 is the third position in the support strand;

[0072] Any such method can be carried out according to the method of the present invention, which is a variant of synthesis method version 4, wherein:

[0073] (i) In the cleavage step of the first cycle (step 2), the universal nucleotide is changed to occupy position n+3 in the support strand of the scaffold polynucleotide, where n+3 is the third nucleotide position in the support strand relative to position n in the direction proximal to the primer strand portion of the auxiliary strand; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1;

[0074] (ii) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide is changed to occupy position n+4 in the support strand and pairs with the nucleotide that is 3 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation ends; where n+4 is the position 4 in the support strand relative to position n in the direction proximal to the primer strand portion of the auxiliary strand;

[0075] (iii) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+3 of the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved at positions n and n-1; and

[0076] (iv) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+4 in the support strand and pairs with the nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end.

[0077] Any such method can be carried out according to the method of the present invention, which is another variant of synthetic method version 4, wherein:

[0078] (i) In the cleavage step of the first cycle (step 2), the universal nucleotide is changed to occupy position n+3+x in the support strand of the scaffold polynucleotide, where position n+3 is the third nucleotide position in the support strand relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1;

[0079] (ii) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide is changed to occupy position n+4+x in the support strand and pairs with the nucleotide that is 3+x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+4 is the position 4 in the support strand relative to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0080] (iii) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+3+x of the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved at positions n and n-1;

[0081] (iv) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+4+x in the support strand and pairs with the nucleotide that is 2+x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; and

[0082] (v) where x is an integer between 1 and 10 or greater, and where x is the same integer in steps (2), (5), (6) and (9).

[0083] Alternatively, any such method as described above may be performed according to the method of synthesis method version 5 specified in the present invention, wherein:

[0084] a) Before and during the cleavage step of the first cycle (step 2), the universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in said cycle, after it is added to the end of the primer strand portion, the position in the synthesis strand will be occupied by the first nucleotide of the predefined sequence, wherein the nucleotide at position n in the support strand is opposite to and pairs with the terminal nucleotide of the auxiliary strand, and wherein position n+1 is the next nucleotide position in the support strand relative to position n in the direction proximal to the primer strand portion of the auxiliary strand;

[0085] b) In the cleavage step of the first cycle (step 2), the support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, where positions n-1 and n-2 are the next nucleotide position and the consecutive nucleotide position in the support strand respectively relative to position n in the direction distal to the primer strand portion of the auxiliary strand;

[0086] c) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the penultimate nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; wherein after the ligation polynucleotide is ligated into the cleaved scaffold polynucleotide, position n is the nucleotide position opposite to the first nucleotide of the predefined sequence, and position n+2 is the second nucleotide position in the support strand relative to position n in the direction proximal to the primer strand portion of the auxiliary strand;

[0087] d) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles:

[0088] i. The universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite to the position in the synthesis strand, and in said cycle, after it is added to the end of the primer strand portion, the position in the synthesis strand will be occupied by the next nucleotide of the predefined sequence; and wherein position n+1 is the next nucleotide position in the support strand relative to position n in the direction proximal to the primer strand portion of the auxiliary strand; and

[0089] ii. The support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, where positions n-1 and n-2 are the next nucleotide position and the consecutive nucleotide position in the support strand, respectively, relative to position n in the direction proximal to the distal / primer strand portion of the auxiliary strand;

[0090] e) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in the cycle is the penultimate nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+2 in the support strand and pairs with the terminal nucleotide of the auxiliary strand; where position n is the nucleotide position when the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide, and this position n will be opposite to the next nucleotide of the predefined sequence in the cycle (step 7), and where position n+2 is the second nucleotide position in the support strand relative to the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0091] Any such method can be performed according to the method of the present invention, which is a variant of the synthetic method version 5, wherein:

[0092] (i) In the cleavage step of the first cycle (step 2), the universal nucleotide is changed to occupy position n+2 in the support strand of the scaffold polynucleotide, where position n+2 is the second nucleotide position in the support strand relative to the direction distal to the proximal / primer strand portion of the auxiliary strand; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1;

[0093] (ii) In the ligation step of the first cycle (step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide is changed to occupy position n+3 in the support strand and pairs with the nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+3 is the position 3 in the support strand relative to the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0094] (iii) In the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+2 of the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved at positions n and n-1; and

[0095] (iv) In the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+3 in the support strand and pairs with the nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end.

[0096] Any such method may be carried out according to the method of the present invention, which is another variant of the synthetic method version 5, wherein:

[0097] (i) In the cleavage step (step 2) of the first cycle, the universal nucleotide is changed to occupy the position n + 2 + x in the support strand of the scaffold polynucleotide, where n + 2 is the second nucleotide position in the support strand relative to the position n in the direction distal to the proximal / primer strand portion of the auxiliary strand; and the support strand of the scaffold polynucleotide is cleaved between positions n and n - 1;

[0098] (ii) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide is changed to occupy the position n + 3 + x in the support strand and pairs with the nucleotide that is 2 + x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n + 3 is the position 3 in the support strand relative to the position n in the direction distal to the proximal / primer strand portion of the auxiliary strand;

[0099] (iii) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles, the universal nucleotide occupies the position n + 2 + x of the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved at positions n and n - 1;

[0100] (iv) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies the position n + 3 + x in the support strand and pairs with the nucleotide that is 2 + x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; and

[0101] (v) where x is an integer between 1 and 10 or greater, and where x is the same integer in steps (2), (5), (6), and (9).

[0102] In any of the methods described above and herein, the nucleotide paired with the first / next nucleotide of the predefined sequence may be a nucleotide complementary to the first / next nucleotide, preferably a naturally complementary nucleotide.

[0103] In any of the methods described above and herein, in any one or more synthetic cycles, or in all synthetic cycles, before step (2) and / or (6), a scaffold polynucleotide may be provided that includes a synthetic strand hybridized thereto and a support strand, and wherein the synthetic strand is provided without the auxiliary strand. In any one or more synthetic cycles, or in all synthetic cycles, before step (2) and / or (6), the synthetic strand may be removed from the scaffold polynucleotide.

[0104] In any of the methods described above and herein, in any one or more synthesis cycles, or in all synthesis cycles, after the step of ligating the double-stranded linker polynucleotide to the cleaved scaffold polynucleotide and before the step of incorporating the next nucleotide of the predetermined nucleotide sequence into the synthetic strand of the scaffold polynucleotide, the auxiliary strand portion of the synthetic strand can be removed from the scaffold polynucleotide. In any such method, the auxiliary strand portion of the synthetic strand can be removed from the scaffold polynucleotide by: (i) heating the scaffold polynucleotide to a temperature of about 80 °C to about 95 °C and separating the auxiliary strand portion from the scaffold polynucleotide; (ii) treating the scaffold polynucleotide with a urea solution, such as 8 M urea, and separating the auxiliary strand portion from the scaffold polynucleotide; (iii) treating the scaffold polynucleotide with formamide or a formamide solution, such as 100% formamide, and separating the auxiliary strand portion from the scaffold polynucleotide; or (iv) contacting the scaffold polynucleotide with a single-stranded polynucleotide molecule comprising a nucleotide sequence region complementary to the sequence of the auxiliary strand portion, thereby competitively inhibiting the hybridization of the auxiliary strand portion to the scaffold polynucleotide.

[0105] In any such method described above and herein, where a universal nucleotide occupies position n and where the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, each cleavage step can comprise a two-step cleavage process, where each cleavage step can comprise a first step that comprises removing the universal nucleotide, thereby forming an abasic site; and a second step that comprises cleaving the support strand at the abasic site. In any such method, the first step can be carried out with a nucleotide excision enzyme. The nucleotide excision enzyme can be 3-methyladenine DNA glycosylase. The nucleotide excision enzyme can be human alkyladenine DNA glycosylase (hAAG) or uracil DNA glycosylase (UDG). In any such method, the second step can be carried out with a chemical that acts as a base. The base can be NaOH. In any such method, the second step can be carried out with an organic chemical having abasic site cleavage activity. The organic chemical can be N,N'-dimethylethylenediamine. In any such method, the second step can be carried out with an enzyme having abasic site lyase activity, such as AP endonuclease, endonuclease III (Nth) or endonuclease VIII.

[0106] In any such method described above and herein, where a universal nucleotide occupies position n and where the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, each cleavage step can comprise a one-step cleavage process that comprises removing the universal nucleotide with a lyase, where the enzyme is endonuclease III, endonuclease VIII, formamidopyrimidine DNA glycosylase (Fpg) or 8-oxoguanine DNA glycosylase (hOGG1).

[0107] In any such method described above and herein, where a universal nucleotide occupies position n+1 and where the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, or in any such method described above and herein, where a universal nucleotide occupies position n and where the support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, the cleavage step may comprise cleaving the support strand with an enzyme. The enzyme may cleave the support strand after the nucleotide adjacent to the universal nucleotide in the direction towards the primer strand portion, thereby creating an overhang in the synthesized strand that includes the first / next nucleotide. Such an enzyme may be endonuclease V.

[0108] In any method described above and herein, the two strands of the synthesized double-stranded polynucleotide may be DNA strands. The synthesized strand and the support strand may be DNA strands. In such a case, the incorporated nucleotides are preferably dNTPs, preferably dNTPs that include a reversible terminator group. In any such method, any one or more or all of the incorporated nucleotides that include a reversible terminator group may include 3'-O-allyl-dNTP or 3'-O-azidomethyl-dNTP.

[0109] In any method described above and herein, the first strand of the synthesized double-stranded polynucleotide may be a DNA strand and the second strand of the synthesized double-stranded polynucleotide may be an RNA strand. The synthesized strand may be an RNA strand and the support strand may be a DNA strand. In such a case, the incorporated nucleotides are preferably NTPs, preferably NTPs that include a reversible terminator group. In any such method, any one or more or all of the incorporated nucleotides that include a reversible terminator group may be 3'-O-allyl-NTP or 3'-O-azidomethyl-NTP.

[0110] In any method described above and herein that involves incorporating nucleotides into a synthesized strand that includes DNA, such as incorporating one or more dNTPs, the enzyme may be a polymerase, preferably a DNA polymerase, more preferably a modified DNA polymerase that has an enhanced ability to incorporate dNTPs that include a reversible terminator group compared to an unmodified polymerase. The polymerase may be a variant of a native DNA polymerase from the Thermococcus species 9°N, preferably species 9°N-7.

[0111] In any method described above and herein that involves incorporating nucleotides into a synthesized strand that includes RNA, such as incorporating one or more NTPs, the enzyme may be a polymerase, preferably an RNA polymerase, such as T3 or T7 RNA polymerase, more preferably a modified RNA polymerase that has an enhanced ability to incorporate NTPs that include a reversible terminator group compared to an unmodified polymerase.

[0112] In any of the methods described above and herein, the first strand of the synthesized double-stranded polynucleotide can be a DNA strand, and the second strand of the synthesized double-stranded polynucleotide can be an RNA strand. Alternatively, the first strand of the synthesized double-stranded polynucleotide can be an RNA strand, and the second strand of the synthesized double-stranded polynucleotide can be a DNA strand.

[0113] In any of the methods described above and herein, the enzyme has terminal transferase activity, optionally wherein the enzyme is terminal nucleotidyl transferase, terminal deoxynucleotidyl transferase, terminal deoxynucleotidyl transferase (TdT), polγ, polμ, or Φ29 DNA polymerase.

[0114] In any of the methods described above and herein, the step of removing the reversible terminator group from the first / next nucleotide can be carried out with tris(carboxyethyl)phosphine (TCEP).

[0115] In any of the methods described above and herein, the step of ligating the double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide is preferably carried out with a ligase. The ligase can be T3 DNA ligase or T4 DNA ligase.

[0116] In any of the methods described above and herein, in steps (1), (5), and / or (9), the auxiliary strand and the support strand portion hybridized therewith can be linked by a hairpin loop.

[0117] In any of the methods described above and herein, in step (1), the synthetic strand comprising the primer strand portion and the support strand portion hybridized therewith can be linked by a hairpin loop.

[0118] In any of the methods described above and herein, in steps (1), (5), and / or (9):

[0119] a) The auxiliary strand and the support strand portion hybridized therewith can be linked by a hairpin loop; and

[0120] b) The synthetic strand comprising the primer strand portion and the support strand portion hybridized therewith can be linked by a hairpin loop.

[0121] In any of the methods described above and herein, at least one or more or all of the ligation polynucleotides can be provided as a single molecule, the single molecule comprising a hairpin loop that links the support strand and the auxiliary strand at the end opposite the complementary ligation end. In any of the methods described above and herein, the ligation polynucleotide for each synthesis cycle can be provided as a single molecule, each single molecule comprising a hairpin loop that links the support strand and the auxiliary strand at the end opposite the complementary ligation end.

[0122] In any of the methods described above and herein, in step (1), a synthetic strand comprising a primer strand portion and a support strand portion hybridized thereto may be tethered to a common surface. The primer strand portion and the support strand portion hybridized thereto may each comprise a cleavable linker, wherein the linker may be cleaved to separate the double-stranded polynucleotide from the surface after synthesis.

[0123] In any of the methods described above and herein, in step (1), the primer strand portion of the synthetic strand and the support strand portion hybridized thereto may be linked by a hairpin loop, and wherein the hairpin loop is tethered to the surface.

[0124] In any of the methods described above and herein, the hairpin loop may be linked to the surface by a cleavable linker, wherein the linker may be cleaved to separate the double-stranded polynucleotide from the surface after synthesis. The cleavable linker may be a UV-cleavable linker.

[0125] In any of the methods described above and herein, the surface to which the polynucleotide is attached may be the surface of a microparticle or a flat surface.

[0126] In any of the methods described above and herein, the surface to which the polynucleotide is attached may comprise a gel. The surface comprises a polyacrylamide surface, such as about 2% polyacrylamide, preferably wherein the polyacrylamide surface is coupled to a solid support such as glass.

[0127] In any of the methods described above and herein, a synthetic strand comprising a primer strand portion and a support strand portion hybridized thereto may be tethered to a common surface by one or more covalent bonds. One or more covalent bonds may be formed between a functional group on the common surface and a functional group on a scaffold molecule, wherein the functional group on the scaffold molecule may be an amino group, a thiol group, a phosphorothioate group, or a thioamide group. The functional group on the common surface may be bromoacetyl, optionally wherein the bromoacetyl is provided on a polyacrylamide surface derivatized with N-(5-bromoacetylpentyl)acrylamide (BRAPA).

[0128] In any of the methods described above and herein, the step of removing the reversible terminator group from the nucleotides of a predefined sequence may be performed before the cleavage step or before the ligation step.

[0129] In any of the methods described above and herein, the reactions associated with any of the synthetic cycles described above and herein may be carried out in droplets within a microfluidic system. The microfluidic system may be an electrowetting system. The microfluidic system may be an electrowetting-on-dielectric system (EWOD).

[0130] In any of the methods described above and herein, after synthesis, the strands of the double-stranded polynucleotide may be separated to provide single-stranded polynucleotides having a predefined sequence.

[0131] In any of the methods described above and herein, after synthesis, the double-stranded polynucleotide or a region thereof is amplified, preferably by PCR amplification.

[0132] The present invention also provides a method for assembling a polynucleotide having a predefined sequence, the method comprising performing any of the synthesis methods described above and herein to synthesize a first polynucleotide having a predefined sequence and one or more additional polynucleotides having a predefined sequence and ligating the first polynucleotide and the one or more additional polynucleotides together. The first polynucleotide and the one or more additional polynucleotides may preferably comprise different predefined sequences. The first polynucleotide and the one or more additional polynucleotides may be double-stranded or may be single-stranded. The first polynucleotide and the one or more other polynucleotides may first be cleaved to generate compatible ends and then ligated together, for example, by ligation. The first polynucleotide and the one or more other polynucleotides may be cleaved at the cleavage site by a restriction enzyme to generate compatible ends.

[0133] Any of the in vitro methods described above and herein for synthesizing a double-stranded polynucleotide having a predefined sequence, and / or any of the in vitro methods described above and herein for assembling a polynucleotide having a predefined sequence can be carried out in droplets within a microfluidic system. In any such method, the assembly method may include an assembly step that includes providing a first droplet comprising a first synthetic polynucleotide having a predefined sequence and a second droplet comprising one or more other synthetic polynucleotides having a predefined sequence, wherein the droplets are brought into contact with each other and wherein the synthetic polynucleotides are ligated together to assemble a polynucleotide comprising the first nucleotide and the one or more other polynucleotides. In any such method, the synthesis step may be carried out by providing a plurality of droplets, each droplet comprising reaction reagents corresponding to the steps of the synthesis cycle, and delivering the droplets sequentially to a scaffold polynucleotide according to the steps of the synthesis cycle. In any such method, after delivering a droplet and before delivering the next droplet, a washing step may be carried out to remove excess reaction reagents. In any such method, the microfluidic system may be an electrowetting system. In any such method, the microfluidic system may be an electrowetting-on-dielectric system (EWOD). In any such method, the synthesis and assembly steps may be carried out within the same system.

[0134] In a related aspect, the present invention further provides the use of universal nucleotides in an in vitro method for synthesizing a double-stranded polynucleotide having a predefined sequence, wherein the universal nucleotides are used to generate polynucleotide cleavage sites during each synthesis cycle, and wherein in each synthesis cycle, the use comprises: providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion and an auxiliary strand portion optionally separated from the primer strand portion by a single-strand break, and wherein the universal nucleotides are disposed in the support strand to provide cleavage sites; cleaving the scaffold polynucleotide at the cleavage sites, thereby removing the universal nucleotides from the scaffold polynucleotide and forming cleavage ends in the scaffold polynucleotide; adding new nucleotides of a predefined sequence comprising reversible terminator groups to the ends of the synthetic strand at the cleavage ends of the scaffold polynucleotide by a polymerase or transferase; ligating a ligation polynucleotide having a support strand and an auxiliary strand hybridized thereto to the cleavage ends of the scaffold polynucleotide, the support strand comprising nucleotides complementary to the new nucleotides in the synthetic strand of the scaffold polynucleotide and further comprising new universal nucleotides to establish new polynucleotide cleavage sites for the next synthesis cycle, wherein after the cleavage or ligation step, the reversible terminator groups are removed from the new nucleotides; and optionally wherein after the ligation step and before the cleavage step of the next cycle, the auxiliary strand is removed. This use of universal nucleotides in a method for synthesizing a double-stranded polynucleotide having a predefined sequence can be implemented using any of the specific methods defined and described above and herein.

[0135] In a related aspect, the present invention further provides an in vitro method for extending a synthetic strand of a polynucleotide molecule with a predetermined nucleotide, the method comprising: providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion and an auxiliary strand portion optionally separated from the primer strand portion by a single-strand break, and wherein a universal nucleotide is disposed in the support strand and defines a polynucleotide cleavage site; cleaving the scaffold polynucleotide at the cleavage site, thereby removing the universal nucleotide from the scaffold polynucleotide and forming a cleavage end in the scaffold polynucleotide; adding a new nucleotide comprising a predefined sequence with a reversible terminator group to the end of the synthetic strand at the cleavage end of the scaffold polynucleotide by a polymerase or a transferase; wherein the cleavage end of the scaffold polynucleotide serves as a ligation receptor site for a ligation polynucleotide having a support strand and an auxiliary strand hybridized thereto, the support strand comprising a nucleotide paired with the predetermined nucleotide in the synthetic strand of the scaffold polynucleotide and further comprising a new universal nucleotide to establish a new polynucleotide cleavage site for the next synthesis cycle, wherein after the cleavage or ligation step, the reversible terminator group is removed from the new nucleotide; and optionally wherein after the ligation step of the next cycle and before the cleavage step, the auxiliary strand is removed. In any such method for extending a synthetic strand of a polynucleotide molecule with a predetermined nucleotide, the method can be implemented using any of the specific methods defined and described above and herein.

[0136] In a related aspect, the present invention further provides an in vitro method for synthesizing a double-stranded polynucleotide having a predefined sequence, the method comprising synthesis cycles, and wherein each synthesis cycle comprises: providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion and an auxiliary strand portion optionally separated from the primer strand portion by a single-strand break, and wherein a universal nucleotide is disposed in the support strand and defines a polynucleotide cleavage site; cleaving the scaffold polynucleotide at the cleavage site, thereby removing the universal nucleotide from the scaffold polynucleotide and forming a cleavage end in the scaffold polynucleotide; adding a new nucleotide comprising a predefined sequence with a reversible terminator group to the end of the synthetic strand at the cleavage end of the scaffold polynucleotide by a polymerase or a transferase; ligating a ligation polynucleotide having a support strand and an auxiliary strand hybridized thereto to the cleavage end of the scaffold polynucleotide, the support strand comprising a nucleotide paired with the new nucleotide in the synthetic strand of the scaffold polynucleotide and further comprising a new universal nucleotide to establish a new polynucleotide cleavage site for the next synthesis cycle; after the cleavage or ligation step, removing the reversible terminator group from the new nucleotide; and optionally after the ligation step of the next cycle and before the cleavage step, removing the auxiliary strand. In any such method for synthesizing a double-stranded polynucleotide having a predefined sequence, the method can be implemented using any of the specific methods defined and described above and herein.

[0137] In a related aspect, the present invention further provides an in vitro method for ligating a ligation polynucleotide comprising a universal nucleotide to a double-stranded polynucleotide during cycles of synthesizing a double-stranded polynucleotide having a predefined sequence, wherein during the synthesis cycles, the double-stranded polynucleotide is extended with a predefined nucleotide and its conjugate; the method comprising providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion and an auxiliary strand portion optionally separated from the primer strand portion by a single-strand break, and wherein the universal nucleotide is disposed in the support strand and defines a polynucleotide cleavage site; cleaving the scaffold polynucleotide at the cleavage site, thereby removing the universal nucleotide from the scaffold polynucleotide and forming a cleavage end in the scaffold polynucleotide; adding a new nucleotide of a predefined sequence comprising a reversible terminator group to the end of the synthetic strand at the cleavage end of the scaffold polynucleotide by a polymerase or transferase, the reversible terminator group ligating a ligation polynucleotide having a support strand and an auxiliary strand hybridized thereto to the cleavage end of the scaffold polynucleotide; the support strand comprises a nucleotide that pairs with the new nucleotide in the synthetic strand of the scaffold polynucleotide and further comprises a new universal nucleotide to establish a new polynucleotide cleavage site for the next synthesis cycle, thereby establishing a new scaffold polynucleotide for the next synthesis cycle; after the cleavage or ligation step, the reversible terminator group is removed from the new nucleotide; and optionally, after the ligation step of the next cycle and before the cleavage step, the auxiliary strand is removed. In any such method for ligating a ligation polynucleotide comprising a universal nucleotide to a double-stranded polynucleotide during cycles of synthesizing a double-stranded polynucleotide having a predefined sequence, the method can be implemented using any of the specific methods defined and described above and herein.

[0138] The present invention further provides a polynucleotide synthesis system for implementing any of the synthesis and / or assembly methods described above and herein, comprising (a) an array of reaction regions, wherein each reaction region comprises at least one scaffold polynucleotide; and (b) means for delivering reaction reagents to the reaction regions, and optionally, (c) means for cleaving the synthesized double-stranded polynucleotide from the scaffold polynucleotide. Such a system can further comprise means for providing the reaction reagents in the form of droplets and means for delivering the droplets to the scaffold polynucleotide according to the synthesis cycles.

[0139] The present invention further provides a kit for use with any of the systems described above and herein and for implementing any of the synthesis methods described above and herein, the kit comprising volumes of reaction reagents corresponding to the steps of the synthesis cycles.

[0140] The present invention also provides a method for preparing a polynucleotide microarray, wherein the microarray comprises a plurality of reaction regions, each region comprising one or more polynucleotides having a predefined sequence, the method comprising:

[0141] a) providing a surface comprising a plurality of reaction regions, each region comprising one or more double-stranded anchor or scaffold polynucleotides, and

[0142] b) performing synthesis cycles in each reaction region according to any of the methods described above and herein, thereby synthesizing one or more double-stranded polynucleotides having a predefined sequence in each region.

[0143] In such methods, after synthesis, the strands of the double-stranded polynucleotide can be separated to provide a microarray, wherein each region comprises one or more single-stranded polynucleotides having a predefined sequence. Description of the Drawings

[0144] The related figures provided herein and described below show some or all of the steps of a synthesis cycle using a method incorporating the methods of the present invention, as well as means for implementing aspects of the method, such as oligonucleotides, surfaces, surface attachment chemistry, linkers, etc. These figures, along with all of their descriptions and all related methods, reagents, and protocols are for illustrative presentation only and should not be construed as limiting.

[0145] Related figures, such as Figure 6 、 7 、8, 9, 10, 13a, 14a, 15a, etc. show some or all of the steps of a synthesis cycle, including incorporation of nucleotides (e.g., nucleotides comprising reversible terminators), cleavage (e.g., cleaving a scaffold polynucleotide into a first portion and a second portion, where the first portion comprises a universal nucleotide and the second portion comprises the incorporated nucleotide), ligation (e.g., ligating a polynucleotide construct comprising a single-stranded portion to the second portion of the cleaved scaffold polynucleotide comprising the incorporated nucleotide, where the single-stranded portion comprises a partner nucleotide complementary to the incorporated nucleotide), and removal of protecting groups (e.g., removing a reversible terminator group from the incorporated nucleotide). These methods are provided for illustrative support only and are not within the scope of the claimed invention. Figures 1 to 5 The method schemes shown are methods of the present invention. As described in Example 13, Figure 51 the data shown relate to methods of the present invention consistent with synthesis method versions 1, 2, and 4 as described herein Figure 1 、 2 and 4.

[0146] Figure 1 . Scheme of Exemplary Method Version 1 of the Present Invention

[0147] Scheme showing the first synthesis cycle of an exemplary method version 1 according to the present invention. The method includes cycles of providing a scaffold polynucleotide, cleavage, incorporation, deprotection, and ligation. The scheme shows the provision of a scaffold polynucleotide (101) including a universal nucleotide in the support strand, the universal nucleotide defining a polynucleotide cleavage site and a single-strand break (“nick”) in the synthetic strand. The scheme shows the cleavage of the support strand by creating a single-strand break in the support strand (102), then adding a thymine nucleotide to the end of the synthetic polynucleotide at the cleavage end (103) of the scaffold polynucleotide, and removing reversible blocking groups during the protection step (104). After ligating the ligation polynucleotide to the cleaved scaffold polynucleotide (105), the newly incorporated thymine nucleotide is paired with its complementary nucleotide adenine (104). After the ligation step, newly incorporated nucleotide pairs (106) are provided for the recombinant scaffold polynucleotide for the next synthesis cycle. The A-T pair is shown for illustrative purposes only and is not limiting; depending on the desired predefined sequence, the A-T pair can be any pair. The nucleotide pair at the H-I position can be any pair. Nucleotide X can be any nucleotide. The figure also shows reference markers corresponding to the second synthesis cycle.

[0148] Figure 2 . Solution of the exemplary method version 2 of the present invention

[0149] Scheme showing the first synthesis cycle of an exemplary method version 2 according to the present invention. The method includes cycles of providing a scaffold polynucleotide, cleavage, incorporation, deprotection, and ligation. The scheme shows the provision of a scaffold polynucleotide (101) including a universal nucleotide in the support strand, the universal nucleotide defining a polynucleotide cleavage site and a single-strand break (“nick”) in the synthetic strand. The scheme shows the cleavage of the support strand by creating a single-strand break in the support strand (102), then adding a thymine nucleotide to the end of the synthetic polynucleotide at the cleavage end (103) of the scaffold polynucleotide, and removing reversible blocking groups during the protection step (104). After ligating the ligation polynucleotide to the cleaved scaffold polynucleotide (105), the newly incorporated thymine nucleotide is paired with its complementary nucleotide adenine (104). After the ligation step, newly incorporated nucleotide pairs (106) are provided for the recombinant scaffold polynucleotide for the next synthesis cycle. The A-T pair is shown for illustrative purposes only and is not limiting; depending on the desired predefined sequence, the A-T pair can be any pair. The nucleotide pairs at the H-I and J-K positions can be any pair. Nucleotide X can be any nucleotide. The figure also shows reference markers corresponding to the second synthesis cycle.

[0150] Figure 3 . Scheme of Exemplary Method Version 3 of the Present Invention

[0151] Scheme of the first synthesis cycle of an exemplary method version 3 according to the present invention. The method includes cycles of providing a scaffold polynucleotide, cleavage, incorporation, deprotection, and ligation. The scheme shows providing a scaffold polynucleotide (101) including a universal nucleotide in a support strand, the universal nucleotide defining a polynucleotide cleavage site and a single-strand break ("nick") in the synthesized strand. The scheme shows cleaving the support strand by creating a single-strand break in the support strand (102), then adding a thymine nucleotide to the end of the synthesized polynucleotide at the cleavage end (103) of the scaffold polynucleotide, and removing a reversible blocking group during a protection step (104). After ligating a ligation polynucleotide to the cleaved scaffold polynucleotide (105), the newly incorporated thymine nucleotide is paired with its complementary nucleotide adenine (104). After the ligation step, newly incorporated nucleotide pairs (106) are provided for the recombinant scaffold polynucleotide for the next synthesis cycle. The A-T pair is shown for illustrative purposes only and is not limiting; depending on the desired predefined sequence, the A-T pair can be any pair. The nucleotide pairs at positions H-I and J-K can be any pair. Nucleotide X can be any nucleotide. The figure also shows reference markers corresponding to the second synthesis cycle.

[0152] Figure 4 . Scheme of the exemplary method version 4 of the present invention

[0153] Scheme of the first synthesis cycle of an exemplary method version 4 according to the present invention. The method includes cycles of providing a scaffold polynucleotide, cleavage, incorporation, deprotection, and ligation. The scheme shows providing a scaffold polynucleotide (101) including a universal nucleotide in a support strand, the universal nucleotide defining a polynucleotide cleavage site and a single-strand break ("nick") in the synthesized strand. The scheme shows cleaving the support strand by creating a single-strand break in the support strand (102), then adding a thymine nucleotide to the end of the synthesized polynucleotide at the cleavage end (103) of the scaffold polynucleotide, and removing a reversible blocking group during a protection step (104). After ligating a ligation polynucleotide to the cleaved scaffold polynucleotide (105), the newly incorporated thymine nucleotide is paired with its complementary nucleotide adenine (104). After the ligation step, newly incorporated nucleotide pairs (106) are provided for the recombinant scaffold polynucleotide for the next synthesis cycle. The A-T pair is shown for illustrative purposes only and is not limiting; depending on the desired predefined sequence, the A-T pair can be any pair. The nucleotide pairs at positions H-I, J-K, and L-M can be any pair. Nucleotide X can be any nucleotide. The figure also shows reference markers corresponding to the second synthesis cycle.

