LIGATION OF OLIGONUCLEOTIDES

1 . A method to produce a target double-stranded (ds) DNA polynucleotide by annealing single-stranded oligonucleotides (ss oligos) and ligating a ds assembly, comprising the following steps:

a) annealing at least 2 ss oligos comprising matching sequences for hybridization to produce a starter ds oligo with two overhangs, and hybridizing one of said overhangs with a nucleotide (NA) building block, thereby obtaining a ds assembly with two overhangs of at least 2 nucleotides (nt), which ds assembly comprises at least one nick in the ds part of the ds assembly;

b) hybridizing to the ds assembly at least one extender ss oligo (ExtON), which comprises a sticky terminal sequence of at least 2 nt on one side, and an identifiable tail on the other side, wherein the sticky terminal sequence is matching for hybridization with an overhang of the ds assembly, thereby obtaining an extended ds assembly;

c) ligating the nucleotides within the extended ds assembly by a ligation reaction, thereby resolving said at least one nick; and

d) following the ligating step c), removing said at least one ExtON from the extended ds assembly, thereby producing the target ds polynucleotide.

2 . The method of claim 1 , wherein in said step a):

i) at least four ss oligos are annealed and hybridized to produce a ds assembly comprising at least two nicks; or

ii) three ss oligos are annealed and hybridized to produce a ds assembly comprising one nick; or

iii) two ss oligos are annealed and hybridized to one ds oligo or ds polynucleotide to produce a ds assembly comprising two nicks.

3 . The method of claim 1 , wherein step a) comprises annealing and hybridizing four ss oligos, by annealing a first and a second pair of ss oligos, wherein each ss oligo pair comprises two ss oligos that comprise a part matching for annealing the ss oligos of the pair, and another part to obtain an overhang of at least 2 nt on each side upon annealing, and wherein one overhang of the first ss oligo pair is matching for hybridization with one overhang of the second ss oligo pair to produce said ds assembly.

4 . The method of claim 1 , wherein steps a) and b), and optionally step c), are carried out in one reaction containment.

5 . The method of claim 1 , wherein two ExtONs are used for hybridization to the ds assembly, wherein the sticky sequences of said two ExtONs are matching for hybridization with the two overhangs of the ds assembly, thereby obtaining an extended ds assembly that comprises an ExtON on each side.

6 . The method of claim 1 , wherein each of the ss oligos comprises at least 6 nt.

7 . The method of claim 1 , wherein the sticky sequence of said at least one ExtON is comprised of the 5′-terminal sequence of the ExtON, and the tail is comprised of or attached to the 3′-terminal sequence of the ExtON.

8 . The method of claim 1 , wherein the sticky sequence of said at least one ExtON is not phosphorylated and/or comprises a 5′ blocking modification that prevents ligation.

9 . The method of claim 1 , wherein the tail of said at least one ExtON comprises a unique molecular identifier (UMI).

10 . The method of claim 1 , wherein said at least one ExtON is removed by dehybridization and optionally isolating said at least one ExtON or the target ds polynucleotide.

11 . The method of claim 1 , wherein the ligation reaction is an enzymatic reaction and wherein the enzyme is a ligase, polymerase, or ribozyme.

12 . The method of claim 1 , which further comprises producing an assembly of the target ds polynucleotide and one or more DNA building blocks in one or more further steps, to synthetize a longer ds polynucleotide according to a DNA template.

13 . A set of extender single-stranded oligonucleotides (ss oligo) (ExtONs), wherein each ExtON comprises a sticky terminal sequence of at least 2 nucleotides (nt) on one side, that is reactive to hybridize with a matching ss sequence of a double-stranded (ds) nucleotide (NA) building block, and an identifiable tail on the other side, wherein the identifiable tail comprises a unique molecular identifier (UMI) that is specific to the set of ExtONs and common to all ExtONs in the set, which set comprises a variety of ExtONs to cover a diversity of said sticky sequences.

14 . The ExtON set of claim 13 , wherein each ExtON is characterized by one or more of the following:

a) the sticky sequence of the ExtON consists of at least 2 nt;

b) the sticky sequence of the ExtON is comprised of the 5′-terminal sequence of the ExtON, and the tail is comprised of or attached to the 3′-terminal sequence of the ExtON;

c) the sticky sequence of the ExtON is not phosphorylated and/or comprises a 5′ blocking modification that prevents ligation;

d) the ExtON consists of an oligonucleotide of at least 8 nt; and

e) the ExtON consists of an oligonucleotide with a length of up to 30 nt.

15 . The method of claim 1 , wherein the NA building block is a ss oligo, a ds oligo or a ds polynucleotide.

16 . The method of claim 4 , wherein steps a) and b), and optionally c), are carried out in a pool reaction.

17 . The method of claim 11 , wherein the enzyme is any one of a T3, T4 or T7 DNA ligase, Taq DNA ligase, or a DNA polymerase.

18 . The method of claim 13 , wherein the variety of ExtONs covers a diversity of at least 10 different sticky sequences.

19 . The method of claim 14 , wherein the sticky sequence of the ExtON comprises a 5′ blocking modification that prevents phosphorylation.

20 . The method of claim 14 , wherein the ExtON consists of an oligonucleotide with a length of up to 20 nt.