Method for the preparation of a nucleic acid library

1. A method for preparing a nucleic acid library comprising a plurality of elements, whereby each element of said nucleic acid library comprises a first stretch of nucleotides, a second stretch of nucleotides and a third stretch of nucleotides, whereby the sequence of the first stretch of nucleotides and of the third stretch of nucleotides are identical in each element of the nucleic acid library and the elements of the nucleic acid library differ in the sequence of the second stretch of nucleotides, the method comprising the following steps:

a) providing a first at least partially double-stranded oligonucleotide comprising

aa) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence corresponding to the sequence of the first or third stretch of nucleotides of the elements of the nucleic acid library; and

ab) a recognition site, or part thereof, for a first type IIS restriction enzyme which cuts outside its recognition site;

b) providing a first oligonucleotide library comprising a plurality of members, whereby each member is a second at least partially double-stranded oligonucleotide comprising

ba) a recognition site, or part thereof, for a second type IIS restriction enzyme which cuts outside its recognition site;

bb) a single-stranded overhang, whereby the single-stranded overhang is the same for each of the members of the first oligonucleotide library and is complementary to the single-stranded overhang of the first at least partially double-stranded oligonucleotide; and

bc) whereby the members of the first oligonucleotide library differ in the sequence of a stretch of nucleotides and the sequence of said stretch of nucleotides corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library, wherein one or more nucleotide positions within the stretch of nucleotides differs at a controlled frequency;

c) ligating the first at least partially double-stranded oligonucleotide with the members of the first oligonucleotide library via their single-stranded overhangs, whereupon first ligation products are formed;

d) cutting the first ligation products with said second type IIS restriction enzyme in the nucleic acid sequence of the members of the first oligonucleotide library, and providing for a plurality of first elongated at least partially double-stranded oligonucleotides and for shortened second at least partially double-stranded oligonucleotides;

e) removing the shortened second at least partially double-stranded oligonucleotides;

f) providing a second oligonucleotide library comprising a plurality of members, whereby each member is a third at least partially double-stranded oligonucleotide comprising

fa) a recognition site, or part thereof, for a third type IIS restriction enzyme which cuts outside its recognition site;

fb) a single-stranded overhang, whereby the single-stranded overhang is different for the members of the second oligonucleotide library and whereby such single-stranded overhangs of the third oligonucleotide are complementary to the single-stranded overhangs of the first elongated at least partially double-stranded oligonucleotides; and

fc) a stretch of nucleotides which is identical in all members and corresponds to the sequence, or part thereof, of the third or first stretch of nucleotides of the elements of the nucleic acid library;

g) ligating the plurality of first elongated at least partially double-stranded oligonucleotides with the members of the second oligonucleotide library, whereby the single-stranded overhang of the first elongated at least partially double-stranded oligonucleotide is complementary to the overhang of the third at least partially double-stranded oligonucleotides, whereupon second ligation products are formed;

h) cutting the second ligation products with the third type IIS restriction enzyme in the nucleic acid sequence of the third at least partially double-stranded oligonucleotides, and providing for a plurality of second elongated at least partially double-stranded oligonucleotides and shortened third at least partially double-stranded oligonucleotides;

i) removing the shortened third at least partially double-stranded oligonucleotides;

j) providing fourth at least partially double-stranded oligonucleotides comprising

ja) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhang of the second elongated at least partially double-stranded oligonucleotide; and

jb) a recognition site, or part thereof, for a fourth type IIS restriction enzyme which cuts outside its recognition site;

k) ligating the fourth oligonucleotides and the plurality of the second elongated at least partially double-stranded oligonucleotides to form third ligation products;

l) cutting the third ligation products with the fourth type IIS restriction enzyme in the nucleic acid sequence of the fourth at least partially double-stranded oligonucleotides, and providing for a plurality of third elongated at least partially double-stranded oligonucleotides and shortened fourth at least partially double-stranded oligonucleotides; and

m) removing the shortened fourth at least partially double-stranded oligonucleotides; and

n) obtaining the nucleic acid library

wherein the nucleic acid library is a library of nucleic acid molecules coding for a peptide, polypeptide, protein, or functional nucleic acid;

wherein the peptide, polypeptide, or protein comprises one or several constant regions, and one or several variable regions;

wherein the one or several of the variable regions is/are encoded by the sequence of the second stretch of nucleotides of the elements of the nucleic acid library; and

wherein the second stretch of nucleotides of the elements of the nucleic acid library is the only difference between said elements.