[0154] Figure 5 .Scheme of the exemplary method version 5 of the present invention

[0155] The described protocol shows a scaffold polynucleotide (101) provided in a support strand that includes universal nucleotides, which define a polynucleotide cleavage site and a single-strand break (“nick”) in the synthetic strand. The protocol shows cleaving the support strand by creating a single-strand break in the support strand (102), then adding thymine nucleotides to the end of the synthetic polynucleotide at the cleavage end (103) of the scaffold polynucleotide, and removing reversible blocking groups during a protection step (104). After ligating a ligation polynucleotide to the cleaved scaffold polynucleotide (105), the newly incorporated thymine nucleotides are paired with their partner nucleotide adenine (104). After the ligation step, a newly incorporated nucleotide pair (106) is provided for the recombinant scaffold polynucleotide for the next synthesis cycle. The A-T pair is shown for illustrative purposes only and is not limiting; depending on the desired predefined sequence, the A-T pair can be any pair. The nucleotide pairs at the H-I, J-K, and L-M positions can be any pair. Nucleotide X can be any nucleotide. The figure also shows reference numerals corresponding to the second synthesis cycle.

[0156] Figure 6 . Scheme of Exemplary Method Version 1

[0157] Scheme showing the first synthesis cycle of exemplary method version 1 according to the Examples section. This method is provided for illustrative support only and is not within the scope of the claimed invention. The method includes cycles of providing a scaffold polynucleotide, incorporation, cleavage, ligation, and removal of protecting groups. The scheme shows the incorporation of thymine nucleotides and their pairing opposite partner adenine nucleotides (104) in the first synthesis cycle (101, 102), and the provision of a scaffold polynucleotide (106) for the next synthesis cycle. This pair is shown for illustrative purposes only and is not limiting; depending on the desired predefined sequence, this pair can be any pair. Nucleotide Z can be any nucleotide. Nucleotide X can be any suitable nucleotide. The figure also shows reference numerals corresponding to the second synthesis cycle.

[0158] Figure 7 . Scheme of Exemplary Method Version 2

[0159] Shows the scheme of the first synthesis cycle of the exemplary method version 2 according to the example section. This method is provided only for illustrative support and is not within the scope of the claimed invention. The method includes cycles of providing a scaffold polynucleotide, incorporation, cleavage, ligation, and deprotection. The scheme shows the incorporation of thymine nucleotides and their pairing opposite the partner adenine nucleotides (204) in the first cycle (201, 202), and the provision of a scaffold polynucleotide including guanine paired with cytosine (206) in the next synthesis cycle. These pairs are for illustrative purposes only and are not restrictive; depending on the desired predefined sequence, they can be any pair. Nucleotide Z can be any nucleotide. Nucleotide X can be any suitable nucleotide. The figure also shows the reference markers corresponding to the second synthesis cycle.

[0160] Figure 8 . Solution of Exemplary Method Version 3

[0161] Shows the scheme of the first synthesis cycle of the exemplary method version 3 according to the example section. This method is provided only for illustrative support and is not within the scope of the claimed invention. The method includes cycles of providing a scaffold polynucleotide, incorporation, cleavage, ligation, and deprotection. The scheme shows the incorporation of thymine nucleotides and their pairing opposite the partner adenine nucleotides (304) in the first cycle (301, 302), and the provision of a scaffold polynucleotide (306) in the next synthesis cycle. Showing this pair is for illustrative purposes only and is not restrictive; depending on the desired predefined sequence, this pair can be any pair. The scheme also shows the cytosine-guanine pair as a component of the scaffold polynucleotide and not part of the predefined sequence. This pair is also shown for illustrative purposes only and is not restrictive; it can be any pair. Nucleotide Z can be any nucleotide. Nucleotide X can be any suitable nucleotide.

[0162] Figure 9 . Scheme of Exemplary Method Version 4

[0163] Shows the scheme of the first synthesis cycle of the exemplary method version 4 according to the example section. This method is provided only for illustrative support and is not within the scope of the claimed invention. The method includes cycles of providing a scaffold polynucleotide, incorporation, cleavage, ligation, and deprotection. The scheme shows the incorporation of thymine nucleotides and their pairing opposite the partner adenine nucleotides (404) in the first cycle (401, 402), and the provision of a scaffold polynucleotide including guanine paired with cytosine (406) in the next synthesis cycle. These pairs are for illustrative purposes only and are not restrictive; depending on the desired predefined sequence, they can be any pair. Nucleotides X, Y, and Z can be any nucleotide.

[0164] Figure 10 . Scheme of Exemplary Method Version 5

[0165] Scheme showing the first synthesis cycle of exemplary method version 5 according to the example section. This method is provided only for illustrative support and is not within the scope of the claimed invention. The method includes cycles of providing a scaffold polynucleotide, incorporation, cleavage, ligation, and deprotection. The scheme shows the incorporation of thymine nucleotides and their pairing opposite the partner adenine nucleotides (504) in the first cycle (501, 502), and the provision of a scaffold polynucleotide including guanine paired with cytosine (506) in the next synthesis cycle. The scheme also shows cytosine-guanine pairs (position n-2) as components of the scaffold polynucleotide and not part of a predefined sequence. These pairs are for illustrative purposes only and not restrictive; depending on the desired predefined sequence, they can be any pair. Nucleotides X, Y, and Z can be any nucleotide.

[0166] Figure 11 . Protocol for surface immobilization of display scaffolds polynucleotides

[0167] The scheme shows (a to h) possible example hairpin loop configurations of the scaffold polynucleotide and its immobilization to a surface.

[0168] The scheme (i and j) shows examples of surface chemistries for attaching polynucleotides to a surface. The examples show a double-stranded embodiment where the two strands are connected by a hairpin, but the same chemistry can be used to attach one or both strands of an unconnected double-stranded polynucleotide.

[0169] Figure 12. Without auxiliary strand - incorporation

[0170] a) Scheme showing the incorporation step with a dangling dashed box.

[0171] b) Evaluation of DNA polymerase for the incorporation of 3'-O-modified dTTP opposite inosine. The figure depicts a gel that shows at 50 °C in Mn 2+Results of 3'-O-modified dTTP incorporation by various DNA polymerases (BST, Deep Vent (Exo-), Therminator I, and Therminator IX) in the presence of ions. Lane 1: Incorporation of 3'-O-allyl-dTTP using Bst DNA polymerase. Lane 2: Incorporation of 3'-O-azidomethyl-dTTP using Bst DNA polymerase. Lane 3: Incorporation of 3'-O-allyl-dTTP using Deepvent (exo-) DNA polymerase. Lane 4: Incorporation of 3'-O-azidomethyl-dTTP using Deep vent (exo-) DNA polymerase. Lane 5: Incorporation of 3'-O-allyl-dTTP using Therminator I DNA polymerase. Lane 6: Incorporation of 3'-O-azidomethyl-dTTP using Therminator I DNA polymerase. Lane 7: Incorporation of 3'-O-allyl-dTTP using TherminatorIX DNA polymerase. Lane 8: Incorporation of 3'-O-azidomethyl-dTTP using Therminator IX DNA polymerase.

[0172] c) Evaluation of DNA polymerases for the relative incorporation of 3'-O-modified dTTP versus inosine. Results of incorporation using various DNA polymerases.

[0173] d) Evaluation of incorporation temperature using Therminator IX DNA polymerase. The figure depicts a gel that shows the results of incorporation of 3'-modified dTTP versus inosine using Therminator IX DNA polymerase at different temperatures in the presence of Mn 2+ ions. Lane 1: Incorporation of 3'-O-allyl dTTP at 37 °C. Lane 2: Incorporation of 3'-O-azidomethyl dTTP at 37 °C. Lane 3: Incorporation of 3'-O-allyl dTTP at 50 °C. Lane 4: Incorporation of 3'-O-azidomethyl dTTP at 50 °C. Lane 5: Incorporation of 3'-O-allyl dTTP at 65 °C. Lane 6: Incorporation of 3'-O-azidomethyl dTTP at 65 °C.

[0174] e) Evaluation of incorporation temperature using Therminator IX DNA polymerase. Results of incorporation performed at different temperatures.

[0175] f) Evaluation of the presence of Mn 2+ during incorporation using Therminator IX DNA polymerase. The figure depicts a gel that shows the results of incorporation of 3'-O-modified dTTP versus inosine at 65 °C. Lane S: Standard. Lane 1: Without Mn 2+Incorporation of 3'-O-allyl-dTTP by the ion. Channel 2: Without Mn 2+ Incorporation of 3'-O-azidomethyl-dTTP by the ion. Channel 3: In the presence of Mn 2 + Incorporation of 3'-O-allyl-dTTP in the presence of the ion. Channel 4: In the presence of Mn 2+ Incorporation of 3'-O-azidomethyl-dTTP in the presence of the ion.

[0176] g) Evaluation of the presence of Mn when using Therminator IX DNA polymerase for incorporation. 2+ Results of incorporation in the presence and absence of Mn 2+ ions.

[0177] h) Oligonucleotides used to study the incorporation step.

[0178] Figure 13. Without auxiliary strand - cleavage

[0179] a) Scheme showing cleavage of hybridized polynucleotide strands in the absence of a helper strand. The cleavage step is shown as a dangling dashed box.

[0180] b) Gel showing cleavage of oligonucleotides with hAAG and 0.2 M NaOH (strong base) at 37 °C and room temperature 24 °C, respectively. Channel 1. Starting oligonucleotide. Channel 2, which serves as a positive control containing two full-length strands, shows a higher yield of the cleavage-to-uncleaved DNA ratio of 90%:10%. Channel 3, which contains the cleavage reaction without a helper strand, shows a low percentage yield of the cleavage-to-uncleaved DNA ratio of 10%:90%.

[0181] c) Gel showing cleavage of oligonucleotides with hAAG and Endo VIII at 37 °C. Channel 2, which serves as a positive control containing two full-length strands, shows a higher yield of the cleavage-to-uncleaved DNA ratio of approximately 90%:10%. Channel 3, which includes the cleavage reaction without a helper strand, shows a low percentage yield of the cleavage-to-uncleaved DNA ratio of approximately 7%:93%.

[0182] d) Summary of cleavage of oligonucleotides with hAAG / Endo VIII and hAAG / chemical base.

[0183] e) Oligonucleotides used to study the cleavage step.

[0184] Figure 14. Without auxiliary strand - ligation

[0185] a) Scheme showing ligation of hybridized polynucleotide strands in the absence of a helper strand. The ligation step is shown as a dangling dashed box.

[0186] b) The gel shows the ligation of oligonucleotides with Quick T4 DNA ligase at room temperature (24 °C) without the auxiliary strand. Lane 1 contains a mixture of 36-mer TAMRA single-stranded oligonucleotides and 18-mer TAMRA single-stranded oligonucleotides. These oligonucleotides are used as reference bands.

[0187] c) Oligonucleotides used to study the ligation step.

[0188] Figure 15. Version 1 chemical method with auxiliary strand - incorporation

[0189] a) The scheme showing the incorporation step is presented with a dashed box hanging.

[0190] b) Oligonucleotides suitable for studying the incorporation step.

[0191] Figure 16. Version 1 chemical method with auxiliary strand - cleavage

[0192] a) The scheme showing the cleavage of hybridized polynucleotide strands in the absence of the auxiliary strand. The cleavage step is presented with a dashed box hanging.

[0193] b) The gel shows the cleavage of oligonucleotides with hAAG and 0.2 M NaOH (strong base) at 37 °C and room temperature 24 °C, respectively. Lane 1. Starting oligonucleotides. Lane 2, as a positive control containing two full-length strands, shows a higher yield with a cleavage to uncleaved DNA ratio of 90%:10%. Lane 3 containing the cleavage reaction without the auxiliary strand shows a low percentage yield with a cleavage to uncleaved DNA ratio of 10%:90%. Lane 4 containing the cleavage reaction with the auxiliary strand shows an equal percentage yield with a cleavage to uncleaved DNA ratio of 50%:50%.

[0194] c) Evaluation of the cleavage of abasic sites by endonuclease VIII. The gel shows the cleavage of oligonucleotides with hAAG and Endo VIII at 37 °C. Lane 2, as a positive control containing two full-length strands, shows a higher yield with a cleavage to uncleaved DNA ratio of approximately 90%:10%. Lane 3 containing the cleavage reaction without the auxiliary strand shows a low percentage yield with a cleavage to uncleaved DNA ratio of approximately 7%:93%. Lane 4 containing the cleavage reaction with the auxiliary strand shows a low percentage yield with a cleavage to uncleaved DNA ratio of 10%:90%.

[0195] d) Evaluation of the cleavage of abasic sites by N,N'-dimethylethylenediamine. The gel shows the cleavage of oligonucleotides with hAAG and 100 mM N,N'-dimethylethylenediamine at 37 °C. Lane 1. Starting oligonucleotides. Lane 2, as a positive control containing two full-length strands, shows 100% cleavage of the DNA. Lane 3 containing the cleavage reaction with the auxiliary strand shows a higher percentage yield with a cleavage to uncleaved DNA ratio of 90%:10%.

[0196] e) Summary of cleavage of oligonucleotides with hAAG / Endo VIII, hAAG / chemical base, and hAAG / substitute chemical base.

[0197] f) Oligonucleotides for studying the cleavage step.

[0198] Figure 17. Version 1 chemical method with auxiliary strand - ligation

[0199] a) Scheme showing the ligation of hybridized polynucleotide strands in the presence of an auxiliary strand. The ligation step is shown hanging in a dashed box.

[0200] b) Gel showing the ligation of oligonucleotides with Quick T4 DNA ligase at room temperature (24 °C) in the presence of an auxiliary strand. Lane 1 contains a mixture of 36-mer TAMRA single-stranded oligonucleotide and 18-mer TAMRA single-stranded oligonucleotide. These oligonucleotides are used as reference bands. In lane 2, an observable ligation product with an expected band size of 36-mer is present after 20 minutes.

[0201] c) Gel showing the ligation of oligonucleotides with Quick T4 DNA ligase at room temperature (24 °C) after incubation overnight in the presence of an auxiliary strand. Lane 1 contains a mixture of 36-mer TAMRA single-stranded oligonucleotide and 18-mer TAMRA single-stranded oligonucleotide. These oligonucleotides serve as reference bands. In lane 2, a fully ligated product with an expected band size of 36-mer is observable.

[0202] d) Oligonucleotides for studying the ligation step.

[0203] Figure 18. Version 2 chemical method with auxiliary strand - incorporation

[0204] a) Scheme showing the incorporation step is shown hanging in an orange dashed box

[0205] b) Gel showing the results of incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 27 °C. Lane 1: Starting material. Lane 2: Incorporated after 1 minute, conversion rate 5%. Lane 3: Incorporated after 2 minutes, conversion rate 10%. Lane 4: Incorporated after 5 minutes, conversion rate 20%. Lane 5: Incorporated after 10 minutes, conversion rate 30%. Lane 6: Incorporated after 20 minutes, conversion rate 35%.

[0206] c) The figure depicts a gel that shows the results of the incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 37°C. Lane 1: Starting material. Lane 2: Incorporated after 1 minute, conversion rate 30%. Lane 3: Incorporated after 2 minutes, conversion rate 60%. Lane 4: Incorporated after 5 minutes, conversion rate 90%. Lane 5: Incorporated after 10 minutes, conversion rate 90%. Lane 6: Incorporated after 20 minutes, conversion rate 90%.

[0207] d) The gel shows the results of the incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 47°C. Lane 1: Starting material. Lane 2: Incorporated after 1 minute, conversion rate 30%. Lane 3: Incorporated after 2 minutes, conversion rate 65%. Lane 4: Incorporated after 5 minutes, conversion rate 90%. Lane 5: Incorporated after 10 minutes, conversion rate 90%. Lane 6: Incorporated after 20 minutes, conversion rate 90%.

[0208] e) The gel shows the results of the incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 27°C. Lane 1: Starting material. Lane 2: Incorporated after 1 minute, conversion rate 70%. Lane 3: Incorporated after 2 minutes, conversion rate 85%. Lane 4: Incorporated after 5 minutes, conversion rate 92%. Lane 5: Incorporated after 10 minutes, conversion rate 96%. Lane 6: Incorporated after 20 minutes, conversion rate 96%.

[0209] f) The gel shows the results of the incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 37°C. Lane 1: Starting material. Lane 2: Incorporated after 1 minute, conversion rate 85%. Lane 3: Incorporated after 2 minutes, conversion rate 95%. Lane 4: Incorporated after 5 minutes, conversion rate 96%. Lane 5: Incorporated after 10 minutes, conversion rate 96%. Lane 6: Incorporated after 20 minutes, conversion rate 96%.

[0210] g) The gel shows the results of the incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 47°C. Lane 1: Starting material. Lane 2: Incorporated after 1 minute, conversion rate 85%. Lane 3: Incorporated after 2 minutes, conversion rate 90%. Lane 4: Incorporated after 5 minutes, conversion rate 96%. Lane 5: Incorporated after 10 minutes, conversion rate 96%. Lane 6: Incorporated after 20 minutes, conversion rate 96%.

[0211] h) Summary of the incorporation of 3'-O-azidomethyl-dTTP in the presence of various temperatures and Mn 2+ ions.

[0212] i) The gel shows at 37°C in Mn 2+Results of incorporation of Therminator IX DNA polymerase with complementary bases opposite 3'-O-modified dNTPs in the presence of. Lane 1: Starting material. Lane 2: Incorporation of 3'-O-azidomethyl-dTTP for 5 minutes. Lane 3: Incorporation of 3'-O-azidomethyl-dATP for 5 minutes. Lane 4: Incorporation of 3'-O-azidomethyl-dCTP for 5 minutes. Lane 5: Incorporation of 3'-O-azidomethyl-dGTP for 5 minutes.

[0213] j) Oligonucleotides for studying the incorporation step.

[0214] Figure 19. Version 2 chemical method with auxiliary strand - cleavage

[0215] a) Scheme showing cleavage of hybridized polynucleotide strands in the presence of a helper strand. The cleavage step is shown hanging in an orange dashed box.

[0216] b) Gel showing cleavage of oligonucleotides with Endo V at 37 °C. Lane 1. Starting oligonucleotide. Lane 2, as a positive control containing two full-length strands, shows a yield of cleaved to uncleaved DNA ratio of 80%:20%. Lane 3, containing the cleavage reaction without the helper strand, shows a much higher yield of cleaved DNA > 99%. Lane 4, containing the cleavage reaction with the helper strand, also shows a DNA cleavage yield of > 99%.

[0217] c) Summary of the cleavage study of endonuclease V.

[0218] d) Oligonucleotides for studying the cleavage step.

[0219] Figure 20. Version 2 chemical method with auxiliary strand - ligation

[0220] a) Scheme showing ligation of hybridized polynucleotide strands in the absence of a helper strand. The ligation step is shown hanging in an orange dashed box.

[0221] b) Oligonucleotides for studying the ligation step.

[0222] Figure 21. Version 2 chemical method with auxiliary strand - deprotection

[0223] a) Scheme showing the protecting group removal step is shown hanging in an orange dashed box.

[0224] b) The figure depicts a gel that shows the results of removing the protecting group 3'-O-azidomethyl by 50 mM TCEP after incorporation of 3'-O-azidomethyl-dTTP. Lane 1: Starting primer Lane 2: In Mn 2+3'-O-azidomethyl-dTTP is incorporated in the presence of. Channel 3: Extend the product in Channel 2 by adding all natural dNTPs. Channel 4: Deprotect the product (0.5 μM) in Channel 2 with 50 mM TCEP. Channel 5: Extend the product in Channel 4 by adding all natural dNTPs.

[0225] c) The figure depicts a gel that shows the result of deprotecting the 3'-O-azidomethyl group with 300 mM TCEP after incorporating 3'-O-azidomethyl-dTTP. Channel 1: Starting primer Channel 2: Incorporate 3-O-azidomethyl-dTTP in the presence of Mn 2+ 3-O-azidomethyl-dTTP is incorporated in the presence of. Channel 3: Extend the product in Channel 2 by adding all natural dNTPs. Channel 4: Deprotect the product (0.5 μM) in Channel 2 with 300 mM TCEP. Channel 5: Extend the product in Channel 4 by adding all natural dNTPs.

[0226] d) The figure depicts a gel that shows the result of deprotecting the 3'-O-azidomethyl group with 50 mM TCEP after incorporating 3'-O-azidomethyl-dCTP. Channel 1: Starting primer Channel 2: Incorporate 3-O-azidomethyl-dCTP in the presence of Mn 2+ 3-O-azidomethyl-dCTP is incorporated in the presence of. Channel 3: Extend the product in Channel 2 by adding all natural dNTPs. Channel 4: Deprotect the product (0.5 μM) in Channel 2 with 300 mM TCEP. Channel 5: Extend the product in Channel 4 by adding all natural dNTPs.

[0227] e) The figure depicts a gel that shows the result of deprotecting the 3'-O-azidomethyl group with 300 mM TCEP after incorporating 3'-O-azidomethyl-dCTP. Channel 1: Starting primer

[0228] Channel 2: Incorporate 3-O-azidomethyl-dCTP in the presence of Mn 2+ 3-O-azidomethyl-dCTP is incorporated in the presence of. Channel 3: Extend the product in Channel 1 by adding all natural dNTPs. Channel 4: Deprotect the product (0.5 μM) in Channel 1 with 300 mM TCEP. Channel 5: Extend the product in Channel 3 by adding all natural dNTPs.

[0229] f) The figure depicts a gel that shows the result of deprotecting the 3'-O-azidomethyl group with 300 mM TCEP after incorporating 3'-O-azidomethyl-dATP.

[0230] Channel 1: Starting primer

[0231] Channel 2: Incorporate 3-O-azidomethyl-dATP in the presence of Mn 2+3'-O-azidomethyl-dATP was incorporated in the lower part. Channel 3: Extend the product in Channel 2 by adding all natural dNTPs. Channel 4: Deprotect the product (0.5 μM) in Channel 2 with 300 mM TCEP. Channel 5: Extend the product in Channel 4 by adding all natural dNTPs.

[0232] g) The figure depicts a gel showing the result of deprotecting the protecting group 3'-O-azidomethyl by 300 mM TCEP after incorporation of 3'-O-azidomethyl-dGTP. Channel 1: Starting primer. Channel 2: Incorporation in the presence of Mn 2+ 3'-O-azidomethyl-dGTP was incorporated in the lower part. Channel 3: Extend the product in Channel 2 by adding all natural dNTPs. Channel 4: Deprotect the product (0.5 μM) in Channel 2 with 300 mM TCEP. Channel 5: Extend the product in Channel 4 by adding all natural dNTPs.

[0233] h) Efficiency of deprotection of TCEP on 0.2 μM DNA.

[0234] i) Oligonucleotides for studying the cleavage step.

[0235] Figure 22. Version 2 chemical method with double - hairpin model - incorporation

[0236] a) A scheme showing the incorporation step with a dangling dashed box.

[0237] b) Evaluate DNA polymerase for the incorporation of 3'-O-modified dTTP relative to its natural counterpart. The figure depicts a gel showing the result of incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 37 °C. Channel 1: Starting material. Channel 2: Incorporation of natural dNTP mixture. Channel 3: Incorporation of 3'-O-azidomethyl-dTTP by Therminator IX DNA polymerase. Channel 4: Extend the product in Channel 3 by adding all natural dNTPs.

[0238] c) Evaluate DNA polymerase for the incorporation of 3'-O-modified dTTP relative to its natural counterpart. Oligonucleotides suitable for studying the incorporation step.

[0239] Figure 23. Version 2 chemical method with double - hairpin model - cleavage

[0240] a) A scheme showing the cleavage of a hairpin oligonucleotide. The cleavage step is shown with a dangling dashed box.

[0241] b) Gel showing cleavage of the hairpin oligonucleotide with Endo V at 37°C. Lane 1. Starting hairpin oligonucleotide. Lane 2 showing the hairpin oligonucleotide after cleavage at 5 minutes shows a high yield of digested DNA, at a ratio of approximately 98%. Lane 3 showing the hairpin oligonucleotide after cleavage at 10 minutes shows a high yield of digested DNA, at a ratio of approximately 99%. Lane 4 showing the hairpin oligonucleotide after cleavage at 30 minutes shows a high yield of digested DNA, at a ratio of approximately 99%, and lane 5 showing the hairpin oligonucleotide after cleavage at 1 hour shows a high yield of digested DNA, at a ratio of approximately 99%.

[0242] c) Oligonucleotides for studying the cleavage step.

[0243] Figure 24. Version 2 chemical method with double - hairpin model - ligation

[0244] a) Scheme showing ligation of the hybridized hairpin. The overhangs are shown in the dashed box for the ligation step.

[0245] b) Gel showing ligation of the hairpin oligonucleotide with Blunt / TADNA ligase at room temperature (24°C) in the presence of the auxiliary strand. Lane 1 contains the starting hairpin oligonucleotide. Lane 2 showing the hairpin oligonucleotide ligated after 1 minute shows a high yield of ligated DNA product, at a ratio of approximately 85%. Lane 3 showing the hairpin oligonucleotide ligated after 2 minutes shows a high yield of digested DNA, at a ratio of approximately 85%. Lane 4 showing the hairpin oligonucleotide ligated after 3 minutes shows a high yield of ligated DNA product, at a ratio of approximately 85%. Lane 5 showing the hairpin oligonucleotide ligated after 4 minutes shows a high yield of ligated DNA product, at a ratio of approximately >85%.

[0246] c) Hairpin oligonucleotides for studying the ligation step.

[0247] Figure 25. Complete cycle of version 2 chemical method - double - hairpin model

[0248] a) Scheme showing the complete cycle involving steps of enzyme incorporation, cleavage, ligation, and removal of protecting groups.

[0249] b) Evaluation of DNA polymerase for incorporation of 3'-O-modified dTTP relative to its native counterpart. The figure depicts a gel showing the results of incorporation of 3'-O-modified dTTP by Therminator IX DNA polymerase at 37°C. Lane 1: Starting material. Lane 2: Incorporation of 3'-O-azidomethyl-dTTP by Therminator IX DNA polymerase. Lane 3: Extension of the product in lane 2 by addition of all native dNTPs. Lane 4: Cleavage of the product in lane 2 by endonuclease V. Lane 5: Ligation of the product in lane 4 by the blunt TA ligase kit.

[0250] c) Oligonucleotides suitable for studying the incorporation step.

[0251] Figure 26. Complete cycle of version 2 chemical method - single - hairpin model using auxiliary strand

[0252] a) Scheme showing a complete cycle involving steps of enzymatic incorporation, cleavage, ligation, and deprotection.

[0253] b) Oligonucleotides suitable for studying the incorporation step.

[0254] Figure 27. Complete cycle of version 3 chemical method - double - hairpin model

[0255] a) Scheme showing a complete cycle involving steps of enzymatic incorporation, cleavage, ligation, and deprotection.

[0256] b) Oligonucleotides suitable for studying the incorporation step.

[0257] Figure 28. Complete double - cycle of version 2 chemical method - double - hairpin model

[0258] a) Scheme showing the first complete cycle involving steps of enzymatic incorporation, deprotection, cleavage, and ligation.

[0259] b) Scheme showing the second complete cycle after the first complete cycle, involving steps of enzymatic incorporation, deprotection, cleavage, and ligation.

[0260] c) The figure depicts a gel showing a complete double - cycle experiment, including: incorporation, deprotection, cleavage, and ligation steps.

[0261] Channel 1. Starting material.

[0262] Channel 2. Extending the starting material with natural dNTPs.

[0263] Channel 3. Incorporation of 3'-O - azidomethyl - dTTP by Therminator IX DNA polymerase.

[0264] Channel 4. Extension of the product in Channel 3 by adding all natural dNTPs.

[0265] Channel 5. Deprotection of the product in Channel 3 with TCEP.

[0266] Channel 6. Extension of the product in Channel 5 by adding all natural dNTPs.

[0267] Channel 7. Cleavage of the product in Channel 5 with endonuclease V.

[0268] Channel 8. Ligation of the product in Channel 7 with blunt - end TA ligase kit.

[0269] Channel 9. Cleavage of the product in Channel 8 with λ exonuclease.

[0270] Channel 10. Starting material for the second cycle - same as the material in Channel 9.

[0271] Channel 11. Incorporation of 3'-O-azidomethyl-dTTP by Therminator IX DNA polymerase.

[0272] Channel 12. Extension of the product in Channel 11 by addition of all natural dNTPs.

[0273] Channel 13. Deprotection of the product in Channel 11 by TCEP.

[0274] Channel 14. Extension of the product in Channel 13 by addition of all natural dNTPs.

[0275] Channel 15. Cleavage of the product in Channel 13 by endonuclease V.

[0276] Channel 16. Ligation of the product in Channel 15 by blunt-end TA ligation kit.

[0277] d) Oligonucleotides for research.

[0278] Figure 29

[0279] Example showing the release mechanism of the scaffold polynucleotide of a polynucleotide of a predefined sequence synthesized according to the method described herein.

[0280] Figure 30

[0281] Schematic diagram of an exemplary method for synthesizing RNA according to the present invention. The exemplary method shows synthesis in the absence of an auxiliary strand.

[0282] Figure 31

[0283] Schematic diagram of an exemplary method for synthesizing RNA according to the present invention. The exemplary method shows synthesis in the presence of an auxiliary strand.

[0284] Figure 32

[0285] Schematic diagram of an exemplary method for synthesizing RNA according to the present invention. The exemplary method shows synthesis in the presence of an auxiliary strand.

[0286] Figure 33

[0287] Schematic diagram of the first complete cycle of an exemplary method for synthesizing DNA according to version 2 of the synthesis method with a single hairpin model, involving the step of denaturing the auxiliary strand before the incorporation step.

[0288] Figure 34

[0289] Schematic of the second complete cycle of an exemplary method for synthesizing DNA according to synthesis method version 2 with a single hairpin model, involving the step of denaturing the auxiliary strand prior to the incorporation step.

[0290] Figure 35

[0291] Schematic of the third complete cycle of an exemplary method for synthesizing DNA according to synthesis method version 2 with a single hairpin model, involving the step of denaturing the auxiliary strand prior to the incorporation step.

[0292] Figure 36

[0293] Oligonucleotides used in the experiments detailed in Example 9.

[0294] Figure 37

[0295] Gel showing the reaction products corresponding to the complete three-cycle experiment detailed in Example 9.

[0296] The figure depicts a gel that shows the results of a complete three-cycle experiment, including: incorporation, deblocking, cleavage, and ligation steps.

[0297] Lane 1: Starting material.

[0298] Lane 2. Extension of the starting material with native dNTPs

[0299] Lane 3: Incorporation of 3'-O-azidomethyl-dTTP by terminator X DNA polymerase.

[0300] Lane 4: Extension of the product in lane 3 by addition of all native dNTPs

[0301] Lane 5: Deblocking of the product in lane 3 by TCEP

[0302] Lane 6: Extension of the product in lane 5 by addition of all native dNTPs

[0303] Lane 7: Cleavage of the product in lane 5 by endonuclease V.

[0304] Lane 8: Ligation of the product in lane 7 by T3 DNA ligase

[0305] Lane 9: Starting material for the second cycle - same as the material in lane 9.

[0306] Lane 10: Extension of the product in lane 9 by addition of all native dNTPs.

[0307] Channel 11: Incorporation of 3'-O-azidomethyl-dTTP by Terminator X DNA polymerase.

[0308] Channel 12: Extension of the product in Channel 11 by addition of all natural dNTPs.

[0309] Channel 13: Deprotection of the product in Channel 11 by TCEP

[0310] Channel 14: Extension of the product in Channel 13 by addition of all natural dNTPs.

[0311] Channel 15: Cleavage of the product in Channel 13 by Endonuclease V

[0312] Channel 16: Ligation of the product in Channel 15 by T3 DNA ligase

[0313] Channel 17: Starting material for the third cycle - same as the material in Channel 16.

[0314] Channel 18: Extension of the product in Channel 17 by addition of all natural dNTPs.

[0315] Channel 19: Incorporation of 3'-O-azidomethyl-dTTP by Terminator X DNA polymerase.

[0316] Channel 20: Extension of the product in Channel 19 by addition of all natural dNTPs.

[0317] Channel 21: Deprotection of the product in Channel 19 by TCEP

[0318] Channel 22: Extension of the product in Channel 21 by addition of all natural dNTPs.

[0319] Channel 23: Cleavage of the product in Channel 21 by Endonuclease V

[0320] Channel 24: Ligation of the product in Channel 23 by T3 DNA ligase

[0321] Figure 38

[0322] Fluorescence signals incorporated with different amounts of BRAPA from the surface of the polyacrylamide gel, which were exposed to FITC-PEG-SH and FITC-PEG-COOH.

[0323] Figure 39

[0324] Measured fluorescence signals from the fluorescein channel on the surface of the polyacrylamide gel, which incorporated different amounts of BRAPA, which were exposed to FITC-PEG-SH and FITC-PEG-COOH.

[0325] Figure 40

[0326] (a) Sequences of hairpin DNAs with no linker attached to different samples.

[0327] (b) Sequences of hairpin DNAs with linker attached to different samples.

[0328] Figure 41

[0329] Fluorescence signals from hairpin DNA oligomers with and without linker immobilized on bromoacetyl-functionalized polyacrylamide surfaces.

[0330] Figure 42

[0331] Measured fluorescence from hairpin DNA oligomers with and without linker immobilized on bromoacetyl-functionalized polyacrylamide surfaces.

[0332] Figure 43

[0333] Fluorescence signals from hairpin DNA oligomers with and without linker immobilized on bromoacetyl-functionalized polyacrylamide surfaces after incorporation of triphosphates.

[0334] Figure 44

[0335] Measured fluorescence from hairpin DNA oligomers with and without linker immobilized on bromoacetyl-functionalized polyacrylamide surfaces after incorporation of triphosphates.

[0336] Figure 45

[0337] (a) Experimental overview and results of each reaction step as detailed in Example 12.

[0338] (b) Oligonucleotides used in the experiments detailed in Example 12.

[0339] Figure 46

[0340] Fluorescence signals from hairpin DNA oligomers before and after cleavage reaction (Example 12).

[0341] Figure 47

[0342] Fluorescence signals measured from hairpin DNA oligomers before and after cleavage reaction (Example 12).

[0343] Figure 48

[0344] Sequence showing the inosine-containing strand and the complementary "helper" strand for the ligation reaction (Example 12).

[0345] Figure 49

[0346] Results related to the fluorescence signal from the hairpin DNA oligomer corresponding to the ligation reaction monitoring (Example 12).

[0347] Figure 50

[0348] Results related to the measured fluorescence from the hairpin DNA oligomer corresponding to the ligation reaction monitoring (Example 12).

[0349] Figure 51

[0350] Regarding the method according to the present invention, such as versions 1, 2, and 4 of the synthesis method of the present invention ( Figure 1 , Figure 2 and Figure 4 and Example 13), results of incorporating 3'-O-modified-dNTPs by Terminator X DNA polymerase using the incorporation step.

[0351] Figure 51 a provides the nucleic acid sequences of the primer strand (the primer strand portion of the synthetic strand; SEQ ID NO: 68) and the template strand (the support strand; SEQ ID NO: 69).

[0352] Figure 51 b depicts a gel showing the results of incorporating 3'-O-modified-dNTPs by Terminator X DNA polymerase in the presence of Mn2+ ions at 37°C.

[0353] Lane 1: Starting oligonucleotide.

[0354] Lane 2: Incorporation of 3'-O-azidomethyl-dTTP (efficiency > 99%)

[0355] Lane 3: Incorporation of 3'-O-azidomethyl-dATP (efficiency > 99%).

[0356] Lane 4: Incorporation of 3'-O-azidomethyl-dCTP (efficiency > 90%).

[0357] Lane 5: Incorporation of 3'-O-azidomethyl-dGTP (efficiency > 99%).

[0358] After addition, the newly added 3'-O-modified-dNTP occupies position n in the primer strand portion. Thus, for example, according to Figure 1 , 2For step 3 of versions 1, 2, and 4 of the method of the present invention as shown in FIGS. 1 and 4, the next nucleotide position in the primer strand portion is designated n-1.

[0359] Explanation of numbers

[0360] Figure 11 、 12a 、13a, 14a, 15a, 16a, 17a, 18a, 19a, 20a, 21a, 22a, 23a, 24a, 25a, 26a, 27a, 28a, 28b, 29, 30, 31, 32, 33, 34, and 35 will be interpreted consistently with the structures depicted in Figure 6 、 7 、8, 9, and 10. Thus, in these figures, each left strand of the double-stranded scaffold polynucleotide molecule refers to the support strand (corresponding to the strand “a” in Figures 6 to 10 ); each right strand of the double-stranded scaffold polynucleotide molecule refers to the synthetic strand (corresponding to the strand “b” in Figures 6 to 10 ); all scaffold polynucleotide molecules contain a lower synthetic strand that corresponds to the strand including the primer strand portion (corresponding to the solid and dashed lines of the strand “b” in Figures 6 to 10 ); before incorporation of the new nucleotide, certain scaffold polynucleotide molecules are shown (e.g., Figure 15a and 23a ), where the synthetic strand corresponds to the strand including the auxiliary strand portion (corresponding to the dashed line of the strand “b” in Figures 6 to 10 ); certain scaffold polynucleotide molecules (e.g., Figure 12a 、 13a and 14a) are shown without an auxiliary strand portion (corresponding to the absence of the dashed line of the strand “b” in Figures 6 to 10 ); and after the ligation step, certain scaffold polynucleotide molecules are shown (e.g., Figure 33 、 34 and 35), where the upper synthetic strand corresponds to the strand including the auxiliary strand portion (corresponding to the dashed line of the strand “b” in Figures 6 to 10 ), and where the auxiliary strand portion is removed before incorporation of the new nucleotide in the next synthesis cycle.

[0361] In addition, in these figures, where relevant, each new nucleotide is shown incorporated with a reversible terminator group, labeled rtNTP and depicted as a small circular structure (corresponding to the small triangular structure in Figures 6 to 10 ), and the terminal phosphate group is labeled “p” and depicted as a small oval structure.

[0362] Figure 11c, 11d, 11g, 11h, 22a, 23a, 24a, 25a, 27a, 28a, 28b, and 29 show scaffold polynucleotide molecules, wherein strands including an auxiliary strand portion and a support strand are joined by a hairpin loop. Figure 11 b, 22a, 23a, 24a, 25a, 26a, 27a, 28a, 28b, 29, 33, 34, and 35 show scaffold polynucleotide molecules, wherein strands including a primer strand portion and a support strand are joined by a hairpin loop.

[0363] For example Figure 27a and 28a The figure of shows a scaffold polynucleotide molecule, wherein strands including an auxiliary strand portion (upper right strand) and a support strand (upper left strand) are joined by a hairpin loop, and in the same molecule, strands including a primer strand portion (lower right strand) and a support strand (lower left strand) are joined by a hairpin loop.

[0364] Figure 51 The data shown in should be interpreted as including the incorporation step according to the method of the present invention, for example, consistent with versions 1, 2, and 4 of the synthesis method of the present invention (respectively Figure 1 , 2 and 4). Detailed Description

[0365] The present invention provides a method for de novo synthesis of polynucleotide molecules according to a predetermined nucleotide sequence. The synthesized polynucleotide is preferably DNA and preferably a double-stranded polynucleotide molecule. Compared with existing synthesis methods, the present invention provides advantages. For example, all reaction steps can be carried out under aqueous conditions at a mild pH, without requiring extensive protection and deprotection procedures. In addition, the synthesis does not rely on replicating a pre-existing template strand including the predetermined nucleotide sequence.

[0366] The inventors have determined that the use of universal nucleotides as defined herein allows the newly incorporated nucleotides to pair correctly with their desired partner nucleotides during each synthesis cycle. The use of universal nucleotides allows for the generation of polynucleotide cleavage sites within the synthesis region that facilitate cleavage and repeated cycles of synthesis. The present invention provides general methods for synthesizing polynucleotides and for assembling large fragments including such synthesized polynucleotides.

[0367] Reference will be made herein to five exemplary method versions of the present invention ( Figures 1 to 5) and certain variants more generally describe certain embodiments of the synthetic methods of the present invention. It should be understood that all of the exemplary methods including five exemplary method versions of the present invention are not intended to limit the present invention. The present invention provides an in vitro method for synthesizing a double-stranded polynucleotide molecule having a predefined sequence, the method comprising performing a synthesis cycle, wherein in each cycle the first polynucleotide strand is extended by incorporating nucleotides of the predefined sequence, and then the second polynucleotide strand hybridized to the first strand is extended by incorporating nucleotides, thereby forming nucleotide pairs with the incorporated nucleotides of the first strand. Preferably, the method is used for synthesizing DNA. The specific methods described herein are provided as embodiments of the present invention.

[0368] Reaction conditions

[0369] In one aspect, the present invention provides a method for synthesizing a double-stranded polynucleotide having a predefined sequence.

[0370] In some embodiments, the synthesis is carried out under conditions suitable for nucleotide hybridization within the double-stranded polynucleotide. Generally, the polynucleotide is contacted with the reagent under conditions that allow nucleotides to hybridize with complementary nucleotides. Conditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, (1995)).

[0371] The incorporation of nucleotides into the polynucleotide can be carried out under suitable conditions, for example, in the presence of a suitable buffer solution at a suitable temperature (e.g., about 65 °C) using a polymerase (e.g., Therminator IX polymerase) or terminal deoxynucleotidyl transferase (TdT) enzyme or a functional variant thereof to incorporate modified nucleotides (3'-O-modified-dNTP). In one embodiment, the buffer solution can include 2 mM Tris-HCL, 1 mM (NH 4 ) 2 SO 4 4, 1 mM KCl, 0.2 mM MgSO 4 4 and 0.01% Triton X-100.

[0372] Cleavage of polynucleotides can be carried out under suitable conditions, for example in the presence of a suitable buffer solution, at a temperature compatible with the enzyme (e.g., 37 °C) using a polynucleotide cleavage enzyme (e.g., an endonuclease). In one embodiment, the buffer solution may include 5 mM potassium acetate, 2 mM Tris-acetate, 1 mM magnesium acetate, and 0.1 mM DTT.

[0373] Ligation of polynucleotides can be carried out under suitable conditions, for example in the presence of a suitable buffer solution, at a temperature compatible with the enzyme (e.g., room temperature) using a ligase (e.g., T4 DNA ligase). In one embodiment, the buffer solution may include 4.4 mM Tris-HCl, 7 mM MgCl 2 , 0.7 mM dithiothreitol, 0.7 mM ATP, 5% polyethylene glycol (PEG6000).

[0374] Removal of protecting groups can be carried out under suitable conditions, for example using a reducing agent (e.g., TCEP). For example, TCEP in Tris buffer (e.g., at a final concentration of 300 mM) can be used for removal of protecting groups.

[0375] Anchor polynucleotide and scaffold polynucleotide

[0376] Double-stranded polynucleotides having a predefined sequence are synthesized by the method of the present invention by incorporating predetermined nucleotides into a pre-existing polynucleotide, which is referred to herein as a scaffold polynucleotide, which can be attached to or capable of being attached to a surface as described herein. As described in more detail herein, the scaffold polynucleotide forms a support structure to accommodate the newly synthesized polynucleotide, and as is apparent from the description herein, it does not include a pre-existing template strand that is replicated as in conventional synthesis methods. If the scaffold polynucleotide is attached to a surface, the scaffold polynucleotide may be referred to as an anchor polynucleotide. The surface attachment chemistry for attaching the scaffold polynucleotide to a surface to form an anchor polynucleotide is described in more detail herein.

[0377] In one embodiment, the scaffold polynucleotide includes a synthetic strand hybridized to a complementary support strand. The synthetic strand includes a polymerase primer strand portion and optionally an auxiliary strand portion separated by a single-strand break or "nick" (e.g., Figures 1 to 5)。The primer strand portion and the auxiliary strand portion of the synthetic strand can both be provided by hybridizing with a complementary support strand. Alternatively, the auxiliary strand portion of the synthetic strand can be provided separately. The primer strand portion of the synthetic strand can be provided first, and then the support strand and the auxiliary strand can be provided. Alternatively, the components of the scaffold polynucleotide can be provided separately. For example, the support strand can be provided first, then the primer strand portion of the synthetic strand can be provided, and then the auxiliary strand can be provided. The support strand can be provided first, then the auxiliary strand portion of the synthetic strand can be provided, and then the primer strand can be provided. The auxiliary strand portion can be provided before the cleavage step. The auxiliary strand portion can be omitted from the scaffold polynucleotide before cleavage and incorporation of new predetermined nucleotides. The auxiliary strand portion can be removed from the scaffold polynucleotide before cleavage and incorporation of new predetermined nucleotides, for example, by denaturation, as described in more detail herein. After mixing the components under suitable conditions, the scaffold polynucleotide is formed upon hybridization of the individual components.

[0378] The new synthesis is initiated by a polymerase or a transferase at the single-strand break site. Thus, the polymerase or transferase will act on the terminal nucleotide of the primer strand portion extending at the single-strand break site. The single-strand break or "nick" between the auxiliary strand portion and the primer strand portion of the synthetic strand is typically initially achieved by providing the two portions of the synthetic strand as separate molecules, which will align with the support strand after hybridization with the support strand. The (5') terminal nucleotide of the auxiliary strand at the single-strand break site is typically provided in the absence of a phosphate group. The absence of the terminal phosphate group prevents the terminal nucleotide of the auxiliary strand portion from ligating to the terminal nucleotide of the primer strand portion at the single-strand break site, thus maintaining the single-strand break. The generation and maintenance of the single-strand break can be achieved by other means. For example, the terminal nucleotide of the auxiliary strand portion can have a suitable blocking group that prevents ligation to the primer strand portion. Preferably, the auxiliary strand lacking a terminal phosphate group is provided at the single-strand break site.

[0379] A scaffold polynucleotide can be provided in which each support and synthetic strand is not connected at adjacent ends. The scaffold polynucleotide can be provided with support and synthetic strands at both ends of the scaffold polynucleotide, which are connected at adjacent ends, for example, by a hairpin loop. The scaffold polynucleotide can be provided with support and synthetic strands at one end of the scaffold polynucleotide or any other suitable linker, which are connected at adjacent ends, for example, by a hairpin loop.

[0380] As described in more detail herein (see Figure 11 ), the scaffold polynucleotide with or without a hairpin can be immobilized on a solid support or surface.

[0381] The term "hairpin" or "hairpin loop" is commonly used in the current technical field. The term "hairpin loop" is also commonly referred to as a "stem loop". These terms refer to a region of secondary structure in a polynucleotide that includes a loop of unpaired nucleobases formed when one strand of the polynucleotide molecule hybridizes to another part of the same strand due to intramolecular base pairing. Thus, a hairpin can resemble a U-shaped structure. An example of such a structure is Figure 11 as shown.

[0382] Nucleotides and universal nucleotides

[0383] The nucleotides that can be incorporated into a synthetic polynucleotide by any of the methods described herein can be nucleotides, nucleotide analogs, and modified nucleotides.

[0384] In any of the synthetic methods of the invention defined and described herein, the nucleotides are preferably incorporated as nucleotides comprising a reversible terminator group as described herein.

[0385] The nucleotides can include natural nucleobases or unnatural nucleobases. The nucleotides can contain natural nucleobases, sugars, and phosphate groups. Natural nucleobases include adenosine (A), thymine (T), uracil (U), guanine (G), and cytosine (C). One component of the nucleotide can be further modified.

[0386] A nucleotide analog is a nucleotide that is structurally modified in the base, sugar, or phosphate or a combination thereof and is still acceptable to a polymerase as a substrate for incorporation into an oligonucleotide strand.

[0387] An unnatural nucleobase can be a nucleobase that will bind, for example, hydrogen bond, to all nucleobases in a target polynucleotide to some extent. The unnatural nucleobase is preferably one that will bind, for example, hydrogen bond, to the nucleobases of nucleotides including nucleoside adenosine (A), thymine (T), uracil (U), guanine (G), and cytosine (C).

[0388] Unnatural nucleotides can be peptide nucleic acids (PNA), locked nucleic acids (LNA), unlocked nucleic acids (UNA), bridged nucleic acids (BNA), or morpholinos, phosphorothioates, or methylphosphonates.

[0389] Non-natural nucleotides can include modified sugars and / or modified nucleobases. Modified sugars include but are not limited to 2'-O-methyl ribose. Modified nucleobases include but are not limited to methylated nucleobases. Methylation of nucleobases is a recognized form of epigenetic modification that has the ability to alter the expression of genes and other elements such as microRNAs. Methylation of nucleobases occurs at discrete loci that consist mainly of dinucleotides composed of CpG motifs, but can also occur at CHH motifs (where H is A, C, or T). Typically, during methylation, a methyl group is added to the fifth carbon of the cytosine base to produce 5-methylcytosine. Thus, modified nucleobases include but are not limited to 5-methylcytosine.

[0390] Nucleotides of a predefined sequence can be incorporated relative to a partner nucleotide to form a nucleotide pair. The partner nucleotide can be a complementary nucleotide. A complementary nucleotide is a nucleotide that is capable of bonding, such as hydrogen bonding, to the nucleotide of the predefined sequence to some extent.

[0391] Typically, nucleotides of a predefined sequence are incorporated into a polynucleotide relative to their natural complementary partner nucleobases. Thus, adenosine can be incorporated opposite thymine, and vice versa. Guanine can be incorporated opposite cytosine, and vice versa. Alternatively, nucleotides of the predefined sequence can be opposite a partner nucleobase to which they will bond, such as hydrogen bond, to some extent.

[0392] Alternatively, the partner nucleotide can be a non-complementary nucleotide. A non-complementary nucleotide is a nucleotide that is not capable of bonding, such as hydrogen bonding, to the nucleotide of the predefined sequence. Thus, nucleotides of the predefined sequence can be incorporated relative to a partner nucleotide to form a mismatch, provided that the synthesized polynucleotide is generally double-stranded and wherein the first strand is linked to the second strand by hybridization.

[0393] The term "relative" should be understood to refer to the normal use of the term in the field of nucleic acid biochemistry and specifically to conventional Watson-Crick base pairing. Thus, a first nucleic acid molecule of sequence 5'-ACGA-3' can form a duplex with a second nucleic acid molecule of sequence 5'-TCGT-3', where the G of the first molecule will be opposite and hydrogen bond to the C of the second molecule. A first nucleic acid molecule of sequence 5'-ATGA-3' can form a duplex with a second nucleic acid molecule of sequence 5'-TCGT-3', where the T of the first molecule is mismatched with the G of the second molecule but is still opposite it and will serve as the partner nucleotide. The principle applies to any nucleotide partner pair relationship disclosed herein, including those involving universal nucleotides.

[0394] In all the methods described herein, the position in the support strand and the relative position in the synthetic strand are designated as position number "n". This position refers to the position of the nucleotide in the support strand of the scaffold polynucleotide that is opposite the nucleotide position in the synthetic strand in any given synthesis cycle, where the synthetic strand will be occupied or will be occupied by the first / next nucleotide of the predefined sequence after adding the first / next nucleotide of the predefined sequence to the end of the primer strand portion in step (3) or in the incorporation step of a subsequent cycle. Position "n" also refers to the position of the support strand of the ligation polynucleotide at the ligation step (5) or in the ligation step of a subsequent cycle, which is the nucleotide position that will be opposite the first / subsequent nucleotide of the predefined sequence after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide in step (5) of the first cycle or in the ligation step of a subsequent cycle.

[0395] Both the position in the support strand and the relative position in the synthetic strand can be referred to as position n.

[0396] For more details on the definition of position "n", reference Figures 1 to 5 is provided in relation to its description relative to the five exemplary synthesis method versions 1 to 5 described in more detail herein.

[0397] Nucleotides and nucleotide analogs can preferably be provided as nucleoside triphosphates. Thus, in any of the methods of the present invention, for the synthesis of DNA polynucleotides, nucleotides can be incorporated, for example, through the action of a DNA polymerase or, for example, through the action of an enzyme having deoxynucleotide terminal transferase activity, from 2'-deoxyribonucleoside-5'-O-triphosphates (dNTPs). In any of the methods of the present invention, for the synthesis of RNA polynucleotides, nucleotides can be incorporated, for example, through the action of an RNA polymerase or, for example, through the action of an enzyme having nucleotide terminal transferase activity, from ribonucleoside-5'-O-triphosphates (dNTPs). The triphosphate can be replaced by a tetraphosphate or a pentaphosphate (generally an oligophosphate). These oligophosphates can be replaced by other alkyl or acyl groups:

[0398]

[0399] X = O, S, NH Z = any alkyl or acyl group

[0400] and their salts

[0401] The methods of the present invention can use universal nucleotides. Universal nucleotides can be used as components of the support strand of the scaffold molecule to facilitate the correct pairing of newly incorporated nucleotides with their desired partner nucleotides during each cycle of synthesis. If desired, universal nucleotides can also be incorporated as components of a predefined nucleotide sequence into the synthetic strand.

[0402] A universal nucleotide is a nucleotide in which the nucleobase will bind, to some extent, to the nucleobase of any nucleotide of a predefined sequence, such as by hydrogen bonding. A universal nucleotide is preferably a nucleotide that will bind, to some extent, to nucleotides including nucleosides adenosine (A), thymine (T), uracil (U), guanine (G), and cytosine (C), such as by hydrogen bonding. Compared to other nucleotides, a universal nucleotide can bind more strongly to some nucleotides. For example, a universal nucleotide including the nucleoside 2'-deoxyinosine (I) will show a preferential pairing order of I-C > I-A > I-G ≈ I-T.

[0403] Examples of possible universal nucleotides are inosine or nitroindole. A universal nucleotide preferably includes one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole, or phenyl (C6-aromatic ring). A universal nucleotide more preferably includes one of the following nucleosides: 2'-deoxyinosine, inosine, 7-deaza-2'-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 4-nitroindole 2'-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2'-deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2'-deoxyribonucleoside, 6-nitroindole ribonucleoside, 3-nitropyrrole 2'-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, acyclic sugar analogs of hypoxanthine, nitroimidazole 2'-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2'-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2'-deoxyribonucleoside, 4-nitrobenzimidazole ribonucleoside, 5-nitroindazole 2'-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2'-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, or phenyl C-2'-deoxyribosyl nucleoside.

[0404] Some examples of universal bases are shown below:

[0405]

[0406] Universal nucleotides incorporating cleavable bases can also be used, including photo-cleavable bases and enzyme-cleavable bases, and some examples of such bases are shown below.

[0407] Photo-cleavable bases:

[0408]

[0409] Base analogs cleavable by endonuclease III:

[0410]

[0411] Base analogs cleavable by formamidopyrimidine DNA glycosylase (Fpg):

[0412]

[0413] Base analogs cleavable by 8-oxoguanine DNA glycosylase (hOGG1):

[0414]

[0415] Base analogs cleavable by hNeil1:

[0416]

[0417] Base analogs cleavable by thymine DNA glycosylase (TDG):

[0418]

[0419] Base analogs cleavable by human alkyladenine DNA glycosylase (hAAG):

[0420]

[0421] Bases cleavable by uracil DNA glycosylase:

[0422]

[0423] Bases cleavable by human single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1):

[0424]

[0425] Bases cleavable by 5-methylcytosine DNA glycosylase (ROS1):

[0426]

[0427] (See S.S. David, S.D. Williams Chemical reviews 1998, 98, 1221 - 1262 and M.I. Ponferrada-Marín, T. Roldán-Arjona, R.R. Ariza, Nucleic Acids Res 2009, 37, 4264 - 4274).

[0428] In any method involving a scaffold polynucleotide, the universal nucleotide most preferably comprises 2'-deoxyinosine.

[0429] Examples of epigenetic bases that can be incorporated using any of the synthetic methods described herein include the following:

[0430]

[0431] Examples of modified bases that can be incorporated using any of the synthetic methods described herein include the following:

[0432]

[0433] Examples of halogenated bases that can be incorporated using any of the synthetic methods described herein include the following:

[0434]

[0435] where R1 = F, Cl, Br, I, alkyl, aryl, fluorescent label, aminopropargyl, aminoallyl.

[0436] Examples of amino-modified bases that can be incorporated using any of the synthetic methods described herein and can be used for, for example, attachment / linker chemistry include the following:

[0437]

[0438] R 1 = for example: fluorophore fluorophore

[0439] where the base = A, T, G or C, with an alkyne or alkene linker.

[0440] Examples of modified bases that can be incorporated using any of the synthetic methods described herein and can be used for, for example, click chemistry include the following:

[0441]

[0442] Examples of biotin-modified bases that can be incorporated using any of the synthetic methods described herein include the following:

[0443]

[0444] where the base = A, T, G or C, with an alkyne or alkene linker.

[0445] Examples of bases with a fluorophore and a quencher that can be incorporated using any of the synthetic methods described herein include the following:

[0446]

[0447] Nucleotide incorporation enzyme

[0448] Enzymes are available that are capable of extending a single-stranded polynucleotide portion of a double-stranded polynucleotide molecule by adding nucleotides and / or capable of extending one strand of a blunt-ended double-stranded polynucleotide molecule. This includes enzymes with template-independent enzyme activities, such as template-independent polymerase or template-independent transferase activities.

[0449] Thus, in any of the methods described herein, the enzyme used to add nucleotides of a predefined sequence (e.g., added to the end of the synthetic strand of a scaffold polynucleotide) has template-independent enzyme activity, such as template-independent polymerase or template-independent transferase activity.

[0450] Any suitable enzyme can be used to add the predefined nucleotides using the methods described herein. Thus, in all of the methods defined and described herein, with reference to the use of a polymerase or transferase, the polymerase or transferase can be replaced with another enzyme capable of performing the same function as the polymerase or transferase in the context of the methods of the present invention.

[0451] Polymerases can be used in the methods described herein. The polymerase can be selected based on its ability to incorporate modified nucleotides, specifically nucleotides having a reversible terminator group attached as described herein. In the exemplary methods described herein, all polymerases acting on DNA must not have 3' to 5' exonuclease activity. The polymerase can have strand displacement activity.

[0452] Thus, preferably, the polymerase is a modified polymerase having an enhanced ability to incorporate nucleotides including a reversible terminator group compared to an unmodified polymerase. More preferably, the polymerase is a genetically engineered variant of a native DNA polymerase from the Thermococcus species 9°N, preferably species 9°N-7. Examples of modified polymerases are the Therminator IX DNA polymerase and Terminator X DNA polymerase available from New England BioLab. This enzyme has an enhanced ability to incorporate 3'-O-modified dNTPs.

[0453] Examples of other polymerases that can be used to incorporate reversible terminator dNTPs in any method of the present invention are DeepVent (exo-), Vent (Exo-), 9°N DNA polymerase, Terminator DNA polymerase, Therminator IX DNA polymerase, Terminator X DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq polymerase.

[0454] Examples of other polymerases that can be used to incorporate reversible terminator NTPs in any of the methods of the invention are T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, polλ, polμ, or Φ29 DNA polymerase.

[0455] For the extension of such polynucleotide synthesis molecules that include DNA, a DNA polymerase can be used. Any suitable DNA polymerase can be used.

[0456] The DNA polymerase can be, for example, Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, Escherichia coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, Taq DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, and enzymes with reverse transcriptase activity, such as M-MuLV reverse transcriptase.

[0457] The DNA polymerase may lack 3' to 5' exonuclease activity. Any such suitable polymerase can be used. Such a DNA polymerase can be, for example, Bst DNA polymerase full length, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3'→5' exo-), M-MuLV reverse transcriptase, Sulfolobus DNA polymerase IV, Taq DNA polymerase.

[0458] The DNA polymerase may have strand displacement activity. Any such suitable polymerase can be used. Such a DNA polymerase can be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow) fragment (3'→5' exo-), M-MuLV reverse transcriptase, phi29 DNA polymerase.

[0459] The DNA polymerase may lack 3' to 5' exonuclease activity and may have strand displacement activity. Any such suitable polymerase can be used. Such a DNA polymerase can be, for example, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, Escherichia coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reverse transcriptase.

[0460] The DNA polymerase may lack 5'-to-3' exonuclease activity. Any such suitable polymerase can be used. Such a DNA polymerase can be, for example, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase, the large (Klenow) fragment of DNA Pol I, the large (Klenow) fragment of DNA Pol I (3'→5' exo-), M-MuLV reverse transcriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, T4 DNA polymerase, T7 DNA polymerase.

[0461] The DNA polymerase may lack both 3'-to-5' and 5'-to-3' exonuclease activities and may have strand displacement activity. Any such suitable polymerase can be used. Such a DNA polymerase can be, for example, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase, the large (Klenow) fragment of DNA Pol I (3'→5' exo-), M-MuLV reverse transcriptase.

[0462] The DNA polymerase can also be a genetically engineered variant. For example, the DNA polymerase can be a genetically engineered variant of the native DNA polymerase of Thermococcus species 9°N, such as species 9°N-7. One such example of a modified polymerase is the Therminator IX DNA polymerase available from New England BioLabs. Other engineered or variant DNA polymerases include Deep Vent (exo-), Vent (Exo-), 9°N DNA polymerase, Therminator DNA polymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase, Sulfolobus DNA polymerase I, and Taq polymerase.

[0463] For the extension of such polynucleotide synthesis molecules including RNA, any suitable enzyme can be used. For example, an RNA polymerase can be used. Any suitable RNA polymerase can be used.

[0464] The RNA polymerase can be T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, the Escherichia coli RNA polymerase holoenzyme.

[0465] The enzyme can have terminal transferase activity. For example, the enzyme can be terminal nucleotidyl transferase or terminal deoxynucleotidyl transferase, and wherein the polynucleotide synthesis molecule is extended to form a polynucleotide molecule including DNA or RNA, preferably DNA. Any one of these enzymes can be used in the method of the present invention where extension of the polynucleotide synthesis molecule is required.

[0466] One such enzyme is terminal nucleotidyl transferase, such as terminal deoxynucleotidyl transferase (TdT) (see, e.g., Motea et al., 2010; Minhaz Ud-Dean, Syst. Synth. Biol., 2008, 2(3-4), 67-73). TdT is capable of catalyzing the addition of nucleotide molecules (nucleoside monophosphates) from nucleoside triphosphate substrates (NTP or dNTP) to a polynucleotide synthesis molecule. TdT is capable of catalyzing the addition of natural and unnatural nucleotides. It is also capable of catalyzing the addition of nucleotide analogs (Motea et al., 2010). The Polλ and polμ enzymes can also be used (Ramadan K et al., J. Mol. Biol., 2004, 339(2), 395-404), such as Φ29 DNA polymerase can be used.

[0467] Techniques for extending single-stranded polynucleotide molecules, both DNA and RNA, in the absence of a template through the action of a terminal transferase (e.g., terminal deoxynucleotidyl transferase; TdT) to produce synthetic single-stranded polynucleotide molecules have been widely discussed in the art. These techniques are disclosed in, for example, patent application publications WO2016 / 034807, WO 2016 / 128731, WO2016 / 139477, and WO2017 / 009663, as well as US2014 / 0363852, US2016 / 0046973, US2016 / 0108382, and US2016 / 0168611. These documents describe the controlled extension of single-stranded polynucleotide synthesis molecules to produce synthetic single-stranded polynucleotide molecules through the action of TdT. The extension through natural and non-natural / artificial nucleotides using such an enzyme is described, such as the extension through modified nucleotides, e.g., the extension of nucleotides incorporating a blocking group. Any terminal transferase and any enzyme fragment, derivative, analog, or functional equivalent thereof disclosed in these documents can be applied to the method of the present invention, provided that the terminal transferase function is preserved in the enzyme.

[0468] Directed evolution techniques, conventional screening, rational or semi-rational engineering / mutagenesis methods, or any other suitable method can be used to modify any such enzyme to provide and / or optimize the desired function. Any other enzyme capable of extending a single-stranded polynucleotide molecule portion, such as a molecule including DNA or RNA, in the absence of a template can be used.

[0469] Thus, in any method defined herein, single-stranded polynucleotide synthetic molecules comprising DNA or blunt-ended double-stranded polynucleotides comprising DNA can be enzymatically extended by an enzyme having template-independent enzyme activity, such as template-independent polymerase or transferase activity. The enzyme can have nucleotide transferase activity, such as deoxynucleotide transferase, such as terminal deoxynucleotidyl transferase (TdT) or an enzymatic fragment, derivative, analogue or functional equivalent thereof. The polynucleotide synthetic molecules extended by the action of such an enzyme comprise DNA.

[0470] In any method defined herein, a single-stranded portion of a polynucleotide synthetic molecule comprising RNA or a blunt-ended double-stranded polynucleotide comprising RNA can be enzymatically extended by an enzyme having nucleotide transferase (e.g., including TdT) or an enzymatic fragment, derivative, analogue or functional equivalent thereof. The polynucleotide synthetic molecules extended by the action of such an enzyme can comprise RNA. For the synthesis of a single-stranded polynucleotide synthetic molecule comprising RNA or a single-stranded portion of a polynucleotide synthetic molecule comprising RNA, any suitable nucleotide transferase can be used. Nucleotide transferases, such as poly(U) polymerase and poly(A) polymerase (e.g., from Escherichia coli) are capable of adding nucleotide monophosphate units to polynucleotide synthetic molecules in a template-independent manner. Any one of these enzymes and any enzymatic fragment, derivative, analogue or functional equivalent thereof can be applied to the methods of the present invention provided that the nucleotide transferase function is preserved in the enzyme. Directed evolution techniques, conventional screening, rational or semi-rational engineering / mutagenesis methods or any other suitable method can be used to modify any such enzyme to provide and / or optimize the desired function.

[0471] Reversible blocking group

[0472] All methods defined and described herein involve reversible blocking groups or reversible terminator groups. The function of these groups is to prevent further extension by the enzyme in a given synthesis cycle, such that only nucleotides of a predefined sequence can be controllably used for extending the synthetic strand, thus preventing non-specific nucleotide incorporation. Any function that achieves the said effect can be used in any method defined and described herein. Reversible blocking groups / reversible terminator groups linked to nucleotides and deblocking steps are the preferred methods for achieving the said effect. However, this effect can be achieved by appropriate alternative means.

[0473] Any suitable reversible blocking group can be linked to a nucleotide to prevent further extension by the enzyme after incorporation of the nucleotide in a given cycle and to limit incorporation to one nucleotide per cycle. In any method of the present invention, the reversible blocking group is preferably a reversible terminator group, which serves to prevent further extension by the polymerase. Examples of reversible terminators are provided below.

[0474] Propargyl reversible terminator:

[0475]

[0476] Allyl reversible terminator:

[0477]

[0478] Cyclooctene reversible terminator:

[0479]

[0480] Cyanoethyl reversible terminator:

[0481]

[0482] Nitrobenzyl reversible terminator:

[0483]

[0484] Disulfide reversible terminator:

[0485]

[0486] Azidomethyl reversible terminator:

[0487]

[0488] Aminoalkoxy reversible terminator:

[0489]

[0490] Nucleoside triphosphates having a bulky group attached to the base can act as alternatives to the reversible terminator group at the 3'-hydroxyl and can prevent further incorporation. The group can remove the protecting group by TCEP or DTT, generating natural nucleotides.

[0491]

[0492] X = O, S, NH, CH 2 Z = bulky group

[0493] For synthesizing DNA polynucleotides by any method according to the present invention, the preferred modified nucleoside is 3'-O-modified-2'-deoxyribonucleoside-5'-O-triphosphate. For synthesizing RNA polynucleotides by any method according to the present invention, the preferred modified nucleoside is 3'-O-modified-ribonucleoside-5'-O-triphosphate.

[0494] Preferred modified dNTPs are modified dNTPs that are 3'-O-allyl-dNTP and 3'-O-azidomethyl-dNTP.

[0495] The 3'-O-allyl-dNTPs are as follows.

[0496]

[0497] The 3'-O-azidomethyl-dNTPs are as follows.

[0498]

[0499] The inventive methods described and defined herein may involve a protecting group removal or deblocking step. Such steps involve removing a reversible blocking group (e.g., a reversible terminator group) by any suitable method, or otherwise reversing the function of the blocking / terminator group to inhibit further extension by an enzyme / polymerase.

[0500] Any suitable reagent can be used to remove the reversible terminator group in the protecting group removal step.

[0501] A preferred protecting group removal reagent is tris(carboxyethyl)phosphine (TCEP). TCEP can be used to remove the reversible terminator group from 3'-O-allyl-nucleotides (in combination with Pd 0 binding) and 3'-O-azidomethyl-nucleotides.

[0502] Examples of protecting group removal reagents are provided below.

[0503] Propargyl reversible terminator:

[0504] Treat with a Pd catalyst - Na 2 PdCl 4 PdCl 2 treatment.

[0505] A ligand can be used, for example: trisodium triphenylphosphine-3,3',3”-trisulfonate.

[0506] Allyl reversible terminator:

[0507] Treat with a Pd catalyst - Na 2 PdCl 4 PdCl 2 treatment.

[0508] A ligand can be used, for example: trisodium triphenylphosphine-3,3',3”-trisulfonate.

[0509] Azidomethyl reversible terminator:

[0510] Treat with a thiol (mercaptoethanol or dithiothreitol) or tris(2-carboxyethyl)phosphine - TCEP.

[0511] Cyanoethyl reversible terminator:

[0512] Treatment with fluoride - ammonium fluoride, tetrabutylammonium chloride (TBAF).

[0513] Nitrobenzyl reversible terminator:

[0514] Exposure to UV light

[0515] Disulfide reversible terminator:

[0516] Treatment with thiol (mercaptoethanol or dithiothreitol) or tris(2 - carboxyethyl)phosphine - TCEP.

[0517] Aminoalkoxy reversible terminator:

[0518] Treatment with nitrite (NO 2 - 、HNO 2 ) at pH = 5.5

[0519] Reversible blocking groups (such as reversible terminator groups) can be removed by a step carried out immediately after the incorporation step and before the cleavage step, provided that unwanted reagents in the incorporation step are removed to prevent further incorporation after removal of the reversible terminator group. Reversible blocking groups (such as reversible terminator groups) can be removed by a step carried out immediately after the cleavage step and before the ligation step. Reversible blocking groups (such as reversible terminator groups) can be removed by a step carried out immediately after the ligation step.

[0520] Synthetic polynucleotide

[0521] A polynucleotide having a predefined sequence synthesized according to the methods described herein is double - stranded. The synthesized polynucleotide is generally double - stranded, and the first strand is linked to the second strand by hybridization. Mismatches and non - hybridized regions can be tolerated as long as the entire first strand is linked to the second strand by hybridization.

[0522] Hybridization can be defined by moderately stringent or stringent hybridization conditions. Moderately stringent hybridization conditions use a pre - wash solution (or other similar hybridization solutions, such as a solution containing approximately 50% formamide with a hybridization temperature of 42°C) with a hybridization buffer containing 5× sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), approximately 50% formamide, 6× SSC, and a hybridization temperature of 55°C, and the washing conditions are carried out at 60°C in 0.5× SSC, 0.1% SDS. Stringent hybridization conditions hybridize in 6× SSC at 45°C, followed by one or more washes at 68°C in 0.1× SSC, 0.2% SDS.

[0523] Double-stranded polynucleotides having a predefined sequence synthesized according to the methods described herein can be retained as double-stranded polynucleotides. Alternatively, the two strands of the double-stranded polynucleotide can be separated to provide single-stranded polynucleotides having a predefined sequence. The conditions (melting) that permit separation of the two strands of the double-stranded polynucleotide are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, (1995)).

[0524] Double-stranded polynucleotides having a predefined sequence synthesized according to the methods described herein can be amplified after synthesis. Any region of the double-stranded polynucleotide can be amplified. All or any region of the double-stranded polynucleotide can be amplified together with all or any region of a scaffold polynucleotide. The conditions that permit amplification of the double-stranded polynucleotide are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, (1995)). Thus, any of the synthetic methods described herein can further include an amplification step, wherein the double-stranded polynucleotide having a predefined sequence or any region thereof is amplified as described above. Amplification can be carried out by any suitable method, such as polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), helicase-dependent amplification (HDA), ramified network amplification method (RAM), etc. Preferably, amplification is carried out by polymerase chain reaction (PCR).

[0525] The double-stranded or single-stranded polynucleotide having a predefined sequence synthesized according to the methods described herein can be of any length. For example, the length of the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450 or at least 500 nucleotides or nucleotide pairs. For example, in terms of length, the polynucleotide can be about 10 to about 100 nucleotides or nucleotide pairs, about 10 to about 200 nucleotides or nucleotide pairs, about 10 to about 300 nucleotides or nucleotide pairs, about 10 to about 400 nucleotides or nucleotide pairs and about 10 to about 500 nucleotides or nucleotide pairs. The polynucleotide can be up to about 1000 or more nucleotides or nucleotide pairs, up to about 5000 or more nucleotides or nucleotide pairs or up to about 100000 or more nucleotides or nucleotide pairs in length.

[0526] Cleavage of scaffold polynucleotide

[0527] In methods that require the presence of a scaffold polynucleotide and in the cleavage step prior to ligation, the choice of the reagent for performing the cleavage step will depend on the specific method used. The cleavage site is defined by the specific position of the universal nucleotides in the support strand and by whether blunt-ended scaffold polynucleotides are required upon cleavage or overhangs in the scaffold polynucleotide are required upon cleavage. Thus, the configuration of the desired cleavage site and the choice of the appropriate cleavage reagent will depend on the specific chemistry employed in the method, which will be readily apparent by reference to the exemplary methods described herein.

[0528] Some examples of DNA lyases that recognize modified bases are shown in the table below.

[0529]

[0530]

[0531] Ligation polynucleotide

[0532] In methods that require the presence of a scaffold polynucleotide and in the post-cleavage ligation step, the choice of the configuration and structure of the ligation polynucleotide also depends on the specific method used. The ligation polynucleotide generally includes a support strand as described herein and an auxiliary strand as described herein. The support strand and the auxiliary strand used in the ligation polynucleotide can be the same as or different from those used in the initial scaffold polynucleotide construct. For example, the requirement for single-nucleotide or dinucleotide overhangs in the support strand at the ligation ends of the ligation polynucleotide will depend on the method used. The appropriate structure can be readily achieved by reference to the exemplary methods described herein.

[0533] Complementary ligation ends of the ligated polynucleotides typically provide a non-phosphorylated terminal nucleotide in the auxiliary strand adjacent to the overhang. This prevents ligation of the auxiliary strand portion of the synthetic strand to the primer strand portion of the synthetic strand, thus maintaining a single-strand break in the synthetic strand. Alternative methods for preventing ligation in the synthetic strand can be used. For example, a blocking moiety can be ligated to the terminal nucleotide in the auxiliary strand. Additionally, as further described herein, the auxiliary strand can be removed from the scaffold molecule, e.g., by denaturation, prior to cleavage and incorporation of the next predetermined nucleotide in the next synthesis cycle.

[0534] Ligation

[0535] In the methods of the invention involving a ligation step, any suitable method can be used to effect the ligation. Preferably, the ligation step will be carried out by a ligase. The ligase can be a modified ligase having enhanced activity towards single-base overhang substrates. The ligase can be T3 DNA ligase or T4 DNA ligase. The ligase can be a blunt TA ligase. For example, a blunt TA ligase is available from New England Biolabs (NEB). This is a ready-to-use premixed solution of T4 DNA ligase, a ligation enhancer, and an optimized reaction buffer, which is specifically formulated to improve ligation and transformation of both blunt and single-base overhang substrates. Molecules, enzymes, chemicals, and methods for ligating (joining) single-stranded and double-stranded polynucleotides are well known to those skilled in the art.

[0536] Solid - phase synthesis

[0537] The synthetic polynucleotides produced according to the synthetic methods of the invention can preferably be synthesized using solid-phase or reversible solid-phase techniques. Various such techniques are known in the art and can be used. Prior to initiating the synthesis of a new double-stranded polynucleotide of a predefined sequence, the scaffold polynucleotide can be immobilized on a surface, e.g., a planar surface such as glass, a gel-based material, or a microparticle such as a bead or a functionalized quantum dot. The material including the surface itself can be bound to a substrate. For example, the scaffold polynucleotide can be immobilized on a gel-based material such as polyacrylamide, and wherein the gel-based material is bound to a support substrate such as glass.

[0538] The polynucleotides can be immobilized or tethered to the surface directly or indirectly. For example, they can be attached directly to the surface by chemical bonding. They can be tethered indirectly to the surface through an intermediate surface, e.g., the surface of a microparticle or a bead, such as in SPRI or in an electrowetting system, as described below. The synthesis cycle can then be initiated and completed while the scaffold polynucleotide incorporating the newly synthesized polynucleotide is immobilized.

[0539] In such methods, the double-stranded scaffold polynucleotide can be immobilized to a surface prior to incorporation of the first nucleotide of the predefined sequence. Thus, such immobilized double-stranded scaffold polynucleotide can act as an anchor to attach the double-stranded polynucleotide of the predefined sequence to the surface during and after synthesis.

[0540] Only one strand of such double-stranded anchor / scaffold polynucleotide can be immobilized on the surface at the same end of the molecule. Alternatively, each of the two strands of the double-stranded anchor / scaffold polynucleotide can be immobilized on the surface at the same end of the molecule. A double-stranded anchor / scaffold polynucleotide can be provided in which each strand is joined at adjacent ends, such as by a hairpin loop at the end opposite the site of de novo synthesis initiation, and the joined ends can be immobilized on the surface (e.g., as schematically depicted in Figure 11 ).

[0541] In methods involving a scaffold polynucleotide, as described herein, the scaffold polynucleotide can be attached to a surface prior to incorporation of the first nucleotide in the predefined sequence. Thus, the synthetic strands comprising a primer strand portion and a support strand portion hybridized thereto can be attached to the surface separately, as Figure 11 shown in (a) and (c). The synthetic strands comprising a primer strand portion and a support strand portion hybridized thereto can be joined at adjacent ends, such as by a hairpin loop, e.g., at the end opposite the site of de novo synthesis initiation, and the joined ends can be tethered to the surface, as Figure 11 shown in (b) and (d). One or the other of the synthetic strands comprising a primer strand portion and a support strand portion hybridized thereto can be attached to the surface separately, as Figure 11 shown in (e) to (h). Preferably, the synthetic strands comprising a primer strand portion and a support strand portion hybridized thereto are attached to the surface.

[0542] Solid - phase synthesis on a flat surface

[0543] Prior to initiation of synthesis of a new double-stranded polynucleotide of the predefined sequence, the synthetic anchor / scaffold polynucleotide can be synthesized by methods known in the art, including those described herein, and tethered to the surface.

[0544] A preformed polynucleotide can be tethered to a surface by methods commonly used to produce nucleic acid microarrays attached to a flat surface. For example, an anchor / scaffold polynucleotide can be produced and then spotted or printed onto a flat surface. Contact printing techniques can be used to deposit the anchor / scaffold polynucleotide on the surface. For example, a solid or hollow tip or needle can be dipped into a solution comprising the preformed scaffold polynucleotide and contacted with the flat surface. Alternatively, an oligonucleotide can be adsorbed onto a microstamp and then transferred to the flat surface by physical contact. Non-contact printing techniques include thermal printing or piezoelectric printing, in which sub-nanoliter-sized microdrops comprising the preformed scaffold polynucleotide can be ejected from a printing tip using methods similar to those used in inkjet and bubble jet printing.

[0545] Single-stranded oligonucleotides can be synthesized directly on a flat surface, for example, using the so-called "on-chip" methods used to produce microarrays. Such single-stranded oligonucleotides can then serve as attachment sites for immobilizing preformed anchor / scaffold polynucleotides.

[0546] On-chip techniques for producing single-stranded oligonucleotides involve photolithography, which involves using UV light directed through a photolithography mask to selectively activate protected nucleotides, allowing subsequent incorporation of new protected nucleotides. Cycles of UV-mediated deprotection and coupling of predetermined nucleotides allow in situ production of oligonucleotides with the desired sequence. As an alternative to using a photolithography mask, oligonucleotides with the desired sequence can be produced by sequentially depositing nucleobases using inkjet printing technology and using cycles of coupling, oxidation, and deprotection to produce oligonucleotides on a flat surface (for a review, see Kosuri and Church, Nature Methods, 2014, 11, 499-507).

[0547] In any of the synthesis methods described herein, including methods involving reversible immobilization as described below, the surface can be made of any suitable material. Generally, the surface can include silicon, glass, or polymeric materials. The surface can include a gel surface, such as a polyacrylamide surface, such as about 2% polyacrylamide, optionally using an N-(5-bromoacetylpentyl)acrylamide (BRAPA)-derivatized polyacrylamide surface, preferably a polyacrylamide surface coupled to a solid support such as glass.

[0548] Reversible immobilization

[0549] Synthetic polynucleotides with a predefined sequence can be synthesized according to the present invention using binding surfaces and structures (such as microparticles and beads) that facilitate reversible immobilization. Solid-phase reversible immobilization (SPRI) methods or modified methods are known in the art and can be used (e.g., see DeAngelis M.M. et al. (1995) Solid-Phase Reversible Immobilization for the Isolation of PCR Products, Nucleic Acids Research, 23(22): 4742-4743).

[0550] The surface can be provided in the form of microparticles, such as paramagnetic beads. The paramagnetic beads can aggregate under the influence of a magnetic field. For example, the paramagnetic surface can have chemical groups, such as carboxyl groups, which will act as binding moieties for nucleic acids under appropriate attachment conditions, as described in more detail below. Nucleic acids can be eluted from these surfaces under appropriate elution conditions. The surfaces of the microparticles and beads can be provided with UV-sensitive polycarbonate. In the presence of a suitable immobilization buffer, nucleic acids can bind to the activated surface.

[0551] The microparticles and beads can be made to move freely in the reaction solution and then reversibly immobilized, for example, by holding the beads in micropores or pits etched into the surface. The beads can be positioned as part of an array, for example, by using unique nucleic acid "barcodes" attached to the beads or by using color coding.

[0552] Thus, before starting the synthesis of a new double-stranded polynucleotide of a predefined sequence, an anchor / scaffold polynucleotide according to the present invention can be synthesized and then reversibly immobilized onto such a binding surface. The polynucleotides synthesized by the method of the present invention can be synthesized while being reversibly immobilized on such a binding surface.

[0553] Microfluidic technology and systems

[0554] The surface can be part of an electrowetting-on-dielectric system (EWOD). The EWOD system provides a dielectric-coated surface that facilitates the microfluidic manipulation of very small liquid volumes in the form of microdroplets (see, for example, Chou, W-L. et al. (2015) Recent Advances in Applications of Droplet Microfluidics, Micromachines, 6:1249-1271.). Droplet volumes can be programmably created, moved, partitioned, and combined on the chip by electrowetting techniques. Thus, the EWOD system provides an alternative means for reversibly immobilizing polynucleotides during and after synthesis.

[0555] Polynucleotides having a predefined sequence can be synthesized in the solid phase by the methods described herein, where the polynucleotides are immobilized on the EWOD surface and the steps required in each cycle are facilitated by electrowetting techniques. For example, in a method involving a scaffold polynucleotide and requiring steps of incorporation, cleavage, ligation, and removal of protecting groups, the reagents required for each step, as well as any required washing steps for removing used and unwanted reagents, can be provided in the form of microdroplets that are transported under the influence of an electric field by electrowetting techniques.

[0556] Other microfluidic platforms that can be used in the synthesis methods of the present invention are available. For example, emulsion-based droplet technologies commonly used for nucleic acid manipulation can be used. In such systems, droplets are formed in an emulsion generated by mixing two immiscible fluids, typically water and oil. Emulsion droplets can be programmably created, moved, split, and combined in a microfluidic network. Hydrogel systems can also be provided. In any of the synthesis methods described herein, the droplets can be operated in any suitable compatible system, such as the EWOD system described above and other microfluidic systems, such as microfluidic systems including structures based on components comprising elastomeric materials.

[0557] The droplets can have any suitable size, provided that they are compatible with the synthesis methods herein. The droplet size will vary depending on the particular system employed and the associated architecture of the system. Thus, the size can be adjusted appropriately. In any of the synthesis methods described herein, the droplet diameter can range from about 150 nm to about 5 mm. Droplet diameters below 1 μm can be verified by methods known in the art, such as by techniques involving capillary ejection methods, such as - as described in Calvo et al. (Nature Physics, 2007, 3, pp737 - 742).

[0558] Sequencing of intermediate or final synthetic products.

[0559] The intermediate products of synthesis or assembly or the final polynucleotide synthesis products can be sequenced as a quality control check to determine whether the desired polynucleotide has been correctly synthesized or assembled. One or more polynucleotides of interest can be removed from the solid-phase synthesis platform and sequenced by any of a variety of known commercially available sequencing techniques, such as using the MinION sold by Oxford Nanopore Technologies Limited. TMNanopore sequencing performed by the device. Sequencing can be carried out on the solid-phase platform itself without transferring the polynucleotide to a separate synthesis device. Sequencing can be conveniently carried out on the same electrowetting-on-dielectric (EWOD) device, such as the EWOD device used for synthesis, whereby the synthesis device includes one or more pairs of measurement electrodes. A droplet comprising the polynucleotide of interest can be brought into contact with one of the electrodes of the pair of electrodes, and the droplet forms a droplet interface bilayer with a second droplet in contact with the second electrode of the pair of electrodes, wherein the droplet bilayer interface includes a nanopore in an amphiphilic membrane. The polynucleotide can be positioned at the nanopore, for example under enzymatic control, and the ionic current passing through the nanopore can be measured under a potential difference between the pair of electrodes during passage of the polynucleotide through the nanopore channel. The ionic current measurements as a function of time can be recorded and used to determine the polynucleotide sequence. Prior to sequencing, the polynucleotide can undergo one or more sample preparation steps to optimize it for sequencing, as disclosed in patent application PCT / GB2015 / 050140. Examples of enzymes, amphiphilic membranes, and nanopores that can be suitably employed are disclosed in the patent applications PCT / GB2013 / 052767 and PCT / GB2014 / 052736. Reagents required for preparing samples of polynucleotides, nanopores, amphiphilic membranes, etc. can be provided to the EWOD device through a sample inlet port. The sample inlet port can be connected to a reagent chamber.

[0560] Surface - attachment chemical method

[0561] Although oligonucleotides are usually chemically linked, they can also be attached to a surface by indirect means such as by affinity interactions. For example, oligonucleotides can be functionalized with biotin and bound to a surface coated with avidin or streptavidin.

[0562] To immobilize polynucleotides to surfaces (such as flat surfaces), microparticles, beads, etc., various surface attachment methods and chemicals can be used. The surface can be functionalized or derivatized to facilitate attachment. Such functionalization is known in the art. For example, the surface can be functionalized with: polyhistidine tags (hexahistidine tags, 6xHis-tags, His6 tags or ), Ni-NTA, streptavidin, biotin, oligonucleotides, polynucleotides (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxyl groups, quaternary amine groups, thiol groups, azide groups, alkyne groups, DIBO, lipids, FLAG-tags (FLAG octapeptide), polynucleotide-binding proteins, peptides, proteins, antibodies or antibody fragments. The surface can be functionalized with a molecule or group that specifically binds to an anchor / scaffold polynucleotide.

[0563] Some examples of chemicals suitable for linking polynucleotides to surfaces are shown in Figure 11i andFigure 11j in

[0564] In any of the methods described herein, a scaffold polynucleotide comprising a synthetic strand can be tethered to a common surface via one or more covalent bonds, the synthetic strand comprising a primer strand portion and a portion of a support strand hybridized thereto. One or more covalent bonds can be formed between a functional group on the common surface and a functional group on the scaffold molecule. The functional group on the scaffold molecule can be, for example, an amino group, a thiol group, a phosphorothioate group, or a thioamide group. The functional group on the common surface can be bromoacetyl, optionally provided on a polyacrylamide surface derivatized with N-(5-bromoacetylpentyl)acrylamide (BRAPA).

[0565] In any method of the present invention, the scaffold polynucleotide can be attached to the surface directly or indirectly via a linker. Any suitable linker that is biocompatible and hydrophilic can be used.

[0566] The linker can be a straight-strand linker or a branched-strand linker.

[0567] The linker can include a hydrocarbon strand. The hydrocarbon strand can include from 2 to about 2000 or more carbon atoms. The hydrocarbon strand can include an alkylene group, such as C2 to about 2000 or more alkylene groups. The hydrocarbon strand can have the general formula -(CH 2 ) n -, where n is from 2 to about 2000 or higher. The hydrocarbon strand can optionally be interrupted by one or more ester groups (i.e., -C(O)-O-) or one or more amide groups (i.e., -C(O)-N(H)-).

[0568] Any linker selected from the group including the following can be used: PEG, polyacrylamide, poly(2-hydroxyethyl methacrylate), poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, such as poly(carboxybetaine methacrylate) (PCBMA), poly[N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine] (PSBMA), sugar polymers, and polypeptides.

[0569] The linker can include polyethylene glycol (PEG) having the following general formula:

[0570] -(CH 2 -CH 2 -O)n-, where n is from 1 to about 600 or greater.

[0571] The linker can include a poly(ethylene glycol) having the general formula -[(CH 2 -CH 2 -O) n -PO 2 - -O] moligoethylene glycol phosphate units, where n is from 1 to about 600 or greater, and m can be 1 - 200 or greater.

[0572] Any of the above linkers can be attached at one end of the linker to a scaffold molecule as described herein and at the other end of the linker to a first functional group, where the first functional group can provide a covalent attachment to a surface. The first functional group can be, for example, an amino group, a thiol group, a phosphorothioate group, or a thioamide group, as further described herein. The surface can be functionalized with an additional functional group to provide a covalent bond with the first functional group. The additional functional group can be, for example, 2-bromoacetamido as further described herein. Optionally, a bromoacetyl group is provided on a N-(5-bromoacetamidopentyl)acrylamide (BRAPA)-derived polyacrylamide surface. A further functional group on the surface can be a bromoacetyl group, optionally where the bromoacetyl group is provided on a N-(5-bromoacetylpentyl)acrylamide (BRAPA)-derived polyacrylamide surface, and where appropriate the first functional group can be, for example, an amino group, a mercapto group, a phosphorothioate group, or a thioamide group. The surface to which the polynucleotide is attached can include a gel. The surface includes a polyacrylamide surface, for example, about 2% polyacrylamide, preferably a polyacrylamide surface coupled to a solid support such as glass.

[0573] In any method of the invention, the scaffold polynucleotide can optionally be linked to the linker through a branched nucleotide incorporated into the scaffold polynucleotide. Any suitable branched nucleotide can be used with any suitable compatible linker.

[0574] Prior to starting the synthesis cycle of the invention, a scaffold polynucleotide can be synthesized in which one or more branched nucleotides are incorporated into the scaffold polynucleotide. The exact position at which one or more branched nucleotides are incorporated into the scaffold polynucleotide and thus can be linked to the linker can vary and can be selected as desired. The position can be, for example, at the end of the support strand and / or the synthesis strand or, for example, in the loop region where the support strand is linked to the synthesis strand in embodiments including a hairpin loop.

[0575] During the synthesis of the scaffold polynucleotide, one or more branched nucleotides can be incorporated into the scaffold polynucleotide, where a blocking group blocks the reactive group of the branched portion. The blocking group can then be removed (deprotected) prior to coupling to the branched portion of the linker, or if the linker includes multiple units, the first unit (molecule) of the linker can be removed.

[0576] During the synthesis of the scaffold polynucleotide, one or more branched nucleotides can be incorporated into the scaffold polynucleotide that has a group suitable for a subsequent "click chemistry" reaction to couple to the branched portion of the linker, or if the linker includes multiple units, to the first unit. An example of such a group is ethynyl.

[0577] Some non-limiting exemplary branched nucleotides are shown below.

[0578]

[0579]

[0580] The linker may optionally include one or more spacer molecules (units), such as an Sp9 spacer, where a first spacer unit is attached to the branched nucleotide.

[0581] The linker may include one or more other spacer groups attached to the first spacer group. For example, the linker may include multiple Sp9 spacer groups, such as. The first spacer group is attached to the branched moiety, and then one or more additional spacer groups are added in sequence to extend the spacer strand including multiple spacer units, which are linked by phosphate groups therebetween.

[0582] Some non-limiting examples of spacer units (Sp3, Sp9, and Sp13) are shown below, which may include a first spacer unit attached to the branched nucleotide, or an additional spacer unit attached to an existing spacer unit that is already attached to the branched nucleotide.

[0583]

[0584] The linker may include one or more ethylene glycol units.

[0585] The linker may include an oligonucleotide, where multiple units are nucleotides.

[0586] In the structures described above, the term 5” is used to distinguish the 5' end of the nucleotide attached to the branched moiety, where 5' has its ordinary meaning in the art. 5” means the position on the nucleotide where the linker can extend. The position of 5” may vary. The position of 5” is typically a position in the nucleobase of the nucleotide. The position of 5” in the nucleobase can vary depending on the nature of the desired branched moiety, as shown in the above structures.

[0587] Microarray

[0588] Any of the polynucleotide synthesis methods described herein can be used to fabricate polynucleotide microarrays (Trevino, V. et al., Mol. Med. 2007 13, pp527-541). Thus, an anchor or scaffold polynucleotide can be attached to multiple individually addressable reaction sites on a surface, and polynucleotides having a predefined sequence can be synthesized in situ on the microarray.

[0589] After synthesis, in each reaction zone, unique sequences can be provided for polynucleotides of a predefined sequence. Barcode sequences can be provided to the anchor or scaffold polynucleotides to facilitate identification.

[0590] In addition to the methods for synthesizing polynucleotides of a predefined sequence, microarray fabrication can be performed using techniques commonly used in the art, including those described herein. For example, known surface attachment methods and chemical methods can be used to tether the anchor or scaffold polynucleotides to a surface, including those described herein.

[0591] After synthesizing the polynucleotides of a predefined sequence, a final cleavage step can be provided to remove any unwanted polynucleotide sequences from the untethered ends.

[0592] The polynucleotides of a predefined sequence can be provided in double-stranded form at the reaction site. Alternatively, after synthesis, the double-stranded polynucleotides can be separated and one strand can be removed, leaving a single-stranded polynucleotide at the reaction site. Selective tethering of the strands can be provided to facilitate the process. For example, in a method involving a scaffold polynucleotide, the synthesized strand can be tethered to the surface and the support strand can be untethered, and vice versa. The synthesized strand can have a non-cleavable linker and the support strand can have a cleavable linker, and vice versa. Separation of the strands can be performed by conventional methods, such as heat treatment.

[0593] Assembly of synthetic polynucleotides

[0594] Polynucleotides having a predefined sequence synthesized by the methods described herein and optionally amplified by the methods described herein can be ligated to one or more other such polynucleotides to produce larger synthetic polynucleotides.

[0595] Ligation of multiple polynucleotides can be achieved by techniques well known in the art. A first polynucleotide synthesized by the methods described herein and one or more additional polynucleotides can be cleaved to produce compatible ends, and then the polynucleotides can be ligated together. Cleavage can be achieved by any suitable method. Generally, a restriction enzyme cleavage site can be generated in the polynucleotide, and then the cleavage step can be performed using a restriction enzyme to release the synthesized polynucleotide from any anchor / scaffold polynucleotide. The cleavage site can be designed as part of the anchor / scaffold polynucleotide. Alternatively, a cleavage site can be generated within the newly synthesized polynucleotide as part of a predefined nucleotide sequence.

[0596] The assembly of polynucleotides is preferably carried out using solid-phase methods. For example, after synthesis, the first polynucleotide can be singly cleaved at a suitable position away from the surface attachment site. Thus, the first polynucleotide will remain attached to the surface, and the single cleavage will generate ends that are compatible for ligation to another polynucleotide. Additional polynucleotides can be cleaved at two suitable positions to generate compatible ends for ligation to other polynucleotides at each end, while releasing the additional polynucleotides from surface attachment. The additional polynucleotides can be ligated to the first polynucleotide compatibly, thereby generating a larger immobilized polynucleotide having a predefined sequence and having ends compatible for ligation to another additional polynucleotide. Thus, iterative cycles of ligation of preselected cleaved synthetic polynucleotides can generate longer synthetic polynucleotide molecules. The order of ligation of the additional polynucleotides will be determined by the desired predefined sequence.

[0597] Thus, the assembly method of the present invention can allow the generation of synthetic polynucleotide molecules that are about one or more Mb in length.

[0598] The assembly and / or synthesis methods of the present invention can be carried out using devices known in the art. Available technologies and devices allow very small volumes of reagents to be selectively moved, dispensed, and combined with other volumes at different positions of an array, typically in the form of droplets, and electrowetting techniques such as electrowetting-on-dielectric (EWOD) can be used, as described above. Suitable electrowetting techniques and systems capable of manipulating droplets that can be used in the present invention are disclosed, for example, in US8653832, US8828336, US20140197028, and US20140202863.

[0599] Solid-phase cleavage can be achieved by providing a cleavable linker in one or both of the primer strand portion and the support strand portion hybridized thereto. The cleavable linker can be, for example, a UV-cleavable linker.

[0600] Examples of cleavage methods involving enzymatic cleavage are shown in Figure 29 The schematic shows a scaffold polynucleotide of a polynucleotide attached to a surface (shown by the black diamond structure) and including a predefined sequence. The scaffold polynucleotide includes a top and a bottom hairpin. In each case, the top hairpin can be cleaved using a cleavage step of a universal nucleotide to define a cleavage site. The bottom hairpin can be removed via a restriction endonuclease at a site engineered into the scaffold polynucleotide or engineered into the newly synthesized polynucleotide of the predefined sequence.

[0601] Thus, as described above, polynucleotides having a predefined sequence can be synthesized while immobilized on an electrowetting surface. The synthesized polynucleotides can be cleaved from the electrowetting surface and moved in the form of droplets under the influence of an electric field. The droplets can be combined at specific reaction sites on the surface where the cleaved synthesized polynucleotides can be delivered for ligation with other cleaved synthesized polynucleotides. The polynucleotides can then be ligated (e.g., by ligation). Using these techniques, populations of different polynucleotides can be synthesized and sequentially ligated according to the desired predefined sequence. Using such a system, a fully automated polynucleotide synthesis and assembly system can be designed. The system can be programmed to receive the desired sequence, supply reagents, perform synthesis cycles and subsequently assemble the desired polynucleotide according to the desired predefined sequence.

[0602] Systems and kits

[0603] The present invention also provides a polynucleotide synthesis system for carrying out any of the synthesis methods described and defined herein and any subsequent amplification and assembly steps described and defined herein.

[0604] Generally, the synthesis cycle reaction will be carried out by incorporating nucleotides of a predefined sequence into a scaffold polynucleotide molecule that is tethered to a surface in the manner described and defined herein. The surface can be any suitable surface as described and defined herein.

[0605] In one embodiment, the reaction of incorporating nucleotides of a predefined sequence into a scaffold polynucleotide molecule involves carrying out any synthesis method on the scaffold polynucleotide in a reaction zone.

[0606] The reaction zone is any region of a suitable substrate to which the scaffold polynucleotide molecule is attached and in which reagents for carrying out the synthesis method can be delivered.

[0607] In one embodiment, the reaction zone can be a single region of a surface that includes a single scaffold polynucleotide molecule, where the single scaffold polynucleotide molecule can be addressed with reagents.

[0608] In another embodiment, the reaction zone can be a single region of a surface that includes multiple scaffold polynucleotide molecules, where the scaffold polynucleotide molecules cannot be individually addressed with reagents that are isolated from one another. Thus, in such an embodiment, the multiple scaffold polynucleotide molecules in the reaction zone are exposed to the same reagents and conditions and thus can produce synthesized polynucleotide molecules having the same or substantially the same nucleotide sequence.

[0609] In one embodiment, a synthesis system for implementing any of the synthesis methods described and defined herein may include a plurality of reaction regions, where each reaction region includes one or more attached scaffold polynucleotide molecules, and where each reaction region can be individually addressed with reagents in isolation from each other reaction region. Such a system can be configured, for example, in the form of an array, such as where the reaction regions are formed on a substrate, typically a planar substrate.

[0610] Systems having a substrate that includes a single reaction region or includes a plurality of reaction regions can be included within, for example, an EWOD system or a microfluidic system, and the system is configured to deliver reagents to the reaction sites. EWOD and microfluidic systems are described in more detail herein. For example, an EWOD system can be configured to deliver reagents to the reaction sites under electrical control. A microfluidic system, such as one that includes microfabricated structures, such as a microfluidic system formed from an elastomer or similar material, can be configured to deliver reagents to the reaction sites under fluid pressure and / or suction control or by mechanical means. The reagents can be delivered by any suitable means, such as through carbon nanotubes that serve as reagent delivery conduits. Any suitable system can be envisioned.

[0611] EWOD, microfluidic, and other systems can be configured to deliver any other desired reagents to the reaction sites, such as enzymes for cleaving the synthesized double-stranded polynucleotides from the scaffold polynucleotides after synthesis, and / or reagents for cleaving the linker to release the entire scaffold polynucleotide from the substrate and / or reagents for amplifying the polynucleotide molecule or any region or portion thereof after synthesis, and / or reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules that are synthesized according to the synthesis methods of the present invention.

[0612] The present invention also provides a kit for implementing any of the synthesis methods described and defined herein. The kit can contain any desired combination of reagents for performing any of the synthesis and / or assembly methods of the present invention described and defined herein. For example, the kit can include any one or more volumes of reaction reagents, the reaction reagents including scaffold polynucleotides, volumes of reaction reagents corresponding to any one or more steps of the synthesis cycles described and defined herein, volumes of reaction reagents including nucleotides having reversible blocking groups or reversible terminator groups, volumes of reaction reagents for amplifying one or more polynucleotide molecules or any region or portion thereof after synthesis, volumes of reaction reagents for assembling larger polynucleotide molecules from smaller polynucleotide molecules synthesized according to the synthesis methods of the present invention, volumes of reaction reagents for cleaving the synthesized double-stranded polynucleotides from the scaffold polynucleotides after synthesis, and volumes of reaction reagents for cleaving one or more linkers to release the intact scaffold polynucleotide from the substrate.

[0613] Synthetic strand

[0614] In the method of synthesizing a polynucleotide or oligonucleotide described herein, including but not limited to those in Figures 1 to 5 and further in versions 1 to 5 of the synthesis method of the present invention described herein, the scaffold polynucleotide has a synthesis strand. During the synthesis cycle, each new nucleotide of a predefined sequence is incorporated into the synthesis strand. Enzymes, such as polymerase or an enzyme with terminal transferase activity, can be used to catalyze the incorporation / addition of each new nucleotide, nucleotide analogue / derivative, or non-nucleotide. The synthesis strand includes a primer strand portion and preferably includes an auxiliary strand portion.

[0615] Auxiliary strand

[0616] An auxiliary strand can be provided in the scaffold polynucleotide to facilitate the binding of the cleavage enzyme in the cleavage step. An auxiliary strand can be provided in the ligation polynucleotide to facilitate the ligation of the ligation polynucleotide to the cleaved scaffold polynucleotide in the ligation step. The auxiliary strand can be omitted, provided that an alternative means is provided to ensure the binding of the cleavage enzyme in the cleavage step and, if necessary, the ligation in the ligation step. In a preferred method of the present invention, the synthesis strand has an auxiliary strand. Preferably, the ligation polynucleotide has an auxiliary strand, and the auxiliary strand is retained in the scaffold polynucleotide in the cleavage step, as Figures 1 to 5 shown.

[0617] There are no special requirements for the parameters of the length, sequence, and structure of the auxiliary strand, provided that the auxiliary strand is suitable for facilitating the binding of the cleavage enzyme in the cleavage step.

[0618] The auxiliary strand can include nucleotides, nucleotide analogues / derivatives, and / or non-nucleotides.

[0619] Preferably, mismatches with the support strand within the sequence region of the auxiliary strand should be avoided, regions rich in GC and AT should be avoided, and in addition, regions of secondary structure, such as hairpins or bulges, should be avoided.

[0620] The length of the auxiliary strand can be 10 bases or more. Optionally, the length of the auxiliary strand can be 15 bases or more, preferably 30 bases or more. However, the length of the auxiliary strand can vary, provided that the auxiliary strand is capable of facilitating cleavage and / or ligation.

[0621] The auxiliary strand must hybridize to the corresponding region of the support strand. If the auxiliary strand can facilitate the binding of the cleavage enzyme in the cleavage step and / or the binding of the ligation enzyme in the ligation step, it is not necessary for the entire auxiliary strand to hybridize to the corresponding region of the support strand. Therefore, mismatches between the auxiliary strand and the corresponding region of the support strand can be tolerated. The auxiliary strand can be longer than the corresponding region of the support strand. The support strand can extend beyond the region corresponding to the auxiliary strand in the direction away from the primer strand. The auxiliary strand can be linked to the corresponding region of the support strand, for example, by a hairpin.

[0622] The auxiliary strand is preferably hybridized with the support strand such that the terminal nucleotide of the auxiliary strand at the cleavage site occupies the next consecutive nucleotide position in the synthetic strand relative to the terminal nucleotide of the primer strand at the cleavage site. Thus, in this configuration, there is no nucleotide position cleavage between the auxiliary strand and the primer strand. However, due to the presence of a single-strand break or cleavage, the auxiliary strand and the primer strand will be physically separated. Preferably, the terminal nucleobase of the auxiliary strand at the cleavage site hybridizes with its partner nucleotide in the support strand.

[0623] The nucleotide in the auxiliary strand that pairs with the universal nucleotide can be any suitable nucleotide. Preferably, pairings that may distort the molecular helical structure should be avoided. Preferably, cytosine serves as the partner of the universal nucleotide. In a particularly preferred embodiment, the universal nucleotide is inosine or an analogue, variant, or derivative thereof, and the partner nucleotide of the universal nucleotide in the auxiliary strand is cytosine.

[0624] Removal of auxiliary strand

[0625] Although it is preferred that the auxiliary strand remains during the synthesis step, in any of the synthetic methods of the present invention described herein (including exemplary method versions 1 to 5), a scaffold polynucleotide is provided in step (1) (i.e., in the first cycle), the scaffold polynucleotide including a synthetic strand and a support strand (101, 201, 301, 401, 501) hybridized thereto, and the synthetic strand can be provided without the auxiliary strand.

[0626] Furthermore, in any one or more cycles of the synthesis, or in all cycles of the synthesis, after the step of ligating the double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide and before the cleavage step of the scaffold polynucleotide (106, 206, 306, 406, 506), the auxiliary strand portion of the synthetic strand can be removed from the scaffold polynucleotide.

[0627] The auxiliary strand portion of the synthetic strand can be removed from the scaffold polynucleotide by any suitable method, including but not limited to: (i) heating the scaffold polynucleotide to a temperature of about 80 °C to about 95 °C and separating the auxiliary strand portion from the scaffold polynucleotide, (ii) treating the scaffold polynucleotide with a urea solution (such as 8 M urea) and separating the auxiliary strand portion from the scaffold polynucleotide, (iii) treating the scaffold polynucleotide with formamide or a formamide solution (such as 100% formamide) and separating the auxiliary strand portion from the scaffold polynucleotide, or (iv) contacting the scaffold polynucleotide with a single-stranded polynucleotide molecule that includes a nucleotide sequence region complementary to the sequence of the auxiliary strand portion, thereby competitively inhibiting the hybridization of the auxiliary strand portion with the scaffold polynucleotide.

[0628] After the step of ligating the duplexed joining polynucleotide to the cleaved scaffold polynucleotide and before the step of cleaving the scaffold polynucleotide, in a method in which the auxiliary strand portion is removed from the scaffold polynucleotide, the cleavage step will comprise cleaving the support strand in the absence of the duplexed region provided by the auxiliary strand. Any suitable enzyme can be selected to perform such a cleavage step, for example any suitable enzyme disclosed herein.

[0629] Primer strand

[0630] The primer strand portion should be suitable to allow an enzyme, such as a polymerase with terminal transferase activity or enzyme-primed synthesis, i.e., to catalyze the addition of new nucleotides at the primer strand terminus at the nick site.

[0631] The primer strand may include a sequence region that can be used to prime new polynucleotide synthesis (e.g., as indicated by the dashed lines in the structures depicted in each of Figures 1 to 5 ). The primer strand may consist of a sequence region that can act to prime new polynucleotide synthesis, and thus the entirety of the primer strand may be a sequence that can act to prime new polynucleotide synthesis as described herein.

[0632] There are no special requirements for the parameters of the length, sequence, and structure of the primer strand, provided that the primer strand is suitable to prime new polynucleotide synthesis.

[0633] The primer strand may include nucleotides, nucleotide analogs / derivatives, and / or non-nucleotides.

[0634] One skilled in the art can readily construct a primer strand capable of priming new polynucleotide synthesis. Thus, within the sequence region of the primer strand that can act to prime new polynucleotides, mismatches with the support strand should be avoided, regions rich in GC and AT should be avoided, and in addition regions of secondary structure, such as hairpins or bulges, should be avoided.

[0635] One skilled in the art can select the length of the sequence region of the primer strand that can be used to prime new polynucleotide synthesis according to the needs and the polymerase used. The length of the region can be 7 bases or more, 8 bases or more, 9 bases or more, or 10 bases or more. Optionally, the length of the region is 15 bases or more, preferably 30 bases or more.

[0636] The primer strand must hybridize to the corresponding region of the support strand. If the primer strand can prime new polynucleotide synthesis, it is not necessary for the entire primer strand to hybridize to the corresponding region of the support strand. Thus, mismatches between the primer strand and the corresponding region of the support strand can be tolerated to a certain extent. Preferably, the primer strand sequence region that can be used to prime new polynucleotide synthesis should include nucleobases complementary to the corresponding nucleobases in the support strand.

[0637] The primer strand can be longer than the corresponding region of the support strand. The support strand can extend beyond the region corresponding to the primer strand in a direction away from the auxiliary strand. The primer strand can be attached to the corresponding region of the support strand, for example, by a hairpin.

[0638] Support strand

[0639] In the methods of the invention, including but not limited to versions 1 to 5 of the synthetic methods of the invention described above, the scaffold polynucleotide comprises a support strand. The support strand hybridizes to the synthetic strand. There are no special requirements for the length, sequence, and structure parameters of the support strand, provided that the support strand is partially compatible with the primer strand and, if included, with the auxiliary strand portion of the synthetic strand, as described above.

[0640] RNA synthesis

[0641] The methods described for DNA synthesis can be adapted for RNA synthesis. In one modification, the synthetic steps described for versions 1 to 5 of the synthetic methods of the invention can be modified. Thus, in each of synthetic methods 1 to 5, the support strand of the scaffold polynucleotide is a DNA strand, as described above. The primer strand portion of the synthetic strand of the scaffold polynucleotide is an RNA strand. If present, the auxiliary strand is preferably an RNA strand. If present, the auxiliary strand can be a DNA strand.

[0642] Nucleotides can be incorporated from ribonucleoside-5'-O-triphosphates (NTPs), which can be modified to include reversible terminator groups, as described above. Preferably, 3'-O-modified ribonucleoside-5'-O-triphosphates are used. Modified nucleotides are incorporated by the action of RNA polymerase.

[0643] Thus, the description of versions 1 to 5 of the synthetic methods of the invention can be applied to RNA synthesis with the necessary modifications, but as described and modified.

[0644] Figure 31 and Figure 32 Describing the reaction flow of RNA synthesis, which is a modification of DNA synthesis methods 1 and 2 of the examples depicted in Figure 6 and Figure 7 respectively. As depicted in Figures 1 to 5 respectively, versions 1 to 5 of the methods of the invention can be modified in the same manner.

[0645] In any method applicable to RNA synthesis, the above descriptions of the support strand, primer strand, auxiliary strand, linking polynucleotide, and universal nucleotide can be applied with necessary modifications, but as modified as described. The cleavage steps and cleavage positions as previously described can be applied with necessary modifications since the support strand including the universal nucleotide is a DNA strand. In a preferred embodiment, SplintR DNA ligase is used for the ligation step.

[0646] Exemplary method

[0647] Exemplary methods for synthesizing polynucleotide or oligonucleotide molecules according to the present invention are described herein and are included in the appended claims.

[0648] In the following five exemplary methods for synthesizing polynucleotide or oligonucleotide molecules according to the present invention, reference will be made to Synthesis Method Versions 1, 2, 3, 4, and 5 and will be explained according to the reaction schemes set forth in Figure 1 、 2 、3, 4, and 5 rather than according to any of the reaction schemes set forth in Figures 6 to 10 or the same description in the Examples section. Figures 6 to 10 The reaction schemes stated in any of Figures 1 to 5 and their description in the following Examples section provide illustrative support for the methods of the present invention based on reaction schemes modified compared to the methods of the present invention. Accordingly, the reference numerals in the text below correspond to those in

[0649] In each of the exemplary methods described below, the structures described in each step may, where appropriate, be referred to specific figures by means of reference numerals. However, such references are not intended to be limited to the structures shown in the figures, and the description of the relevant structures corresponds to the entire description provided herein, including but not limited to those specifically illustrated.

[0650] Five non-limiting exemplary methods are described below, Method Versions 1 to 5 (see, for example, Figures 1 to 5 respectively). In step (1) of each of these exemplary methods, a scaffold polynucleotide is provided (see the structures depicted in step 1 of each of Figures 1 to 5 ) (101, 201, 301, 401, 501), which includes a synthetic strand (see the strand labeled "b" in the structures depicted in step 1 of each of Figures 1 to 5 ) hybridized to a complementary support strand (see the strand labeled "a" in the structures depicted in step 1 of each of Figures 1 to 5 ).

[0651] The scaffold polynucleotide is double-stranded and provides a support structure to accommodate regions of synthetic polynucleotides as it is synthesized de novo. The scaffold polynucleotide includes a synthetic strand that includes a primer strand portion (see the dotted portion of the strand labeled "b" in the structures depicted in step 1 of each of Figures 1 to 5 ) separated by a single-strand break or "nick" and a helper strand portion (see the dashed portion of the strand labeled "b" in the structures depicted in step 1 of each of Figures 1 to 5 ). As described in more detail herein, in certain of the exemplary method versions 1 to 5 and variants thereof described herein, the helper strand can be removed, for example, by denaturation, prior to the cleavage step (2).

[0652] Both the primer strand portion and the helper strand portion of the synthetic strand are provided by hybridization to a complementary support strand.

[0653] In each of the five methods, a universal nucleotide (labeled "Un" in the structure depicted in Figures 1 to 5 ) is provided in the support strand that facilitates cleavage of the scaffold polynucleotide (102, 202, 302, 402, 502). The role of the universal nucleotide will be apparent from the detailed description of each of the methods below.

[0654] Cleavage of the scaffold polynucleotide (step 2) results in the loss of the helper strand (if present immediately prior to cleavage) and the loss of the support strand that includes the universal nucleotide. Cleavage leaves the cleaved double-stranded scaffold polynucleotide in situ, which at the cleavage site includes the cleavage end of the support strand and the end of the primer strand portion of the synthetic strand that includes the pre-cleavage nick site.

[0655] The end of the primer strand portion of the synthetic strand provides a primer site for initiation of synthesis by an enzyme having transferase activity. Thus, the enzyme will act on the terminal nucleotide of the extending primer strand portion. Thus, this terminal nucleotide will generally define the 3' end of the primer strand portion, for example allowing extension by a polymerase or transferase that catalyzes extension in the 5' to 3' direction. The opposite end of the synthetic strand that includes the primer strand portion will thus generally define the 5' end of the synthetic strand, and the terminal nucleotide of the support strand adjacent to the 5' end of the synthetic strand will thus generally define the 3' end of the support strand.

[0656] The terminal nucleotide of the helper strand portion of the synthetic strand located at the site of the single-strand break will generally define the 5' end of the helper strand portion, and thus the opposite end of the helper strand portion of the synthetic strand will generally define the 3' end of the synthetic strand.

[0657] A single-strand break or “nick” between the auxiliary strand portion and the primer strand portion of the synthetic strand is typically achieved by providing the (5')-terminal nucleotide of the auxiliary strand in a form that will prevent its ligation to the primer strand during the ligation step. This can typically be achieved by providing the (5')-terminal nucleotide of the auxiliary strand without a phosphate group. The break is typically achieved by assembling the scaffold polynucleotide with separate components including: (i) a support strand; (ii) the auxiliary strand portion of the synthetic strand, which typically has a non-phosphorylated (5')-terminal nucleotide; and (iii) the synthetic strand portion including the primer strand portion. After mixing the components under suitable conditions, the scaffold polynucleotide forms upon hybridization of the separate components.

[0658] In certain methods described herein, the auxiliary strand may optionally be removed from the scaffold polynucleotide prior to the cleavage step, e.g., by denaturation and release from its previously hybridized support strand.

[0659] Cleavage causes removal of the support strand including the universal nucleotide and the auxiliary strand (if present immediately prior to cleavage) hybridized to the support strand.

[0660] In step (3) of the method described, the first nucleotide in the predetermined nucleotide sequence is incorporated into the synthetic strand by the action of an enzyme having transferase activity, such as a polymerase or a terminal nucleotidyl transferase (102, 202, 302, 402, 502). The first nucleotide bears a reversible terminator group (depicted as the small triangle of the nucleotide incorporated in step 3 of each of Figures 1 to 5 ), which prevents further extension by the enzyme. Thus, only a single nucleotide is incorporated in step (3).

[0661] Nucleotides including any suitable reversible terminator group can be used. Preferred nucleotides having a reversible terminator group are 3'-O-allyl-dNTP and / or 3'-O-azidomethyl-dNTP as described herein.

[0662] In step (4) of the method described, a protecting group removal step is carried out to remove the reversible terminator group from the incorporated first nucleotide of the predetermined nucleotide sequence (104, 204, 304, 404, 504). Alternatively, the protecting group removal step can be carried out after the ligation step (5) described below.

[0663] In step (5) of the method, a ligation step (105, 205, 305, 405, 505) is performed, in which a ligation polynucleotide is ligated to a cleaved double-stranded scaffold polynucleotide that includes a first nucleotide of a predetermined nucleotide sequence. The ligation polynucleotide includes a support strand and an auxiliary strand that hybridizes to the support strand. The ligation polynucleotide includes complementary ligation ends that are complementary to the cleaved ends of the cleaved double-stranded scaffold polynucleotide that includes the first nucleotide. The support strand of the ligation polynucleotide includes a partner nucleotide of the first nucleotide of the predetermined nucleotide sequence at the complementary ligation ends, where after ligation, the partner nucleotide is disposed opposite the first nucleotide of the predetermined nucleotide sequence to form a nucleotide pair. The support strand of the ligation polynucleotide also includes a universal nucleotide at the complementary ligation ends, which will facilitate cleavage in the next cycle of synthesis. The terminal nucleotide of the auxiliary strand of the ligation polynucleotide at the complementary ligation ends is provided such that the auxiliary strand cannot be ligated to the primer strand. This is typically achieved by providing the terminal nucleotide of the auxiliary strand without a phosphate group. Thus, after ligating the support strand of the ligation polynucleotide to the support strand of the cleaved double-stranded scaffold polynucleotide, a single-stranded break or "nick" is provided in the synthetic strand between the primer strand portion and the auxiliary strand.

[0664] After ligating the ligation polynucleotide to the cleaved double-stranded scaffold polynucleotide, the intact double-stranded scaffold polynucleotide reforms, which includes the newly incorporated nucleotide pair and the universal nucleotide for facilitating cleavage in the next synthesis cycle.

[0665] Repeated cycles of synthesis including the same steps as described above are performed to produce a synthetic polynucleotide.

[0666] The specific method is described in more detail below.

[0667] Synthesis method version 1

[0668] In a first exemplary version of the synthesis method of the present invention, a double-stranded scaffold polynucleotide is provided, which includes a universal nucleotide ([ Figure 1 step 1; 101) disposed in the support strand. In each cycle of synthesis, the scaffold polynucleotide is cleaved at a cleavage site defined by a sequence that includes the universal nucleotide ([ Figure 1 step 2; 102, 107).

[0669] Cleavage of the scaffold polynucleotide (step 2) results in the loss of the auxiliary strand (if present immediately prior to cleavage) and the loss of the support strand that includes the universal nucleotide. The cleavage leaves the cleaved double-stranded scaffold polynucleotide in situ, which includes the cleavage end of the support strand at the cleavage site and the end of the primer strand portion of the synthetic strand that includes the pre-cleavage nick site. The cleavage produces a blunt-ended cleaved double-stranded scaffold polynucleotide at the cleavage site, without overhangs.

[0670] In step (3) of the method, a first nucleotide in a predetermined nucleotide sequence is added to the end of the primer strand portion of the synthesis strand by the action of an enzyme having transferase activity, such as a polymerase or a terminal nucleotide transferase (103). The first nucleotide has a reversible terminator group that prevents further extension by the enzyme. Thus, only a single nucleotide is incorporated in step (3).

[0671] In step (4) of the method, a deprotection step is carried out to remove the terminator group from the newly incorporated nucleotide. In principle, the deprotection step can be carried out after the ligation step (5). Preferably, the deprotection step is carried out as step (4).

[0672] In step (5) of the method, a ligation polynucleotide (see the structure depicted in the upper left of the upper part of Figure 1 ) is ligated to the cleaved scaffold polynucleotide. The ligation incorporates a partner nucleotide into the scaffold polynucleotide and allows the newly incorporated nucleotide to pair with the partner nucleotide (step 5; 105 of Figure 1 ), thus completing the synthesis cycle.

[0673] In a first exemplary version of the synthesis method of the present invention, the scaffold polynucleotide is provided in step (1) (101) as described above. In the method, the universal nucleotide in the support strand of the scaffold polynucleotide is positioned opposite and pairs with the terminal nucleotide of the auxiliary strand at the single-strand break site (labeled "X" in the structure of Figure 1 ; see the structure depicted in step 1 of Figure 1 ).

[0674] In step (2) of the method, the scaffold polynucleotide is cleaved (102) at the cleavage site. The cleavage site is defined by a sequence including the universal nucleotide in the support strand. The cleavage causes a double-strand break in the scaffold polynucleotide. The cleavage of the scaffold polynucleotide (step 2) results in the loss of the auxiliary strand (if present immediately before cleavage) and the loss of the support strand including the universal nucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-strand scaffold polynucleotide includes the cleavage end of the support strand and the end of the primer strand portion of the synthesis strand including the pre-cleavage nick site at the cleavage site.

[0675] In this exemplary method, the synthesis strand already has a single-strand break or "nick", so only the support strand needs to be cleaved to provide a double-strand break in the scaffold polynucleotide.

[0676] In this exemplary method version, the cleavage results in a blunt-ended cleaved double-strand scaffold polynucleotide with no overhangs in either the synthesis strand or the support strand.

[0677] In the method, the universal nucleotide occupies position "n" in the support strand prior to the cleavage step. To obtain such blunt-ended cleaved double-stranded scaffold polynucleotides when the universal nucleotide occupies position n in the support strand, the support strand is cleaved at a specific position relative to the universal nucleotide. The support strand of the scaffold polynucleotide is cleaved between nucleotide positions n and n-1.

[0678] "n" means the nucleotide position in the support strand that is opposite to the position in the synthetic strand that will be occupied by nucleotides of a predefined sequence after addition to the end of the synthetic strand cleaved in the synthetic cycle. "n" also means the position of the nucleotide in the synthetic strand in the synthetic cycle that will be occupied by nucleotides of a predefined sequence when added to the end of the primer strand portion of the synthetic strand. Thus, in the cleavage step, the universal nucleotide at position n in the support strand is opposite to the position that will be occupied by nucleotides of a predefined sequence incorporated in the synthetic cycle. In steps (1) and (2), the position that will be occupied by nucleotides of a predefined sequence incorporated in the synthetic cycle corresponds to the terminal nucleotide of the auxiliary strand portion of the synthetic strand (shown as "X" in Figure 1 ).

[0679] "n-1" refers to the next nucleotide position in the distal direction of the auxiliary strand / proximal direction of the primer strand (the nucleotide labeled "H" at position n-1, as schematically shown in Figure 1 steps (1) and (2) of Figure 1 ) relative to the position occupied or previously occupied by the universal nucleotide. n-1 can also refer to the corresponding position in the synthetic strand. Thus, in the cleavage step, position n-1 in the support strand is opposite to the position occupied by the terminal nucleotide of the primer strand portion of the synthetic strand (i.e., the nucleotide labeled "I" at position n-1, as schematically shown in

[0680] step (2) of Figure 1 ). In the method according to version 1, the nucleotide pair occupying positions H and I can be any nucleotide pair.

[0681] After the support strand between nucleotide positions n and n-1 is cleaved, the universal nucleotide, the auxiliary strand (if present prior to cleavage), and the portion of the support strand hybridized to the auxiliary strand are removed from the remaining cleaved scaffold polynucleotide (see the structure shown in the upper right corner of Figure 1 ).

[0682] Thus, in method version 1, the universal nucleotide occupies position n in the support strand at steps (1) and (2), and the support strand is cleaved between nucleotide positions n and n-1.

[0683] Preferably, the support strand is cleaved by cleaving the phosphodiester bond between nucleotide positions n and n-1 (the first phosphodiester bond of the support strand relative to the position of the universal nucleotide in the direction of the distal side of the auxiliary strand / proximal side of the primer strand).

[0684] The support strand can be cleaved by cleaving one ester bond of the phosphodiester bond between nucleotide positions n and n-1.

[0685] Preferably, the support strand is cleaved by cleaving the first ester bond relative to nucleotide position n. This will have the effect of retaining the terminal phosphate group on the support strand of the cleaved scaffold polynucleotide at the cleavage position.

[0686] When the universal nucleotide occupies position n, any suitable mechanism can be employed to effect cleavage of the support strand between nucleotide positions n and n-1.

[0687] As described above, cleavage of the support strand between nucleotide positions n and n-1 can be effected by the action of an enzyme.

[0688] As described above, cleavage of the support strand between nucleotide positions n and n-1 can be effected as a two-step cleavage process.

[0689] The first cleavage step of the two-step cleavage process can include removing the universal nucleotide from the support strand, thus forming an abasic site at position n, and the second cleavage step can include cleaving the support strand between the abasic site, at positions n and n-1.

[0690] Example 2 describes one mechanism for cleaving the support strand at a cleavage site defined by a sequence comprising a universal nucleotide, the universal nucleotide occupying position n in the support strand. The cleavage mechanism described in Example 2 is exemplary and other mechanisms can be employed, provided that a blunt-ended cleavage of the double-stranded scaffold polynucleotide as described above is achieved.

[0691] In the first cleavage step of the two-step cleavage process, the universal nucleotide is removed from the support strand while leaving the phosphosugar backbone intact. This can be effected by the action of an enzyme that can specifically excise a single universal nucleotide from a double-stranded polynucleotide. In an exemplary cleavage method, the universal nucleotide is inosine, and inosine is excised from the support strand by the action of an enzyme, thus forming an abasic site. In an exemplary cleavage method, the enzyme is 3-methyladenine DNA glycosylase, particularly human alkyladenine DNA glycosylase (hAAG). Other enzymes, molecules or chemicals can be used, provided that an abasic site is formed. The nucleotide excision enzyme can be UDG DNA glycosylase.

[0692] In the second cleavage step, by performing a single-strand break, the two-step cleavage process support strand is cleaved at the abasic site. In an exemplary method, the support strand is cleaved by the action of a chemical such as NaOH as a base. Alternatively, an organic chemical such as N,N'-dimethylethylenediamine can be used. Alternatively, an enzyme having abasic site cleavage enzyme activity such as AP endonuclease 1, endonuclease III (type N), or endonuclease VIII can be used. Other enzymes, molecules, or chemicals can be used provided that the support strand is cleaved at the abasic site as described above.

[0693] Thus, in embodiments where the universal nucleotide is at position n of the support strand in (1) and (2) and the support strand is cleaved between positions n and n-1, the first cleavage step can be carried out with a nucleotide excision enzyme. Examples of such enzymes are 3-methyladenine DNA glycosylase such as human alkyladenine DNA glycosylase (hAAG). The second cleavage step can be carried out with a chemical such as NaOH as a base. The second step can be carried out with an organic chemical such as N,N'-dimethylethylenediamine having abasic site cleavage activity. The second step can be carried out with an enzyme such as endonuclease VIII having abasic site cleavage enzyme activity.

[0694] As described above, the cleavage of the support strand between nucleotide positions n and n-1 can be carried out as a single-step cleavage process. Examples of enzymes that can be used in any such method include endonuclease III, endonuclease VIII, formamidopyrimidine DNA glycosylase (Fpg), and 8-oxoguanine DNA glycosylase (hOGG1).

[0695] In this exemplary method version of the present invention and all versions, in order to allow the incorporation of the next nucleotide in the next synthesis cycle, the reversible terminator group must be removed from the first nucleotide (deprotection step; 104). This can be carried out at various stages of the first cycle. Generally, and preferably, it will be carried out as step (4) of the method before the ligation step (5), as shown in step 4 of Figure 1 (104). However, the deprotection step can be carried out at any step after the incorporation of the new nucleotide, for example, after the ligation step (5). Regardless of at which stage the deprotection step is carried out, the enzyme and the remaining unincorporated first nucleotide should be removed first to prevent multiple incorporations of the first nucleotide. Preferably, the enzyme and the unincorporated first nucleotide are removed before the ligation step (step (5)).

[0696] The removal of the reversible terminator group from the first nucleotide can be carried out by any suitable method. For example, the removal can be carried out by using a chemical such as tris(carboxyethyl)phosphine (TCEP).

[0697] In step (5) of the method, a double-stranded linking polynucleotide (105) is ligated to the cleaved scaffold polynucleotide. The linking polynucleotide comprises a support strand and an auxiliary strand. The linking polynucleotide further comprises complementary linking ends, which in the support strand comprise a universal nucleotide and a single overhanging nucleotide that overhangs the terminal nucleotide of the auxiliary strand and is a partner nucleotide of the first nucleotide of a predefined sequence. The linking polynucleotide further comprises a terminal nucleotide in the auxiliary strand adjacent to the overhanging end, which terminal nucleotide is configured such that it cannot be ligated to another polynucleotide strand (at the position marked "X" in the structure depicted in the upper left part of Figure 1 ). Typically, this terminal nucleotide will lack a phosphate group. Typically, as described above, this terminal nucleotide of the auxiliary strand will define the 5' end of the auxiliary strand.

[0698] The complementary linking ends are configured such that they will ligate compatibly with the overhanging ends of the cleaved scaffold polynucleotide product when subjected to suitable ligation conditions after the incorporation step (3). After ligating the support strand, the first nucleotide pairs with its partner nucleotide.

[0699] Ligation of the support strand can be carried out in any suitable manner. The ligation will result in ligation of only the support strand, maintaining a single-strand break between the first nucleotide (i.e., the primer strand portion) in the synthetic strand and the terminal nucleotide of the auxiliary strand adjacent to the first nucleotide.

[0700] Ligation can generally be carried out by an enzyme having ligase activity. For example, ligation can be carried out with T3 DNA ligase or T4 DNA ligase or a functional variant or equivalent thereof. Use of such enzymes will result in maintenance of the single-strand break in the synthetic strand because the terminal nucleotide of the auxiliary strand is provided such that it cannot act as a substrate for the ligase, e.g., due to the absence of a terminal phosphate group.

[0701] Ligation of the linking polynucleotide to the cleaved scaffold polynucleotide completes the first synthesis cycle, wherein the scaffold polynucleotide of step (1) is effectively reconstituted, except that the first nucleotide of the predefined nucleotide sequence is incorporated into the scaffold polynucleotide opposite its partner nucleotide.

[0702] In step (5) of this exemplary method (105), in the complementary ligation ends of the polynucleotides, the universal nucleotide in the support strand is positioned opposite and pairs with the terminal nucleotide of the auxiliary strand. It should be noted that in the case of ligating polynucleotides, the universal nucleotide is at position n+1 and is at the position (position n) immediately adjacent to the terminal nucleotide of the support strand. In the case of ligating polynucleotides, position n has the same meaning as described above for the scaffold polynucleotide in steps (1) and (2). Thus, in step (5), "n" refers to the nucleotide position in the support strand after ligation, which is opposite to the position in the synthetic strand, and in that synthetic cycle, that position in the synthetic strand is now occupied by the nucleotides of the predefined sequence after adding it to the end of the primer strand portion of the synthetic strand. "n" also refers to the nucleotide position in the synthetic strand after ligation, and in that synthetic cycle, that position in the synthetic strand is now occupied by the nucleotides of the predefined sequence after adding it to the end of the primer strand portion of the synthetic strand.

[0703] It should be noted that the universal nucleotide occupies position n ( Figure 1 101 and 102) in the scaffold polynucleotide during steps (1) and (2), while the universal nucleotide occupies position n+1 ( Figure 1 105) in the ligated polynucleotide in the same synthetic cycle. This is because in that synthetic cycle, the double-stranded scaffold polynucleotide cleaved in step (5) has the nucleotides of the predefined sequence after adding it, and because the ligated polynucleotide has nucleotides that pair with the nucleotides of the predefined sequence; and furthermore because as defined herein, "n" always refers to the nucleotide position in the synthetic strand that is occupied (or will be occupied) by the nucleotides of the predefined sequence incorporated in that cycle or to the nucleotide position in the support strand opposite thereto. Thus, at the end of any given cycle of synthesis, immediately after the ligation step (5), the position occupied by the universal nucleotide will be n+1 and will have moved from n, which the universal nucleotide occupied in step (1). The product of the ligation step (5) ( Figure 1The structure labeled "ligation product" in [description] will be the scaffold polynucleotide for the next synthesis cycle. When the ligation product is considered the scaffold polynucleotide (106) for the next synthesis cycle, it should be understood that the positions occupied by the universal nucleotides will now also be referred to as position n (106), rather than n+1 (105). In the next cycle (108), after adding it to the end of the cleaved synthesis strand, since the positions occupied by the universal nucleotides in the next synthesis cycle (106) will be the nucleotide positions in the support strand, which are opposite to the positions in the synthesis strand that will be occupied by the next nucleotide of the predefined sequence. Thus, in Method Version 1, the position occupied by the universal nucleotides at the start of any given cycle (101, 106, etc.) of synthesis is always referred to as position n, and the nucleotides newly incorporated into the cycle always occupy the position referred to as n.

[0704] After completing the first synthesis cycle, the second and subsequent cycles are performed using the same method steps.

[0705] As in step (2) of the first synthesis cycle of Method Version 1, in step (6) the scaffold polynucleotide is cleaved (107) at the cleavage site. The cleavage site is defined by a sequence including the universal nucleotide in the support strand. Cleavage causes a double-strand break in the scaffold polynucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-stranded scaffold polynucleotide at the cleavage site includes the cleaved end of the support strand and the end of the primer strand portion of the synthesis strand including the pre-cleavage nick site. Cleavage causes the removal of the support strand including the universal nucleotide and the auxiliary strand hybridized to the support strand (if present immediately before cleavage).

[0706] The cleavage step can be carried out as described above for step (2) of the first cycle.

[0707] In step (7), as in step (3) of the first cycle, the next nucleotide is added to the end of the cleaved primer strand portion of the synthesis strand.

[0708] In step (8), the reversible terminator group is removed from the next nucleotide (deprotection step; 109). As described above for the first cycle, this can be carried out at various stages. Generally, and preferably, it will be carried out as step (8) of the method before the ligation step (9). However, the deprotection step can be carried out at any step after incorporating the new nucleotide, for example, after the ligation step (9).

[0709] As described above regarding the first synthesis cycle, the deprotection of the reversible terminator group can be carried out in the next cycle (109) and subsequent cycles.

[0710] In step (9) of the next cycle, the double-stranded ligation polynucleotide is ligated (110) to the cleaved scaffold polynucleotide. The ligation polynucleotide for step (9) of the next and subsequent synthesis cycles can be configured, and the ligation step can be performed as described above for step (5) of the first synthesis cycle.

[0711] Repeat the synthesis cycle as many times as needed as described above to synthesize a double-stranded polynucleotide having a predetermined nucleotide sequence.

[0712] Synthesis method version 2

[0713] A second exemplary version of the synthesis method of the present invention is performed in a manner similar to the first version, except for the modified structural arrangement of the positions of the universal nucleotides relative to the positions occupied by the new nucleotides to be incorporated in a given synthesis cycle and relative to the nicks and cleavage sites.

[0714] Thus, as in version 1, cleavage of the scaffold polynucleotide (step 2, 202) in version 2 results in the loss of the auxiliary strand (if present immediately before cleavage) and the loss of the support strand including the universal nucleotides. Cleavage leaves the cleaved double-stranded scaffold polynucleotide in place, which at the cleavage site includes the cleavage end of the support strand and the primer strand portion of the synthetic strand including the pre-cleavage nick site. Cleavage produces a blunt-ended cleaved double-stranded scaffold polynucleotide at the cleavage site, without overhangs.

[0715] In step (3) of the method, the first nucleotide in the predetermined nucleotide sequence is added to the end of the primer strand portion of the synthetic strand by the action of an enzyme having transferase activity, such as a polymerase or a terminal nucleotide transferase (203). The first nucleotide has a reversible terminator group that prevents further extension by the enzyme. Thus, in step (3), only a single nucleotide is incorporated.

[0716] In step (4) of the method, a deprotection step (204) is performed to remove the terminator group from the newly incorporated nucleotide. In principle, the deprotection step can be performed after the ligation step (5). The deprotection step is preferably performed as step (4).

[0717] In step (5) of the method, the ligation polynucleotide (see the structure depicted in the upper left of Figure 2 is ligated to the cleaved scaffold polynucleotide. Ligation incorporates the partner nucleotide into the scaffold polynucleotide and allows the newly incorporated nucleotide to pair with the partner nucleotide ( Figure 2 step 5; 205), thus completing the synthesis cycle.

[0718] In a second exemplary version of the synthesis method of the present invention, the scaffold polynucleotide is provided in step (1) (201) as described above. In this method, the universal nucleotide in the support strand of the scaffold polynucleotide is positioned relative to the penultimate nucleotide of the auxiliary strand adjacent to the single-strand break site (labeled "X" in the structure of Figure 2 ), and is paired with it (see the structure depicted in step 1 of Figure 2 ).

[0719] In step (2) of the method, the scaffold polynucleotide is cleaved (202) at the cleavage site. The cleavage site is defined by a sequence including the universal nucleotide in the support strand. The cleavage causes a double-strand break in the scaffold polynucleotide. Cleavage of the scaffold polynucleotide (step 2) results in the loss of the auxiliary strand (if present immediately before cleavage) and the loss of the support strand including the universal nucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-strand scaffold polynucleotide includes at the cleavage site the cleavage end of the support strand and the end of the primer strand portion of the synthetic strand including the pre-incision site before cleavage.

[0720] The synthetic strand already has a single-strand break or "nick" in this exemplary method, so only cleavage of the support strand is required to provide a double-strand break in the scaffold polynucleotide.

[0721] In this illustrative method, the cleavage produces a blunt-ended cleaved double-strand scaffold polynucleotide with no overhangs at the ends of the synthetic or support strands.

[0722] In this method, the universal nucleotide occupies position "n + 1" in the support strand before the cleavage step. To obtain such a blunt-ended cleaved double-strand scaffold polynucleotide when the universal nucleotide occupies position n + 1 in the support strand, the support strand is cleaved at a specific position relative to the universal nucleotide. The support strand of the scaffold polynucleotide is cleaved between nucleotide positions n and n - 1.

[0723] "n" means the nucleotide position in the support strand corresponding to the position in the synthetic strand that will be occupied by a nucleotide of a predefined sequence after it is added to the end of the synthetic strand cleaved in the synthesis cycle. "n" also means the position of the nucleotide in the synthetic strand in the synthesis cycle that will be occupied by a nucleotide of a predefined sequence when it is added to the end of the primer strand portion of the synthetic strand. Thus, in the cleavage step, the universal nucleotide is at position n + 1 in the support strand, which is opposite to the position occupied by the penultimate nucleotide of the auxiliary strand adjacent to the nick site (shown at position "X" in Figure 2 ).

[0724] In steps (1) and (2), the positions occupied by the nucleotides of the predefined sequence incorporated in the synthesis cycle correspond to the terminal nucleotides of the auxiliary strand portion of the synthetic strand (inFigure 2 shown as “I” in

[0725] Relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand, “n+1” means the next nucleotide position in the support strand (as schematically shown in steps (1) and (2) of Figure 2 , the nucleotide labeled “Un” at position n+1, which occupies the position immediately adjacent to the position labeled “H” at position n in the direction distal to the primer strand). n+1 can also refer to the corresponding position in the synthetic strand (in Figure 2 , labeled “X” at position n+1 in steps (1) and (2)). Thus, in the cleavage step, position n+1 in the support strand is opposite to the position occupied by the penultimate nucleotide of the auxiliary strand adjacent to the cleavage site.

[0726] Relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand, “n-1” means the next nucleotide position in the support strand (as schematically shown in steps (1) and (2) of Figure 2 , the position labeled “J” at position n-1). n-1 can also refer to the corresponding position in the synthetic strand (i.e., as schematically shown in step (2) of Figure 2 , labeled “K” at position n-1). Thus, in the cleavage step, position n-1 in the support strand is opposite to the position occupied by the terminal nucleotide of the primer strand portion of the synthetic strand.

[0727] In the method according to version 2, the nucleotide pairs occupying positions H and I and J and K can be any nucleotide pairs.

[0728] After the support strand between nucleotide positions n and n-1 is cleaved, the universal nucleotide, the auxiliary strand (if present), and the portion of the support strand hybridized to the auxiliary strand are removed from the remaining cleaved scaffold polynucleotide (see the structure shown in the upper right corner of the upper part of Figure 2 ).

[0729] The phosphate group should continue to be attached to the terminal nucleotide of the support strand of the cleaved scaffold polynucleotide at the cleavage site (as depicted in the structure shown in the middle of the lower part of Figure 2 ). This ensures that the support strand of the ligation polynucleotide can be ligated to the support strand of the cleaved scaffold polynucleotide in the ligation step.

[0730] Thus, in method version 2, the universal nucleotide occupies position n+1 in the support strand in steps (1) and (2), and the support strand is cleaved between nucleotide positions n and n-1.

[0731] Preferably, the support strand is cleaved by cleavage of the phosphodiester bond between nucleotide positions n and n-1 (the second phosphodiester bond of the support strand, in the distal direction of the auxiliary strand / proximal direction of the primer strand, relative to the position of the universal nucleotide).

[0732] The support strand can be cleaved by cleaving one ester bond of the phosphodiester bond between nucleotide positions n and n-1.

[0733] Preferably, the support strand is cleaved by cleaving the first ester bond relative to nucleotide position n. This will have the effect of retaining the terminal phosphate group on the support strand of the cleaved scaffold polynucleotide at the cleavage position.

[0734] When the universal nucleotide occupies position n+1, any suitable mechanism can be employed to effect cleavage of the support strand between nucleotide positions n and n-1.

[0735] As described above, cleavage of the support strand between nucleotide positions n and n-1 can be effected by the action of an enzyme.

[0736] As described above, when the universal nucleotide occupies position n+1 in the support strand, cleavage of the support strand between nucleotide positions n and n-1 can be effected by the action of an enzyme, such as endonuclease V.

[0737] A mechanism for cleavage of the support strand between nucleotide positions n and n-1 at a cleavage site defined by a sequence comprising a universal nucleotide occupying position n+1 in the support strand is described in a similar manner in Example 3. The mechanism described is exemplary and other mechanisms can be employed, provided that the cleavage arrangement described above is achieved.

[0738] In this exemplary mechanism, an endonuclease is employed. In the exemplary method, the enzyme is endonuclease V. Other enzymes, molecules or chemicals can be used, provided that when the universal nucleotide occupies position n+1 in the support strand, the support strand is cleaved between nucleotide positions n and n-1.

[0739] In this exemplary method version of the present invention and all versions, in order to allow incorporation of the next nucleotide in the next synthesis cycle, the reversible terminator group must be removed from the first nucleotide (deprotection step; 204). This can be carried out at various stages of the first cycle. Generally, and preferably, it will be carried out as step (4) of the method before the ligation step (5), as Figure 2(204) as shown in step 4. However, the protecting group removal step can be carried out at any step after the incorporation of the new nucleotide, for example, after the ligation step (5). Regardless of at which stage the protecting group removal step is carried out, the enzyme and the remaining unincorporated first nucleotide should be removed first to prevent multiple incorporations of the first nucleotide. Preferably, the enzyme and the unincorporated first nucleotide are removed before the ligation step (step (5)).

[0740] The removal of the reversible terminator group from the first nucleotide can be carried out by any suitable means. For example, it can be carried out by using a chemical substance (e.g., tris(carboxyethyl)phosphine (TCEP)).

[0741] In step (5) of the method, the double-stranded ligation polynucleotide is ligated (205) to the cleaved scaffold polynucleotide. The ligation polynucleotide includes a support strand and an auxiliary strand. The ligation polynucleotide further includes complementary ligation ends, which include a universal nucleotide and a single overhanging nucleotide in the support strand, the single overhanging nucleotide overhanging the terminal nucleotide of the auxiliary strand and being the partner nucleotide of the first nucleotide of a predefined sequence. The ligation polynucleotide further includes a terminal nucleotide in the auxiliary strand adjacent to the overhanging end, which is configured such that it cannot be ligated to another polynucleotide strand (at the position marked "I" in the structure depicted in the upper left part of Figure 2 . Generally, this terminal nucleotide will lack a phosphate group. Generally, as described above, the terminal nucleotide of this auxiliary strand will define the 5' end of the auxiliary strand.

[0742] In the complementary ligation ends of the ligation polynucleotide, the universal nucleotide in the support strand is oppositely positioned (at the position marked "X" in Figure 2 step (5)) and paired with the penultimate nucleotide of the auxiliary strand. The terminal nucleotide of the auxiliary strand (at the position marked "I" at position n + 1 in Figure 2 step (5)) is paired with the nucleotide in the support strand that occupies the position between the universal nucleotide at position n + 2 and the partner nucleotide at position n (at the position marked "H" at position n + 1 in Figure 2 step (5)).

[0743] The complementary ligation ends are configured such that they will ligate compatibly with the overhanging ends of the cleaved scaffold polynucleotide product when subjected to suitable ligation conditions after the incorporation step (3). After ligating the support strand, the first nucleotide becomes paired with its partner nucleotide.

[0744] The ligation of the support strand can be carried out by any suitable means. The ligation will result in the ligation of only the support strand, maintaining a single-strand break between the first nucleotide (i.e., the primer strand portion) in the synthetic strand and the terminal nucleotide of the auxiliary strand adjacent to the first nucleotide.

[0745] Ligation can generally be carried out by an enzyme having ligase activity. For example, ligation can be carried out with T3 DNA ligase or T4 DNA ligase or a functional variant or equivalent thereof. The use of such enzymes will result in the maintenance of the single-strand break in the synthetic strand, since the terminal nucleotides of the auxiliary strand are provided in such a way that they cannot act as substrates for the ligase, for example due to the absence of terminal phosphate groups.

[0746] Ligation of the polynucleotide to the cleaved scaffold polynucleotide completes the first synthesis cycle, in which the scaffold polynucleotide of step (1) is effectively reconstituted, except that the first nucleotide of the predetermined nucleotide sequence is incorporated into the scaffold polynucleotide opposite its cognate nucleotide.

[0747] In step (5) of this exemplary method (205), in the complementary ligation ends of the ligated polynucleotide, the universal nucleotide in the support strand is positioned opposite (at the position marked "X") and paired with the penultimate nucleotide of the auxiliary strand. It should be noted that in the case of the ligated polynucleotide, the universal nucleotide is at position n+2, i.e., two positions (position n) removed from the terminal nucleotide of the support strand. In the case of the ligated polynucleotide, position n has the same meaning as described above for the scaffold polynucleotide in steps (1) and (2). Thus, in step (5), "n" refers to the nucleotide position in the support strand after ligation, which is opposite the position in the synthetic strand, and due to the cycle of synthesis, the position in the synthetic strand is now occupied by the nucleotide of the predefined sequence after addition to the end of the primer strand portion of the synthetic strand. "n" also refers to the nucleotide position in the synthetic strand after ligation, and in that synthesis cycle, the position in the synthetic strand is now occupied by the nucleotide of the predefined sequence after addition to the end of the primer strand portion of the synthetic strand.

[0748] It should be noted that the universal nucleotide occupies position n+1 in the scaffold polynucleotide during steps (1) and (2) Figure 2 at 201 and 202), while the universal nucleotide occupies position n+2 in the ligated polynucleotide in the same synthesis cycle Figure 2of 205). This is because in that synthesis cycle, the double-stranded scaffold polynucleotide cleaved in step (5) has nucleotides of a predefined sequence after its addition, and because the ligation polynucleotide has nucleotides that pair with the nucleotides of the predefined sequence; and furthermore because, as defined herein, "n" always refers to the nucleotide position in the synthetic strand that is occupied (or will be occupied) by the nucleotides of the predefined sequence incorporated into the cycle or to the nucleotide position opposite thereto in the support strand. Thus, at the end of any given cycle of synthesis, immediately following the ligation step (5), the position occupied by the universal nucleotide will be n+2 and will have moved from n+1, and the universal nucleotide will occupy that position in step (1). The product of the ligation step (5) ( Figure 2 the structure labeled "ligation product" in) will be the scaffold polynucleotide for the next synthesis cycle. When the ligation product is considered as the scaffold polynucleotide for the next synthesis cycle (206), it should be understood that the position occupied by the universal nucleotide is now again referred to as position n+1 (206) rather than n+2 (205). Thus, in method version 2, the position occupied by the universal nucleotide at the start of any given cycle of synthesis (201, 206, etc.) is always referred to as position n+1, and the newly incorporated nucleotide in the cycle always occupies the position referred to as n.

[0749] After completion of the first synthesis cycle, the second and subsequent cycles are performed using the same method steps.

[0750] As in step (2) of the first synthesis cycle of method version 2, in step (6) the scaffold polynucleotide is cleaved (207) at the cleavage site. The cleavage site is defined by a sequence including the universal nucleotide in the support strand. The cleavage produces a double-stranded break in the scaffold polynucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-stranded scaffold polynucleotide includes at the cleavage site the cleavage end of the support strand and the primer strand portion of the synthetic strand including the pre-cleavage nick site. The cleavage causes removal of the support strand including the universal nucleotide and the auxiliary strand hybridized to the support strand (if present immediately prior to cleavage).

[0751] The cleavage step can be carried out as described above for step (2) of the first cycle.

[0752] In step (7), as in step (3) of the first cycle, the next nucleotide is added to the end of the cleaved primer strand portion of the synthetic strand.

[0753] In step (8), the reversible terminator group is removed from the next nucleotide (deprotection step; 209). As described above for the first cycle, this can be performed at various stages. Generally, and preferably, it will be performed as step (8) of the method prior to the ligation step (9). However, the deprotection step can be performed at any step after incorporation of the new nucleotide, for example, after the ligation step (9).

[0754] As described above for the first synthesis cycle, deprotection of the reversible terminator group can be carried out in the next cycle (209) and subsequent cycles.

[0755] In step (9) of the next cycle, the double-stranded ligation polynucleotide is ligated (210) to the cleaved scaffold polynucleotide. The ligation polynucleotide for step (9) of the next and subsequent synthesis cycles can be configured, and the ligation step can be carried out as described above for step (5) of the first synthesis cycle.

[0756] As described above, the synthesis cycles are repeated as needed multiple times to synthesize a double-stranded polynucleotide having a predetermined nucleotide sequence.

[0757] Synthesis method version 3

[0758] A third exemplary version of the synthesis method of the present invention is performed in a manner similar to the first version, except for a modified structural arrangement of the position of the universal nucleotide relative to the cleavage site.

[0759] Thus, as in version 1, cleavage of the scaffold polynucleotide in version 3 (step 2, 302) results in the loss of the auxiliary strand (if present immediately prior to cleavage) and the loss of the support strand including the universal nucleotide. Cleavage leaves the cleaved double-stranded scaffold polynucleotide in situ, which at the cleavage site includes the cleavage end of the support strand and the primer strand portion of the synthetic strand including the pre-cleavage nick site. Cleavage produces a blunt-ended cleaved double-stranded scaffold polynucleotide with a single nucleotide overhang at the cleavage site.

[0760] In step (3) of the method, the first nucleotide in the predetermined nucleotide sequence is added to the end of the primer strand portion of the synthetic strand by the action of an enzyme having transferase activity, such as a polymerase or terminal nucleotidyl transferase (303). The first nucleotide bears a reversible terminator group that prevents further extension by the enzyme. Thus, in step (3), only a single nucleotide is incorporated.

[0761] In step (4) of the method, a deprotection step (304) is carried out to remove the terminator group from the newly incorporated nucleotide. In principle, the deprotection step can be carried out after the ligation step (5). The deprotection step is preferably carried out as step (4).

[0762] In step (5) of the method, a linking polynucleotide (see the structure depicted in the upper left portion of Figure 3 ) is linked to the cleaved scaffold polynucleotide. The linking incorporates the partner nucleotide into the scaffold polynucleotide and allows the newly incorporated nucleotide to pair with the partner nucleotide ( Figure 3 , step 5; 305), thus completing the synthesis cycle.

[0763] In a third exemplary version of the synthesis method of the present invention, the scaffold polynucleotide is provided in step (1) (301) as described above. In this method, the universal nucleotide in the support strand of the scaffold polynucleotide is positioned relative to and pairs with the terminal nucleotide of the auxiliary strand adjacent to the single-strand break site (labeled "X" in the structure of Figure 3 ) (see the structure depicted in step 1 of Figure 3 ).

[0764] In step (2) of the method, the scaffold polynucleotide is cleaved (302) at the cleavage site. The cleavage site is defined by a sequence including the universal nucleotide in the support strand. The cleavage results in a double-strand break in the scaffold polynucleotide. Cleavage of the scaffold polynucleotide (step 2) causes the loss of the auxiliary strand (if present immediately before cleavage) and the loss of the support strand including the universal nucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-strand scaffold polynucleotide includes at the cleavage site the cleavage end of the support strand and the end of the primer strand portion of the synthetic strand including the pre-cleavage nick site.

[0765] The synthetic strand already has a single-strand break or "nick" in this exemplary method, so only cleavage of the support strand is required to provide a double-strand break in the scaffold polynucleotide.

[0766] In this exemplary method version, the cleavage produces a cleaved double-strand scaffold polynucleotide with single-nucleotide overhangs. Thus, the terminal nucleotide of the primer strand portion of the synthetic strand overhangs the terminal nucleotide of the support strand.

[0767] In this method, the universal nucleotide occupies position "n" in the support strand before the cleavage step. To obtain such a cleaved double-strand scaffold polynucleotide in which the terminal nucleotide of the primer strand portion of the synthetic strand overhangs the supported terminal nucleotide when the universal nucleotide occupies position n in the support strand, the support strand of the scaffold polynucleotide is cleaved at a specific position relative to the universal nucleotide and relative to the nick site. The support strand of the scaffold polynucleotide is cleaved between nucleotide positions n - 1 and n - 2.

[0768] "n" means the nucleotide position in the support strand opposite the position in the synthetic strand, which position in the synthetic strand will be occupied by the nucleotides of a predefined sequence after the end of the synthetic strand cleaved in the synthetic cycle is added thereto. "n" also means the position of the nucleotide in the synthetic strand in the synthetic cycle that will be occupied by the nucleotides of a predefined sequence when it is added to the end of the primer strand portion of the synthetic strand. Thus, in the cleavage step, the universal nucleotide is at position n in the support strand, which position is opposite to the position occupied by the terminal nucleotide of the auxiliary strand adjacent to the cleavage site (shown at position "X" in Figure 3 ).

[0769] In steps (1) and (2), the positions occupied by the nucleotides of the predefined sequence incorporated in the synthetic cycle correspond to the terminal nucleotides of the auxiliary strand portion of the synthetic strand (shown at position "X" in Figure 3 ).

[0770] Relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand, "n-1" means the next nucleotide position in the support strand (as schematically shown in steps (1) and (2) of Figure 3 , the position marked "H" at position n-1). n-1 may also refer to the corresponding position in the synthetic strand (i.e., as schematically shown in step (2) of Figure 3 , the position marked "I" at position n-1). Thus, in the cleavage step, position n-1 in the support strand is opposite to the position occupied by the terminal nucleotide of the primer strand portion of the synthetic strand. Thus, in Figure 3 steps (1) and (2), the nucleotides at positions "J" and "K" occupy position n-2.

[0771] In the method according to version 3, the nucleotide pairs occupying positions H and I and J and K can be any nucleotide pairs.

[0772] After the support strand between nucleotide positions n-1 and n-2 is cleaved, the universal nucleotide, the auxiliary strand (if present), and the portion of the support strand hybridized to the auxiliary strand are removed from the remaining cleaved scaffold polynucleotide (see the structure shown in the upper right corner of Figure 3 ).

[0773] The phosphate group should continue to be attached to the terminal nucleotide of the support strand of the cleaved scaffold polynucleotide at the cleavage site (as depicted in the structure shown in the middle of the lower part of Figure 3 ). This ensures that the support strand of the ligation polynucleotide can be ligated to the support strand of the cleaved scaffold polynucleotide in the ligation step.

[0774] Thus, in method version 3, the universal nucleotide occupies position n in the support strand at steps (1) and (2), and the support strand is cleaved between nucleotide positions n-1 and n-2.

[0775] Preferably, the support strand is cleaved by cleaving the phosphodiester bond between nucleotide positions n-1 and n-2 (the second phosphodiester bond of the support strand relative to the position of the universal nucleotide in the direction of the distal end of the auxiliary strand / proximal end of the primer strand).

[0776] The support strand can be cleaved by cleaving one ester bond of the phosphodiester bond between nucleotide positions n-1 and n-2.

[0777] Preferably, the support strand is cleaved by cleaving the first ester bond relative to nucleotide position n-1. This will have the effect of retaining the terminal phosphate group on the support strand of the cleaved scaffold polynucleotide at the cleavage position.

[0778] When the universal nucleotide occupies position n, any suitable mechanism can be employed to effect cleavage of the support strand between nucleotide positions n-1 and n-2.

[0779] As described above, cleavage of the support strand between nucleotide positions n-1 and n-2 can be effected by the action of an enzyme.

[0780] As described above, when the universal nucleotide occupies position n in the support strand, cleavage of the support strand between nucleotide positions n-1 and n-2 can be effected by the action of an enzyme, such as endonuclease V.

[0781] In Example 3, a mechanism for cleaving the support strand between nucleotide positions n-1 and n-2 at the cleavage site defined by a sequence comprising a universal nucleotide occupying position n in the support strand is described in a similar manner. The described mechanism is exemplary and other mechanisms can be employed, provided that the arrangement of the single nucleotide overhang described above is achieved.

[0782] In this exemplary mechanism, an endonuclease is employed. In the exemplary method, the enzyme is endonuclease V. Other enzymes, molecules or chemicals can be used, provided that when the universal nucleotide occupies position n in the support strand, the support strand is cleaved between nucleotide positions n-1 and n-2.

[0783] In this exemplary method version of the present invention and all versions, in order to allow the incorporation of the next nucleotide in the next synthesis cycle, the reversible terminator group must be removed from the first nucleotide (deprotection step; 304). This can be performed at various stages of the first cycle. Generally, and preferably, it will be performed as step (4) of the method before the ligation step (5), as Figure 3(as shown in step 4 of (304). However, the protecting group removal step can be performed at any step after the incorporation of the new nucleotide, for example, after the ligation step (5). Regardless of at which stage the protecting group removal step is performed, the enzyme and the remaining unincorporated first nucleotide should be removed first to prevent multiple incorporations of the first nucleotide. Preferably, the enzyme and the unincorporated first nucleotide are removed before the ligation step (step (5)).

[0784] The reversible terminator group can be removed from the first nucleotide by any suitable means. For example, it can be removed by using a chemical such as tris(carboxyethyl)phosphine (TCEP).

[0785] In step (5) of the method, the double-stranded ligation polynucleotide is ligated (305) to the cleaved scaffold polynucleotide. The ligation polynucleotide includes a support strand and an auxiliary strand. The ligation polynucleotide further includes complementary ligation ends, which include a universal nucleotide and two overhanging nucleotides in the support strand, and the overhanging nucleotides overhang the terminal nucleotide of the auxiliary strand. The penultimate nucleotide of the support strand of the ligation polynucleotide at the complementary ligation end is the partner nucleotide of the first nucleotide of the predefined sequence. The ligation polynucleotide further includes a terminal nucleotide in the auxiliary strand adjacent to the overhanging end, and the terminal nucleotide is configured such that it cannot be ligated to another polynucleotide strand (at the position marked "X" in the structure depicted in the upper left part of Figure 3 ). Generally, this terminal nucleotide will lack a phosphate group. Generally, as described above, this terminal nucleotide of the auxiliary strand will define the 5' end of the auxiliary strand.

[0786] In the complementary ligation ends of the ligation polynucleotide, the universal nucleotide in the support strand is oppositely positioned (n + 1) with respect to the terminal nucleotide of the auxiliary strand (at the position marked "X" in Figure 3 step (5)) and pairs with it. The partner nucleotide of the first nucleotide of the predefined sequence occupies the next nucleotide position (position n) relative to the universal nucleotide in the distal direction of the auxiliary strand, and is positioned between the universal nucleotide and the terminal nucleotide of the support strand of the ligation polynucleotide (at the position marked "H" at position n - 1 in Figure 3 step (5)).

[0787] The complementary ligation ends are configured such that they will ligate compatibly with the overhanging ends of the cleaved scaffold polynucleotide product after the incorporation step (3) when suitable ligation conditions are encountered. After the support strand is ligated, the first nucleotide pairs with its partner nucleotide.

[0788] The attachment of the support strand can be effected by any suitable means. The attachment will result in an attachment of only the support strand, maintaining a single-strand break between the first nucleotide in the synthetic strand (i.e., the primer strand portion) and the terminal nucleotide of the auxiliary strand adjacent to the first nucleotide.

[0789] The attachment can generally be effected by an enzyme having ligase activity. For example, the attachment can be effected with T3 DNA ligase or T4 DNA ligase or a functional variant or equivalent thereof. The use of such an enzyme will result in the maintenance of the single-strand break in the synthetic strand, since the terminal nucleotide of the auxiliary strand is provided such that it cannot act as a substrate for the ligase, e.g., due to the absence of a terminal phosphate group.

[0790] The attachment of the polynucleotide to the cleaved scaffold polynucleotide completes the first synthesis cycle, where the scaffold polynucleotide of step (1) is effectively reconstituted, except that the first nucleotide of the predefined nucleotide sequence is incorporated into the scaffold polynucleotide opposite its cognate nucleotide.

[0791] In step (5) of this exemplary method (305), in the complementary attachment termini of the polynucleotide to be attached, the universal nucleotide in the support strand is positioned opposite (position marked "X") and paired with the terminal nucleotide of the auxiliary strand. It should be noted that in the case of the polynucleotide to be attached, the universal nucleotide is at position n+1 and at two positions (position n-1) removed from the terminal nucleotide of the support strand. In the case of the polynucleotide to be attached, position n has the same meaning as described above for the scaffold polynucleotide in steps (1) and (2). Thus, in step (5), "n" refers to the nucleotide position in the support strand after attachment, which is opposite the position in the synthetic strand, which position in the synthetic strand is now occupied by the nucleotide of the predefined sequence after addition to the end of the primer strand portion of the synthetic strand. "n" also refers to the nucleotide position in the synthetic strand after attachment, where in that synthesis cycle, that position in the synthetic strand is now occupied by the nucleotide of the predefined sequence after addition to the end of the primer strand portion of the synthetic strand.

[0792] It should be noted that the universal nucleotide occupies position n in the scaffold polynucleotide during steps (1) and (2) Figure 3 of 301 and 302), while the universal nucleotide occupies position n+1 in the polynucleotide to be attached in the same synthesis cycle Figure 3of 305). This is because in that synthesis cycle, the double-stranded scaffold polynucleotide cleaved in step (5) has nucleotides of a predefined sequence after its addition, and because the ligation polynucleotide has nucleotides that pair with the nucleotides of the predefined sequence; and furthermore because, as defined herein, "n" always refers to the nucleotide position in the synthetic strand, the nucleotide position in the synthetic strand that is occupied (or will be occupied) by the nucleotides of the predefined sequence incorporated in the cycle or refers to the nucleotide position opposite thereto in the support strand. Thus, at the end of any given cycle of synthesis, immediately following the ligation step (5), the position occupied by the universal nucleotide will be n+1 and will have moved from n, and the universal nucleotide will occupy that position in step (1). The product of the ligation step (5) ( Figure 3 the structure labeled "ligation product" in) will be the scaffold polynucleotide for the next synthesis cycle. When the ligation product is considered the scaffold polynucleotide for the next synthesis cycle (306), it should be understood that the position occupied by the universal nucleotide is now again referred to as position n (306) rather than n+1 (305). Thus, in method version 3, the position occupied by the universal nucleotide at the start of any given cycle of synthesis (301, 306, etc.) is always referred to as position n, and the newly incorporated nucleotides in the cycle always occupy the position referred to as n.

[0793] After completion of the first synthesis cycle, the second and subsequent cycles are performed using the same method steps.

[0794] As in step (2) of the first synthesis cycle of method version 3, in step (6) the scaffold polynucleotide is cleaved at the cleavage site (307). The cleavage site is defined by a sequence including the universal nucleotide in the support strand. The cleavage causes a double-strand break in the scaffold polynucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-stranded scaffold polynucleotide at the cleavage site includes the cleavage end of the support strand and the end of the primer strand portion of the synthetic strand including the pre-cleavage nick site. The cleavage causes removal of the support strand including the universal nucleotide and the auxiliary strand hybridized to the support strand (if present immediately prior to cleavage).

[0795] The cleavage step can be carried out as described above for step (2) of the first cycle.

[0796] In step (7), as in step (3) of the first cycle, the next nucleotide is added to the end of the cleaved primer strand portion of the synthetic strand.

[0797] In step (8), the reversible terminator group is removed from the next nucleotide (deprotection step; 309). As described above for the first cycle, this can be performed at various stages. Generally, and preferably, it will be performed as step (8) of the method prior to the ligation step (9). However, the deprotection step can be at any step after incorporation of the new nucleotide, for example, after the ligation step (9).

[0798] As described above with respect to the first synthesis cycle, deprotection of the reversible terminator group can be carried out in the next cycle (309) and subsequent cycles.

[0799] In step (9) of the next cycle, the double-stranded ligation polynucleotide is ligated (310) to the cleaved scaffold polynucleotide. The ligation polynucleotide of step (9) of the next and subsequent synthesis cycles can be configured, and the ligation step can be carried out as described above for step (5) of the first synthesis cycle.

[0800] As described above, the synthesis cycle is repeated as needed multiple times to synthesize a double-stranded polynucleotide having a predetermined nucleotide sequence.

[0801] Synthesis method version 4

[0802] A fourth exemplary version of the synthesis method of the present invention is performed in a manner similar to the first version, except for a modified structural arrangement of the positions of the universal nucleotides relative to the positions occupied by the new nucleotides to be incorporated in a given synthesis cycle and relative to the nicking and cleavage sites.

[0803] Thus, as in version 1, cleavage of the scaffold polynucleotide (step 2, 402) in version 4 results in the loss of the auxiliary strand (if present immediately prior to cleavage) and the loss of the support strand including the universal nucleotides. Cleavage leaves the cleaved double-stranded scaffold polynucleotide in place, which at the cleavage site includes the cleavage end of the support strand and the primer strand portion of the synthetic strand including the pre-cleavage nicking site. Cleavage produces a blunt-ended cleaved double-stranded scaffold polynucleotide at the cleavage site, without overhangs.

[0804] In step (3) of the method, the first nucleotide in the predetermined nucleotide sequence is added to the end of the primer strand portion of the synthetic strand by the action of an enzyme having transferase activity, such as a polymerase or terminal nucleotidyl transferase (403). The first nucleotide bears a reversible terminator group that prevents further extension by the enzyme. Thus, in step (3), only a single nucleotide is incorporated.

[0805] In step (4) of the method, a deprotection step (404) is carried out to remove the terminator group from the newly incorporated nucleotide. In principle, the deprotection step can be carried out after the ligation step (5). Preferably, the deprotection step is carried out as step (4).

[0806] In step (5) of the method, a ligating polynucleotide (see the structure depicted in the upper left portion of Figure 4 is ligated to the cleaved scaffold polynucleotide. Ligation incorporates the partner nucleotide into the scaffold polynucleotide and allows the newly incorporated nucleotide to pair with the partner nucleotide ( Figure 4 step 5; 405) of, thus completing the synthesis cycle.

[0807] In a fourth exemplary version of the synthesis method of the present invention, the scaffold polynucleotide is provided in step (1) (401) as described above. In this method, the universal nucleotide in the support strand of the scaffold polynucleotide is opposite and paired with the nucleotide ( Figure 4 labeled "X" in the structure of), which is two positions removed from the terminal nucleotide of the auxiliary strand ( Figure 4 labeled "K" in the structure depicted in step (1) of) adjacent to the single-strand break site.

[0808] In step (2) of the method, the scaffold polynucleotide is cleaved (402) at the cleavage site. The cleavage site is defined by a sequence including the universal nucleotide in the support strand. Cleavage causes a double-strand break in the scaffold polynucleotide. Cleavage of the scaffold polynucleotide (step 2) results in the loss of the auxiliary strand (if present immediately before cleavage) and the loss of the support strand including the universal nucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-strand scaffold polynucleotide includes the cleavage end of the support strand and the end of the primer strand portion of the synthetic strand including the pre-cleavage nick site at the cleavage site.

[0809] The synthetic strand already has a single-strand break or "nick" in this exemplary method, so only cleavage of the support strand is required to provide a double-strand break in the scaffold polynucleotide.

[0810] In this exemplary method version, cleavage produces a blunt-ended cleaved double-strand scaffold polynucleotide without overhanging ends.

[0811] In this method, the universal nucleotide occupies position "n + 2" in the support strand before the cleavage step. To obtain such a blunt-ended cleaved double-strand scaffold polynucleotide when the universal nucleotide occupies position n + 2 in the support strand, the support strand is cleaved at a specific position relative to the universal nucleotide and relative to the nick site. The support strand of the scaffold polynucleotide is cleaved between nucleotide positions n and n - 1.

[0812] "n" means the nucleotide position in the support strand that is opposite to the position in the synthetic strand, and the position in the synthetic strand will be occupied by the nucleotides of a predefined sequence after being added to the end of the synthetic strand that will be cleaved in the synthetic cycle. "n" also means the position of the nucleotide in the synthetic strand in the synthetic cycle that will be occupied by the nucleotides of a predefined sequence when it is added to the end of the primer strand portion of the synthetic strand. Thus, in the cleavage step, the universal nucleotide is at position n+2 in the support strand, which is opposite to the nucleotide that is two positions removed from the terminal nucleotide of the auxiliary strand adjacent to the single-strand break site (shown at position "X" in step (2) of Figure 4 ).

[0813] In steps (1) and (2), the positions occupied by the nucleotides of the predefined sequence incorporated in the synthetic cycle correspond to the nucleotides that are two positions removed from the terminal nucleotide of the auxiliary strand adjacent to the single-strand break site (shown at position "X" in step (2) of Figure 4 ).

[0814] Relative to position n in the direction of the proximal side of the auxiliary strand / distal side of the primer strand, "n+1" means the next nucleotide position in the support strand (as schematically shown in steps (1) and (2) of Figure 4 , the nucleotide marked as "H" at position n+1, which occupies the position adjacent to the position marked as "J" at position n in the direction of the distal side of the primer strand). n+1 can also refer to the corresponding position in the synthetic strand (in steps (1) and (2) of Figure 4 , marked as "I" at position n+1). Thus, in the cleavage step, position n+1 in the support strand is opposite to the position occupied by the penultimate nucleotide of the auxiliary strand adjacent to the nicking site.

[0815] Relative to position n in the direction of the proximal side of the auxiliary strand / distal side of the primer strand, "n-1" means the next nucleotide position in the support strand (as schematically shown in steps (1) and (2) of Figure 4 , the position marked as "L" at position n-1). n-1 can also refer to the corresponding position in the synthetic strand (i.e., as schematically shown in steps (1) and (2) of Figure 4 , marked as "M" at position n-1). Thus, in the cleavage step, position n-1 in the support strand is opposite to the position occupied by the terminal nucleotide of the primer strand portion of the synthetic strand. Thus, in steps (1) and (2) of Figure 4 , the nucleotides at positions "J" and "K" occupy position n.

[0816] In the method according to version 4, the nucleotide pairs occupying positions H and I; J and K; and L and M can be any nucleotide pairs.

[0817] After the support strand between nucleotide positions n and n-1 is cleaved, the universal nucleotide, the auxiliary strand (if present), and the portion of the support strand hybridized to the auxiliary strand are removed from the remaining cleaved scaffold polynucleotide (see the structure shown in the upper right corner of the upper part of Figure 4 ).

[0818] The phosphate group should continue to be attached to the terminal nucleotide of the support strand of the cleaved scaffold polynucleotide at the cleavage site (as depicted in the structure shown in the middle of the lower part of Figure 4 ). This ensures that the support strand of the ligation polynucleotide can be ligated to the support strand of the cleaved scaffold polynucleotide during the ligation step.

[0819] Thus, in method version 4, the universal nucleotide occupies position n+2 in the support strand in steps (1) and (2), and the support strand is cleaved between nucleotide positions n and n-1.

[0820] Preferably, the support strand is cleaved by cleavage of the phosphodiester bond between nucleotide positions n and n-1 (the third phosphodiester bond of the support strand in the distal direction of the auxiliary strand / proximal direction of the primer strand, relative to the position of the universal nucleotide).

[0821] The support strand can be cleaved by cleaving one ester bond of the phosphodiester bond between nucleotide positions n and n-1.

[0822] Preferably, the support strand is cleaved by cleaving the first ester bond relative to nucleotide position n. This will have the effect of retaining the terminal phosphate group on the support strand of the cleaved scaffold polynucleotide at the cleavage position.

[0823] When the universal nucleotide occupies position n+2, any suitable mechanism can be employed to effect cleavage of the support strand between nucleotide positions n and n-1.

[0824] As described above, cleavage of the support strand between nucleotide positions n and n-1 can be effected by the action of an enzyme.

[0825] In this exemplary method version of the present invention and all versions, in order to allow incorporation of the next nucleotide in the next synthesis cycle, the reversible terminator group must be removed from the first nucleotide (deprotection step; 404). This can be carried out at various stages of the first cycle. Generally, and preferably, it will be carried out as step (4) of the method before the ligation step (5), as Figure 4As shown in step 4 of (404). However, the protecting group removal step can be carried out at any step after the incorporation of the new nucleotide, for example, after the ligation step (5). Regardless of at which stage the protecting group removal step is carried out, the enzyme and the remaining unincorporated first nucleotide should be removed first to prevent multiple incorporations of the first nucleotide. Preferably, the enzyme and the unincorporated first nucleotide are removed before the ligation step (step (5)).

[0826] The reversible terminator group can be removed from the first nucleotide by any suitable method. For example, it can be removed by using a chemical such as tris(carboxyethyl)phosphine (TCEP).

[0827] In step (5) of the method, the double-stranded ligation polynucleotide is ligated (405) to the cleaved scaffold polynucleotide. The ligation polynucleotide includes a support strand and an auxiliary strand. The ligation polynucleotide further includes complementary ligation ends, which include a universal nucleotide and a single overhanging nucleotide in the support strand, and the overhanging nucleotide overhangs the terminal nucleotide of the auxiliary strand. The overhanging nucleotide is the partner nucleotide of the first nucleotide of the predefined sequence. The ligation polynucleotide further includes a terminal nucleotide in the auxiliary strand adjacent to the overhanging end, which is configured such that it cannot be ligated to another polynucleotide strand (at the position marked "K" in the structure depicted in the upper left part of Figure 4 . Generally, this terminal nucleotide will lack a phosphate group. Generally, as described above, this terminal nucleotide of the auxiliary strand will define the 5' end of the auxiliary strand.

[0828] In the complementary ligation ends of the ligation polynucleotide, the universal nucleotide in the support strand is positioned opposite to and pairs with the nucleotide (n + 3) (at the position marked "X" in Figure 4 step (5)), which is two positions removed from the terminal nucleotide of the auxiliary strand. The partner nucleotide of the first nucleotide of the predefined sequence occupies the position of the terminal nucleotide of the support strand of the ligation polynucleotide (position n). The nucleotide removed one position from the terminal nucleotide of the support strand of the ligation polynucleotide is marked as "J" at position n + 1 in Figure 4 step (5). The nucleotide removed two positions from the terminal nucleotide of the support strand of the ligation polynucleotide is marked as "H" at position n + 2 in Figure 4 step (5).

[0829] The complementary ligation ends are configured such that they will ligate compatibly with the overhanging ends of the cleaved scaffold polynucleotide product after the incorporation step (3) when suitable ligation conditions are experienced. After the support strand is ligated, the first nucleotide pairs with the partner nucleotide.

[0830] The attachment of the support strand can be effected in any suitable manner. The attachment will result in the attachment of only the support strand, maintaining a single-strand break between the first nucleotide of the synthetic strand (i.e., the primer strand portion) and the terminal nucleotide of the auxiliary strand adjacent to the first nucleotide.

[0831] The attachment can generally be effected by an enzyme having ligase activity. For example, the attachment can be effected with T3 DNA ligase or T4 DNA ligase or a functional variant or equivalent thereof. The use of such an enzyme will result in the maintenance of the single-strand break in the synthetic strand, since the terminal nucleotide of the auxiliary strand is provided such that it cannot act as a substrate for the ligase, for example due to the absence of a terminal phosphate group.

[0832] The attachment of the polynucleotide to the cleaved scaffold polynucleotide completes the first synthesis cycle, wherein the scaffold polynucleotide of step (1) is effectively reconstituted, except that the first nucleotide of the predetermined nucleotide sequence is incorporated into the scaffold polynucleotide opposite its partner nucleotide.

[0833] In step (5) of this exemplary method (305), in the complementary attachment ends of the polynucleotide to be attached, the universal nucleotide in the support strand is positioned opposite a nucleotide that is two positions removed from the terminal nucleotide of the auxiliary strand adjacent to the single-strand break site (shown at position "X" in Figure 4 step (5)). It should be noted that in the case of the polynucleotide to be attached, the universal nucleotide is at position n+3 and three positions are removed from the terminal nucleotide of the support strand (position n). In the case of the polynucleotide to be attached, position n has the same meaning as described above for the scaffold polynucleotide in steps (1) and (2). Thus, in step (5), "n" refers to the nucleotide position in the support strand after attachment, which is opposite the position in the synthetic strand, and due to the cycle of synthesis, that position in the synthetic strand is now occupied by a nucleotide of the predefined sequence after addition to the end of the primer strand portion of the synthetic strand. "n" also refers to the nucleotide position in the synthetic strand after attachment, and in that synthesis cycle, that position in the synthetic strand is now occupied by a nucleotide of the predefined sequence after addition to the end of the primer strand portion of the synthetic strand.

[0834] It should be noted that the universal nucleotide occupies position n+2 in the scaffold polynucleotide during steps (1) and (2) ( Figure 4 401 and 402 of Figure 4of 405). This is because in that synthesis cycle, the double-stranded scaffold polynucleotide cleaved in step (5) has nucleotides of a predefined sequence after its addition, and because the ligation polynucleotide has nucleotides that pair with the nucleotides of the predefined sequence; and furthermore because, as defined herein, "n" always refers to the nucleotide position in the synthetic strand that is occupied (or will be occupied) by the nucleotides of the predefined sequence incorporated into the cycle or to the nucleotide position in the support strand opposite thereto. Thus, at the end of any given cycle of synthesis, immediately following the ligation step (5), the position occupied by the universal nucleotide will be n+3 and will have moved from n+2, and the universal nucleotide will occupy that position in step (1). The product of the ligation step (5) ( Figure 4 the structure labeled "ligation product" in) will be the scaffold polynucleotide for the next synthesis cycle. When the ligation product is considered as the scaffold polynucleotide for the next synthesis cycle (406), it should be understood that the position occupied by the universal nucleotide is now again referred to as position n+2 (406) rather than n+3 (405). Thus, in method version 4, the position occupied by the universal nucleotide at the start of any given cycle of synthesis (401, 406, etc.) is always referred to as position n+2, and the newly incorporated nucleotide in the cycle always occupies the position referred to as n.

[0835] After completion of the first synthesis cycle, the second and subsequent cycles are performed using the same method steps.

[0836] As in step (2) of the first synthesis cycle of method version 4, in step (6) the scaffold polynucleotide is cleaved at the cleavage site (407). The cleavage site is defined by a sequence including the universal nucleotide in the support strand. The cleavage creates a double-stranded break in the scaffold polynucleotide. The cleavage leaves the scaffold polynucleotide in place, and the cleaved double-stranded scaffold polynucleotide includes at the cleavage site the cleavage end of the support strand and the primer strand portion of the synthetic strand including the pre-incision site. The cleavage causes removal of the support strand including the universal nucleotide and the auxiliary strand hybridized to the support strand (if present immediately prior to cleavage).

[0837] The cleavage step can be carried out as described above for step (2) of the first cycle.

[0838] In step (7), as in step (3) of the first cycle, the next nucleotide is added to the end of the cleaved primer strand portion of the synthetic strand.

[0839] In step (8), the reversible terminator group is removed from the next nucleotide (deprotection step; 409). As described above for the first cycle, this can be performed at various stages. Generally, and preferably, it will be performed as step (8) of the method prior to the ligation step (9). However, the deprotection step can be at any step after incorporation of the new nucleotide, for example, after the ligation step (9).

[0840] As described above with respect to the first synthesis cycle, deprotection of the reversible terminator group can be carried out in the next cycle (409) and subsequent cycles.

[0841] In step (9) of the next cycle, the double-stranded ligation polynucleotide is ligated (410) to the cleaved scaffold polynucleotide. The ligation polynucleotide of step (9) of the next and subsequent synthesis cycles can be configured and the ligation step can be carried out as described above for step (5) of the first synthesis cycle.

[0842] As described above, the synthesis cycle is repeated as many times as necessary to synthesize a double-stranded polynucleotide having a predetermined nucleotide sequence.

[0843] Variant method based on synthesis method version 4

[0844] It should be understood that synthesis method version 4 is a variant of synthesis method version 2. Both methods require cleavage of the support strand of the scaffold polynucleotide between positions n and n - 1. Prior to and during the cleavage steps (steps 2 and 6) of the first and second cycles in version 2, a universal nucleotide occupies position n + 1 in the support strand of the scaffold polynucleotide. Compared to prior to and during the cleavage steps (steps 2 and 6) of the first and second cycles in version 4, a universal nucleotide occupies position n + 2 in the support strand of the scaffold polynucleotide. Thus, synthesis method version 4 is the same as synthesis method version 2, except that in synthesis method version 4, the universal nucleotide occupies a position further removed from position n in the direction distal to the primer strand portion.

[0845] Other variants of synthesis method version 4 are envisioned, where the support strand of the scaffold polynucleotide is cleaved between positions n and n - 1, and where in each other variant method, the universal nucleotide occupies a position incrementally further removed from position n in the direction distal to the primer strand portion, starting at position n + 3 and increasing incrementally according to the formula n + 3 + x, where x is an integer between 1 and 10 or greater.

[0846] Accordingly, there is provided an "n + 3" variant of synthesis method version 4, where the method is carried out in the same manner as synthesis method version 4 described above, except for the following variant.

[0847] Before and during the cleavage step of the first cycle (Steps 1 and 2), the universal nucleotide is changed to occupy position n+3 in the support strand of the scaffold polynucleotide, where n+3 is the third nucleotide position in the support strand in the direction distal to the proximal / primer strand portion of the auxiliary strand; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1.

[0848] In the ligation step of the first cycle (Step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide is changed to occupy position n+4 in the support strand and pairs with the nucleotide that is 3 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+4 is position 4 in the support strand with respect to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand.

[0849] Before and during the cleavage step of the second cycle (Steps 5 and 6) and during the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+3 in the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved at positions n and n-1.

[0850] Finally, in the ligation step of the second cycle (Step 9) and in the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+4 in the support strand and pairs with the nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end.

[0851] In addition to the "n+3" method described above, a "n+3+x" variant of Synthesis Method Version 4 is provided, where the method is performed in the same manner as Synthesis Method Version 4 described above, except for the following variant.

[0852] Before and during the cleavage step of the first cycle (Steps 1 and 2), the universal nucleotide is changed to occupy position n+3+x in the support strand of the scaffold polynucleotide, where n+3 is the third nucleotide position in the support strand in the direction distal to the proximal / primer strand portion of the auxiliary strand; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1.

[0853] In the ligation step of the first cycle (Step 5), the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide is changed to occupy position n+4+x in the support strand and pairs with the nucleotide that is 3+x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+4 is position 4 in the support strand with respect to position n in the direction distal to the proximal / primer strand portion of the auxiliary strand.

[0854] Before and during the cleavage step of the second cycle (step 6) and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+3+x of the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved at positions n and n-1;

[0855] Finally, in the ligation step of the second cycle (step 9) and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+4+x in the support strand and pairs with the nucleotide that removes 2+x positions from the terminal nucleotide of the auxiliary strand at the complementary ligation end.

[0856] In all of these methods, x is an integer between 1 and 10 or greater, and wherein in steps (1), (2), (5), (6) and (9), x is the same integer.

[0857] As in synthesis method 4, in the case of the variant method based on version 4, it should be noted that the universal nucleotide occupies position n+3 (or n+3+x, depending on the specific variant method) in the scaffold polynucleotide during steps (1) and (2), while the universal nucleotide occupies position n+4 (or n+4+x, depending on the specific variant method) in the ligation polynucleotide in the same synthesis cycle. This is because in that cycle of synthesis, the double-stranded scaffold polynucleotide cleaved in step (5) has nucleotides of a predefined sequence afte...

Claims

1. An in vitro method for synthesizing a double-stranded polynucleotide having a predefined sequence, the method comprising performing synthesis cycles, wherein each cycle comprises cleaving the double-stranded polynucleotide and extending the cleaved double-stranded polynucleotide by incorporating nucleotide pairs, wherein the end of the first strand of the cleaved double-stranded polynucleotide is extended by adding nucleotides of the predefined sequence, and the end of the second strand of the cleaved double-stranded polynucleotide hybridized to the first strand is extended by adding partner nucleotides, thereby forming nucleotide pairs with the incorporated nucleotides of the first strand; wherein each cycle comprises extending the first strand by adding the nucleotides of the predefined sequence together with a reversibly blocking group attached thereto, and then extending the second strand, wherein the reversibly blocking group is removed before or after the second strand is extended, further wherein in each cycle, the nucleotides are incorporated into a cleaved scaffold polynucleotide; and wherein each cycle comprises: (1) providing a scaffold polynucleotide; (2) cleaving the scaffold polynucleotide at a cleavage site; (3) adding nucleotides of the predefined sequence to the cleaved scaffold polynucleotide by the action of a nucleotide transferase or polymerase, the nucleotides comprising reversibly terminating groups that prevent further extension by the enzyme; (4) removing the reversibly terminating groups from the nucleotides of the predefined sequence; and (5) ligating a ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising partner nucleotides of the nucleotides of the predefined sequence, wherein after ligation, the nucleotides of the predefined sequence pair with the partner nucleotides.

2. The method according to claim 1, wherein step (1) comprises providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion, and the support strand comprises universal nucleotides; wherein step (2) comprises cleaving the scaffold polynucleotide at a cleavage site defined by a sequence comprising the universal nucleotides in the support strand, wherein cleavage comprises cleaving the support strand and removing the universal nucleotides from the scaffold polynucleotide; and wherein in step (5), the ligation polynucleotide comprises a support strand that comprises the partner nucleotides and universal nucleotides that define a cleavage site for the next cycle, and wherein the ligation polynucleotide is ligated to the support strand of the cleaved scaffold polynucleotide, thereby forming the nucleotide pairs.

3. The method according to claim 1, the method comprises: (1) providing a scaffold polynucleotide comprising a synthetic strand and a support strand hybridized thereto, wherein the synthetic strand comprises a primer strand portion and an auxiliary strand portion separated by a single-strand break, and the support strand comprises universal nucleotides; (2) Cleave the scaffold polynucleotide at a cleavage site defined by a sequence comprising the universal nucleotide in the support strand, wherein cleavage comprises cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double-stranded scaffold polynucleotide, the cleaved double-stranded scaffold polynucleotide comprising a support strand and a synthetic strand comprising the primer strand portion; (3) Extend the end of the primer strand portion of the synthetic strand of the cleaved double-stranded scaffold polynucleotide together with the first nucleotide of the predefined sequence by the action of a nucleotide transferase or polymerase, the first nucleotide comprising a reversible terminator group that blocks further extension by the enzyme; (4) Remove the reversible terminator group from the first nucleotide; (5) Ligate a double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising a support strand and a helper strand hybridized thereto and further comprising complementary ligation ends, the ligation ends comprising: (i) a universal nucleotide in the support strand and a partner nucleotide of the first nucleotide, wherein the partner nucleotide of the first nucleotide protrudes from the helper strand; and (ii) a terminal nucleotide lacking a phosphate group in the helper strand; wherein after ligating the support strand, the first nucleotide pairs with the partner nucleotide; (6) Cleave the scaffold polynucleotide at a cleavage site defined by a sequence comprising the universal nucleotide in the support strand, wherein cleavage comprises cleaving the support strand and removing the universal nucleotide from the scaffold polynucleotide to provide a cleaved double-stranded scaffold polynucleotide, the cleaved double-stranded scaffold polynucleotide comprising a support strand and a synthetic strand comprising a primer strand portion; (7) Extend the end of the primer strand portion of the synthetic strand of the cleaved double-stranded scaffold polynucleotide together with the next nucleotide of the predefined nucleotide sequence by the action of a nucleotide transferase or polymerase, the next nucleotide comprising a reversible terminator group that blocks further extension by the enzyme; (8) Remove the reversible terminator group from the next nucleotide; and (9) Ligate a double-stranded ligation polynucleotide to the cleaved scaffold polynucleotide, the ligation polynucleotide comprising a support strand and a helper strand hybridized thereto and further comprising complementary ligation ends, the ligation ends comprising: (i) a universal nucleotide in the support strand and a partner nucleotide of the next nucleotide, wherein the partner nucleotide of the next nucleotide protrudes from the helper strand; and (ii) a terminal nucleotide lacking a phosphate group in the helper strand; wherein after ligating the support strand, the next nucleotide pairs with the partner nucleotide; (10) Repeat steps 6 to 9 multiple times to provide the double-stranded polynucleotide having the predefined nucleotide sequence.

4. The method according to claim 3, wherein: a) Before the cleavage step (step 2) of the first cycle and during said cleavage step, said universal nucleotide occupies position n in said support strand of said scaffold polynucleotide, where position n is the nucleotide position in said support strand opposite to the position in said synthesis strand, and in said cycle, after adding said first nucleotide of said predefined sequence to the end of said primer strand portion, the position in said synthesis strand will be occupied by said first nucleotide, wherein the nucleotide at position n in said support strand is opposite to and pairs with the terminal nucleotide of said auxiliary strand; b) In said cleavage step (step 2) of said first cycle, said support strand of said scaffold polynucleotide is cleaved between position n and n - 1, where position n - 1 is the next nucleotide position in said support strand relative to position n in the direction of the distal side of said auxiliary strand / proximal side of said primer strand portion; c) In said ligation step (step 5) of said first cycle, the complementary ligation ends of said ligation polynucleotide are structured such that the partner nucleotide for said first nucleotide of said predefined sequence is the terminal nucleotide of said support strand and occupies position n, where said universal nucleotide occupies position n + 1 in said support strand and pairs with the terminal nucleotide of said auxiliary strand, where position n is the nucleotide position that will be opposite to said first nucleotide of said predefined sequence after ligating said ligation polynucleotide to said cleaved scaffold polynucleotide in step 5; d) In said cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles: i. Said universal nucleotide occupies position n in said support strand of said scaffold polynucleotide, where position n is the nucleotide position in said support strand opposite to the position in said synthesis strand, and in said cycle, after adding the next nucleotide of said predefined sequence to the end of said primer strand portion, the position in said synthesis strand will be occupied by said next nucleotide; and ii. Said support strand of said scaffold polynucleotide is cleaved between position n and n - 1, where position n - 1 is the next nucleotide position in said support strand relative to position n in the direction of the distal side of said auxiliary strand / proximal side of said primer strand portion; and e) In said ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary joining ends of said ligation polynucleotide are structured such that the partner nucleotide for the next nucleotide of said predefined sequence in said cycle is the terminal nucleotide in said support strand and occupies position n, and said universal nucleotide occupies position n + 1 in said support strand and pairs with the terminal nucleotide of said auxiliary strand; where position n is the nucleotide position that will be opposite to the next nucleotide of said predefined sequence incorporated in said cycle after ligating said ligation polynucleotide to said cleaved scaffold polynucleotide (step 7).

5. The method according to claim 3, wherein: a) Before and during the cleavage step (step 2) of the first cycle, the universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite the position in the synthesis strand, and in the cycle, after adding the first nucleotide of the predefined sequence to the end of the primer strand portion, the position in the synthesis strand will be occupied by the first nucleotide, where the nucleotide at position n in the support strand is opposite and pairs with the terminal nucleotide of the auxiliary strand, and where position n+1 is the next nucleotide position in the support strand relative to position n in the direction of the proximal side of the auxiliary strand / the distal side of the primer strand portion; b) In the cleavage step (step 2) of the first cycle, the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where position n-1 is the next nucleotide position in the support strand relative to position n in the direction of the distal side of the auxiliary strand / the proximal side of the primer strand portion; c) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the terminal nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; where position n is the nucleotide position opposite the first nucleotide of the predefined sequence after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide in step 5, and position n+2 is the second position in the support strand relative to position n in the direction of the proximal side of the auxiliary strand / the distal side of the primer strand portion; d) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles: i. The universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite the position in the synthesis strand, and in the cycle, after adding the next nucleotide of the predefined sequence to the end of the primer strand portion, the position in the synthesis strand will be occupied by the next nucleotide; and where position n+1 is the next nucleotide position in the support strand relative to position n in the direction of the proximal side of the auxiliary strand / the distal side of the primer strand portion; And ii. The support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where position n-1 is the next nucleotide position in the support strand relative to position n in the direction of the distal side of the auxiliary strand / the proximal side of the primer strand portion; and e) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in the cycle is the terminal nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; wherein position n is the nucleotide position that will be opposite the next nucleotide of the predefined sequence incorporated in the cycle after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide (step 7), and position n+2 is the second position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion.

6. The method according to claim 3, wherein: a) Before and at the cleavage step (step 2) of the first cycle, the universal nucleotide occupies position n in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite the position in the synthesis strand that will be occupied by the first nucleotide of the predefined sequence in the cycle after adding the first nucleotide of the predefined sequence to the end of the primer strand portion, wherein the nucleotide at position n in the support strand is opposite and pairs with the terminal nucleotide of the auxiliary strand; b) In the cleavage step (step 2) of the first cycle, the support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, where positions n-1 and n-2 are the next nucleotide position and the subsequent nucleotide position in the support strand, respectively, relative to position n in the direction distal to the auxiliary strand / proximal to the primer strand portion; c) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the penultimate nucleotide of the support strand and occupies position n, where the universal nucleotide occupies position n+1 in the support strand and pairs with the terminal nucleotide of the auxiliary strand; wherein position n is the nucleotide position that will be opposite the first nucleotide of the predefined sequence after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide in step 5, and wherein position n+1 is the next nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; d) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles: i. The universal nucleotide occupies position n in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand that is opposite the position in the synthesis strand which, in the cycle, will be occupied by the next nucleotide of the predefined sequence after the next nucleotide of the predefined sequence is added to the end of the primer strand portion; and ii. The support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, where positions n-1 and n-2 are the next nucleotide position and the subsequent nucleotide position in the support strand, respectively, relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; and e) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in the cycle is the penultimate nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+1 in the support strand and pairs with the terminal nucleotide of the auxiliary strand; where position n is the nucleotide position that will be opposite the next nucleotide of the predefined sequence incorporated in the cycle after the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide (step 7), and relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion, position n+1 is the next nucleotide position in the support strand.

7. The method according to claim 3, wherein: a) Before and during the cleavage step (step 2) of the first cycle, the universal nucleotide occupies position n+2 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand that is opposite the position in the synthesis strand which, in the cycle, will be occupied by the first nucleotide of the predefined sequence after the first nucleotide of the predefined sequence is added to the end of the primer strand portion, where the nucleotide at position n in the support strand is opposite and pairs with the terminal nucleotide of the auxiliary strand, and where relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion, position n+2 is the second nucleotide position in the support strand; b) In the cleavage step (step 2) of the first cycle, the support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where position n-1 is the next nucleotide position in the support strand relative to position n in the direction distal to the auxiliary strand / proximal to the primer strand portion; c) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the terminal nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n+3 in the support strand and pairs with a nucleotide that is two positions removed from the terminal nucleotide of the auxiliary strand in the direction distal to the primer strand portion; where position n is the nucleotide position opposite the first nucleotide of the predefined sequence after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide in step 5, and relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion, position n+3 is the third position in the support strand; d) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles: i. The universal nucleotide occupies position n+2 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite the position in the synthesis strand that, in the cycle, will be occupied by the next nucleotide of the predefined sequence after adding the next nucleotide of the predefined sequence to the end of the primer strand portion; and where relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion, n+2 is the second nucleotide position in the support strand; and ii. The support strand of the scaffold polynucleotide is cleaved between positions n and n-1, where relative to position n in the direction distal to the auxiliary strand / proximal to the primer strand portion, n-1 is the next nucleotide position in the support strand; and e) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in the cycle is the terminal nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+3 in the support strand and pairs with a nucleotide that is two positions removed from the terminal nucleotide of the auxiliary strand in the direction distal to the primer strand portion; where position n is the nucleotide position that will be opposite the next nucleotide of the predefined sequence incorporated in the cycle after ligating the ligation polynucleotide to the cleaved scaffold polynucleotide (step 7), and relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion, position n+3 is the third position in the support strand.

8. The method according to claim 7, wherein: (i) In the cleavage step (step 2) of the first cycle, the universal nucleotide is changed to occupy position n+3 in the support strand of the scaffold polynucleotide, where n+3 is the third nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1; (ii) In the ligation step (step 5) of the first cycle, the complementary ligation end of the ligation polynucleotide is structured such that the universal nucleotide is changed to occupy position n+4 in the support strand and pairs with a nucleotide that is 3 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+4 is position 4 in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; (iii) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+3 in the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1; and (iv) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation end of the ligation polynucleotide is structured such that the universal nucleotide occupies position n+4 in the support strand and pairs with a nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end.

9. The method according to claim 3, wherein: (i) In the cleavage step (step 2) of the first cycle, the universal nucleotide is changed to occupy position n+3+x in the support strand of the scaffold polynucleotide, where n+3 is the third nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1; (ii) In the ligation step (step 5) of the first cycle, the complementary ligation end of the ligation polynucleotide is structured such that the universal nucleotide is changed to occupy position n+4+x in the support strand and pairs with a nucleotide that is 3+x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+4 is position 4 in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; (iii) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+3+x in the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1; (iv) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+4+x in the support strand of the scaffold polynucleotide and pairs with the nucleotide that removes 2+x positions from the terminal nucleotide of the auxiliary strand at the complementary ligation end; and (v) where x is an integer between 1 and 10 or greater, and where x is the same integer in steps (2), (5), (6), and (9).

10. The method according to claim 3, wherein: a) Before and during the cleavage step (step 2) of the first cycle, the universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite the position in the synthesis strand, and in the cycle, after the first nucleotide of the predefined sequence is added to the end of the primer strand portion, the position in the synthesis strand will be occupied by the first nucleotide, where the nucleotide at position n in the support strand is opposite and pairs with the terminal nucleotide of the auxiliary strand, and where position n+1 is the next nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; b) In the cleavage step (step 2) of the first cycle, the support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, where positions n-1 and n-2 are the next nucleotide position and the subsequent nucleotide position in the support strand, respectively, relative to position n in the direction distal to the auxiliary strand / proximal to the primer strand portion; c) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the first nucleotide of the predefined sequence is the penultimate nucleotide in the support strand and occupies position n, where the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; where position n is the nucleotide position opposite the first nucleotide of the predefined sequence after the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide, and where position n+2 is the second nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; d) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles: i. The universal nucleotide occupies position n+1 in the support strand of the scaffold polynucleotide, where position n is the nucleotide position in the support strand opposite the position in the synthesis strand that, in the cycle, will be occupied by the next nucleotide of the predefined sequence after the next nucleotide of the predefined sequence is added to the end of the primer strand portion; and where n+1 is the next nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; and ii. The support strand of the scaffold polynucleotide is cleaved between positions n-1 and n-2, where positions n-1 and n-2 are the next nucleotide position and the subsequent nucleotide position, respectively, in the support strand relative to position n in the direction distal to the auxiliary strand / proximal to the primer strand portion; e) In the ligation step (step 9) of the second cycle and the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the partner nucleotide of the next nucleotide of the predefined sequence in the cycle is the penultimate nucleotide in the support strand and occupies position n, and the universal nucleotide occupies position n+2 in the support strand and pairs with the penultimate nucleotide of the auxiliary strand; where position n is the nucleotide position that will be opposite the next nucleotide of the predefined sequence incorporated in the cycle after the ligation polynucleotide is ligated to the cleaved scaffold polynucleotide (step 7), and where n+2 is the second nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion.

11. The method according to claim 10, wherein: (i) In the cleavage step (step 2) of the first cycle, the universal nucleotide instead occupies position n+2 in the support strand of the scaffold polynucleotide, where n+2 is the second nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1; (ii) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide instead occupies position n+3 in the support strand and pairs with the nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+3 is position 3 in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; (iii) In the cleavage step (step 6) of the second cycle and the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+2 in the support strand of the scaffold polynucleotide, and the support strand of the scaffold polynucleotide is cleaved between positions n and n-1; and (iv) In the ligation step (step 9) of the second cycle and in the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+3 in the support strand of the support polynucleotide and pairs with a nucleotide that is 2 positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end.

12. The method according to claim 10, wherein: (i) In the cleavage step (step 2) of the first cycle, the universal nucleotide instead occupies position n+2+x in the support strand of the support polynucleotide, where n+2 is the second nucleotide position in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; and the support strand of the support polynucleotide is cleaved between positions n and n-1; (ii) In the ligation step (step 5) of the first cycle, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide instead occupies position n+3+x in the support strand and pairs with a nucleotide that is 2+x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; where position n+3 is position 3 in the support strand relative to position n in the direction proximal to the auxiliary strand / distal to the primer strand portion; (iii) In the cleavage step (step 6) of the second cycle and in the cleavage steps of all subsequent cycles, the universal nucleotide occupies position n+2+x in the support strand of the support polynucleotide, and the support strand of the support polynucleotide is cleaved between positions n and n-1; (iv) In the ligation step (step 9) of the second cycle and in the ligation steps of all subsequent cycles, the complementary ligation ends of the ligation polynucleotide are structured such that the universal nucleotide occupies position n+3+x in the support strand and pairs with a nucleotide that is 2+x positions removed from the terminal nucleotide of the auxiliary strand at the complementary ligation end; and (v) where x is an integer between 1 and 10 or greater, and where x is the same integer in steps (2), (5), (6) and (9).

13. The method according to claim 1, wherein the partner nucleotide that pairs with the first nucleotide of the predefined sequence is a nucleotide complementary to the first nucleotide.

14. The method according to claim 3, wherein in any one or more synthesis cycles, or in all synthesis cycles, prior to step (2) and / or (6), the support polynucleotide is provided, which comprises a synthetic strand and a support strand hybridized thereto, and wherein the synthetic strand is provided without an auxiliary strand.

15. The method according to claim 3, wherein in any one or more synthesis cycles, or in all synthesis cycles, prior to step (2) and / or (6), the synthetic strand is removed from the support polynucleotide.

16. The method according to claim 15, wherein the auxiliary strand portion of the synthetic strand is removed from the scaffold polynucleotide by: (i) heating the scaffold polynucleotide to a temperature of 80 °C to 95 °C and separating the auxiliary strand portion from the scaffold polynucleotide; (ii) treating the scaffold polynucleotide with a urea solution and separating the auxiliary strand portion from the scaffold polynucleotide; (iii) treating the scaffold polynucleotide with formamide or a formamide solution and separating the auxiliary strand portion from the scaffold polynucleotide; or (iv) contacting the scaffold polynucleotide with a single-stranded polynucleotide molecule comprising a nucleotide sequence region complementary to the sequence of the auxiliary strand portion, thereby competitively inhibiting the hybridization of the auxiliary strand portion to the scaffold polynucleotide.

17. The method according to claim 2, wherein each cleavage step comprises a two-step cleavage process, wherein each cleavage step comprises a first step that comprises removing the universal nucleotide, thereby forming an abasic site; and a second step that comprises cleaving the support strand at the abasic site.

18. The method according to claim 17, wherein the first step is carried out with a nucleotide excision enzyme.

19. The method according to claim 18, wherein the nucleotide excision enzyme is 3-methyladenine DNA glycosylase.

20. The method according to claim 19, wherein the nucleotide excision enzyme is: i. human alkyladenine DNA glycosylase (hAAG); or ii. uracil DNA glycosylase (UDG).

21. The method according to any one of claims 17 to 20, wherein the second step is carried out with a chemical substance that is a base.

22. The method according to claim 21, wherein the base is NaOH.

23. The method according to claim 17, wherein the second step is carried out with an organic chemical substance having abasic site cleavage activity.

24. The method according to claim 23, wherein the organic chemical substance is N,N'-dimethylethylenediamine.

25. The method according to claim 17, wherein the second step is carried out with an enzyme having abasic site lyase activity, wherein the enzyme having abasic site lyase activity is (i) AP endonuclease 1; (ii) endonuclease III (Nth); or (iii) endonuclease VIII.

26. The method according to claim 1, wherein each cleavage step comprises a single-step cleavage process that comprises removing the universal nucleotide with a lyase enzyme, wherein the enzyme is (i) endonuclease III; (ii) endonuclease VIII; (iii) formamidopyrimidine DNA glycosylase (Fpg); or (iv) 8-oxoguanine DNA glycosylase (hOGG1).

27. The method according to claim 2, wherein the cleavage step comprises cleaving the support strand with an enzyme.

28. The method according to claim 27, wherein the enzyme cleaves the support strand after the nucleotide, the nucleotide being adjacent to the universal nucleotide, thereby generating an overhang in the synthesized strand.

29. The method according to claim 27, wherein the enzyme is endonuclease V.

30. The method according to claim 2, wherein the cleavage step comprises cleaving the support strand with an enzyme.

31. The method according to claim 30, wherein the enzyme cleaves the support strand after the nucleotide, the nucleotide being adjacent to the universal nucleotide, thereby generating an overhang in the synthesized strand.

32. The method according to claim 30, wherein the enzyme is endonuclease V.

33. The method according to claim 1, wherein both strands of the synthesized double-stranded polynucleotide are DNA strands.

34. The method according to claim 2, wherein the synthesized strand and the support strand are DNA strands.

35. The method according to claim 33, wherein the incorporated nucleotide is a dNTP.

36. The method according to claim 35, wherein the incorporated nucleotide is a dNTP comprising a reversible terminator group.

37. The method according to claim 36, wherein one or more of the incorporated nucleotides comprising a reversible terminator group are 3'-O-allyl-dNTP.

38. The method according to claim 36, wherein one or more of the incorporated nucleotides comprising a reversible terminator group are 3'-O-azidomethyl-dNTP.

39. The method according to claim 1, wherein the first strand of the synthesized double-stranded polynucleotide is a DNA strand and the second strand of the synthesized double-stranded polynucleotide is an RNA strand.

40. The method according to claim 2, wherein the synthesized strand is an RNA strand and the support strand is a DNA strand.

41. The method according to claim 40, wherein the incorporated nucleotide is an NTP.

42. The method according to claim 41, wherein the incorporated nucleotide is an NTP comprising a reversible terminator group.

43. The method according to claim 42, wherein the incorporated nucleotide comprising a reversible terminator group is 3'-O-allyl-NTP.

44. The method according to claim 42, wherein the incorporated nucleotide comprising a reversible terminator group is 3'-O-azidomethyl-NTP.

45. The method according to claim 1, wherein the enzyme is a polymerase, and the polymerase is a DNA polymerase.

46. The method according to claim 45, wherein the polymerase is a variant of the native DNA polymerase from the species Thermococcus 9°N.

47. The method according to claim 1, wherein the enzyme is a polymerase, and the polymerase is an RNA polymerase.

48. The method according to claim 1, wherein the enzyme has terminal transferase activity.

49. The method according to claim 1, wherein the step of removing the reversible terminator group from the first nucleotide is carried out with tris(carboxyethyl)phosphine (TCEP).

50. The method according to claim 1, wherein the step of ligating the double-stranded linking polynucleotide to the cleaved scaffold polynucleotide is carried out using a ligase.

51. The method according to claim 50, wherein the ligase is T3 DNA ligase or T4 DNA ligase.

52. The method according to claim 3, wherein in step (1), (5) and / or (9), the portion of the auxiliary strand and the support strand hybridized therewith are linked by a hairpin loop.

53. The method according to claim 2, wherein in step (1), the synthetic strand comprising the primer strand portion and the portion of the support strand hybridized therewith are linked by a hairpin loop.

54. The method according to claim 3, wherein in step (1), (5) and / or (9): a) the portion of the auxiliary strand and the support strand hybridized therewith are linked by a hairpin loop; and b) the synthetic strand comprising the primer strand portion and the portion of the support strand hybridized therewith are linked by a hairpin loop.

55. The method according to claim 3, wherein at least one of the linking polynucleotides is provided as a single molecule, the single molecule comprising a hairpin loop linking the support strand and the auxiliary strand at the end opposite the complementary linking end.

56. The method according to claim 3, wherein the linking polynucleotides for each synthesis cycle are provided as single molecules, each single molecule comprising a hairpin loop linking the support strand and the auxiliary strand at the end opposite the complementary linking end.

57. The method according to claim 3, wherein in step (1), the synthetic strand comprising the primer strand portion and the portion of the support strand hybridized therewith are tethered to a common surface.

58. The method according to claim 57, wherein the primer strand portion and the portion of the support strand hybridized therewith each comprise a cleavable linker, wherein the linker is cleavable to separate the double-stranded polynucleotide from the surface after synthesis.

59. The method according to claim 3, wherein in step (1), the primer strand portion of the synthetic strand and the portion of the support strand hybridized therewith are linked by a hairpin loop, and wherein the hairpin loop is tethered to a surface.

60. The method according to claim 59, wherein the hairpin loop is tethered to the surface by a cleavable linker, wherein the linker is cleavable to separate the double-stranded polynucleotide from the surface after synthesis.

61. The method according to claim 58, wherein the cleavable linker is a UV-cleavable linker.

62. The method according to claim 57, wherein the surface is a microparticle.

63. The method according to claim 57, wherein the surface is a planar surface.

64. The method according to claim 63, wherein the surface comprises a gel.

65. The method according to claim 64, wherein the surface comprises a polyacrylamide surface.

66. The method according to claim 57, wherein the synthetic strand comprising the primer strand portion and the portion of the support strand hybridizing therewith are tethered to the common surface by one or more covalent bonds.

67. The method according to claim 66, wherein the one or more covalent bonds are formed between a functional group on the common surface and a functional group on the scaffold molecule, and the functional group on the scaffold molecule is an amino group, a thiol group, a thiophosphoryl group or a thioamide group.

68. The method according to claim 67, wherein the functional group on the common surface is bromoacetyl.

69. The method according to claim 1, wherein the step of removing the reversible terminator group from the nucleotides of the predefined sequence is carried out before or after the ligation step.

70. The method according to claim 1, wherein the synthesis cycle is carried out in droplets within a microfluidic system.

71. The method according to claim 70, wherein the microfluidic system is an electrowetting system.

72. The method according to claim 71, wherein the microfluidic system is a dielectrophoretic electrowetting system (EWOD).

73. The method according to claim 1, wherein after synthesis, the strands of the double-stranded polynucleotide are separated to provide single-stranded polynucleotides having a predefined sequence.

74. The method according to claim 1, wherein after synthesis, the double-stranded polynucleotide or a region thereof is amplified by PCR.

75. A method of assembling a polynucleotide having a predefined sequence, the method comprising performing the method according to claim 1 to synthesize a first polynucleotide having a predefined sequence and one or more additional polynucleotides having a predefined sequence, and joining the first polynucleotide and the one or more additional polynucleotides together.

76. The method according to claim 75, wherein the first polynucleotide and the one or more additional polynucleotides are double-stranded.

77. The method according to claim 75, wherein the first polynucleotide and the one or more additional polynucleotides are single-stranded.

78. The method according to claim 75, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved to produce compatible ends and joined together by ligation.

79. The method according to claim 78, wherein the first polynucleotide and the one or more additional polynucleotides are cleaved at the cleavage site by a restriction enzyme.

80. The method according to claim 71, wherein the synthesis and / or assembly step is carried out in droplets within a microfluidic system.

81. The method according to claim 80, wherein the assembly step comprises providing a first droplet comprising a first synthetic polynucleotide having a predefined sequence and a second droplet comprising one or more additional synthetic polynucleotides having a predefined sequence, wherein the droplets are brought into contact with each other and wherein the synthetic polynucleotides are joined together, thereby assembling a polynucleotide comprising the first and one or more additional polynucleotides.

82. The method according to claim 81, wherein the synthesis step is carried out as follows: by providing a plurality of droplets, each droplet comprising a reaction reagent corresponding to a synthesis cycle step, and sequentially delivering the droplets to the support polynucleotide according to the synthesis cycle step.

83. The method according to claim 82, wherein after delivering the droplet and before delivering the next droplet, a washing step is carried out to remove excess reaction reagent.

84. The method according to claim 82, wherein the microfluidic system is an electrowetting system.

85. The method according to claim 84, wherein the microfluidic system is an electrowetting-on-dielectric system (EWOD).

86. The method according to claim 82, wherein the synthesis and assembly steps are carried out within the same system.

87. A polynucleotide synthesis system for carrying out the method according to any one of claims 1 to 86, which comprises: (a) a plurality of reaction regions, each reaction region comprising at least one support polynucleotide; and (b) means for delivering the reaction reagent to the reaction region; and (c) means for cleaving the synthesized double-stranded polynucleotide from the support polynucleotide.

88. The system according to claim 87, further comprising means for providing the reaction reagent in the form of droplets and means for delivering the droplets to the support polynucleotide according to the synthesis cycle.

89. A kit for use with the system according to claim 87 and for carrying out the method according to any one of claims 1 to 86, the kit comprising a volume of reaction reagent corresponding to the steps of the synthesis cycle.

90. A method for preparing a polynucleotide microarray, wherein the microarray comprises a plurality of reaction regions, each region comprising one or more polynucleotides having a predefined sequence, the method comprises: a) providing a surface comprising a plurality of reaction regions, each region comprising one or more double-stranded anchors or support polynucleotides, and b) carrying out a synthesis cycle in each reaction region according to the method of claim 1, thereby synthesizing one or more double-stranded polynucleotides having a predefined sequence at each region.

91. The method according to claim 90, wherein after synthesis, the strands of the double-stranded polynucleotide are separated to provide a microarray, wherein each region comprises one or more single-stranded polynucleotides having a predefined sequence.

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