2. The method according to claim 1 , further comprising:

n) repeating steps j) to m), whereby the fourth at least partially double-stranded oligonucleotides of step j) are a further at least partially double-stranded oligonucleotide, the third ligation product in step k) is a further ligation product, the plurality of third elongated at least partially double-stranded oligonucleotides in step l) is a plurality of further elongated at least partially double-stranded oligonucleotides, and the shortened fourth at least partially double-stranded oligonucleotides of step l) are shortened further at least partially double-stranded oligonucleotides.

3. The method according to claim 2 , whereby after ligation step k) the further ligation product is cleaved in the nucleic acid sequence of the further elongated at least partially double-stranded oligonucleotides.

4. The method according to claim 1 , wherein the first type IIS restriction enzyme is selected from the group consisting of BpiI, BbsI, Esp3I, BsmBI, Eco31I, BsaI, BfuAI, FokI, BseRI, BbvI and BsgI.

5. The method according to claim 1 , wherein the second, third, fourth, fifth and any further type IIS restriction enzyme is each and individually selected from the group consisting of Earl, Eam1104I, SapI, LguI, Ksp632I and BspQI.

6. The method according to claim 1 , wherein the nucleic acid is a single-stranded nucleic acid and the length of the second stretch of nucleotides of the elements of the nucleic acid library consists of two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides or multiples of three nucleotides.

7. The method according to claim 1 , wherein the nucleic acid is a double-stranded nucleic acid and the length of the second stretch of nucleotides of the elements of the nucleic acid library consists of two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides or multiples of three nucleotides.

8. The method according to claim 1 , wherein the second stretch of nucleotides of the elements of the nucleic acid library is coding for one or several amino acids.

9. The method according to claim 1 , wherein the second at least partially double-stranded oligonucleotides, the third at least partially double-stranded oligonucleotides, the fourth at least partially double-stranded oligonucleotides, the fifth and the further at least partially double-stranded oligonucleotides comprise a modification, whereby such modification allows for the immobilization of the oligonucleotides to a surface.

10. The method according to claim 9 , whereby the modification is selected from the group consisting of a biotin residue, a digoxigenin residue, a fluorescein isothiocyanate residue, an amino compound and a succinyl ester.

11. The method according to claim 1 , wherein the nucleic acid is selected from the group comprising aptamers, promoters, ribozymes, and RNAimediating molecules.

12. The method according to claim 1 , wherein the peptide, polypeptide and protein is selected from the group consisting of peptide aptamers, enzymes, restriction enzymes, DNA binding domains, vaccines, antibodies, pharmaceutically active proteins, anticalines, DARPins, nanobodies, and AdNectines.

13. The method according to claim 1 , wherein the controlled frequency comprises an equimolar distribution.

14. The method according to claim 1 , wherein the controlled frequency comprises a non-equimolar or biased distribution.

15. A method for preparing a nucleic acid library comprising a plurality of elements, whereby each element of said nucleic acid library comprises a first stretch of nucleotides, a second stretch of nucleotides and a third stretch of nucleotides, whereby the sequence of the first stretch of nucleotides and of the third stretch of nucleotides are identical in each element of the nucleic acid library and the elements of the nucleic acid library differ in the sequence of the second stretch of nucleotides, the method comprising the following steps:

a) providing a first at least partially double-stranded oligonucleotide comprising

aa) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence corresponding to the sequence of the first or third stretch of nucleotides of the elements of the nucleic acid library; and

ab) a recognition site, or part thereof, for a first type IIS restriction enzyme which cuts outside its recognition site

b) providing a first oligonucleotide library comprising a plurality of members, whereby each member is a second at least partially double-stranded oligonucleotide comprising

ba) a recognition site, or part thereof, for a second type IIS restriction enzyme which cuts outside its recognition site, and a single-stranded overhang, whereby the single-stranded overhang is the same for each of the members of the first oligonucleotide library and is complementary to the single-stranded overhang of the first at least partially double-stranded oligonucleotide; and

bb) a stretch of nucleotides whereby the members of the first oligonucleotide library differ in the sequence of said stretch of nucleotides and the sequence of the said stretch of nucleotides of the members of the first oligonucleotide library corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library, wherein one or more nucleotide positions within the stretch of nucleotides differs at a controlled frequency;

c) ligating the first at least partially double-stranded oligonucleotide with the first oligonucleotide library via their single-stranded overhangs, whereupon first ligation products are formed;

d) cutting the first ligation products with said second type IIS restriction enzyme in the nucleic acid sequence of the member of the first oligonucleotide library, and providing for a plurality of first elongated at least partially double-stranded oligonucleotides and for shortened second at least partially double-stranded oligonucleotides, whereby such plurality of first elongated at least partially double-stranded oligonucleotides consist of various molecule species which differ in the sequence of the single-stranded overhang;

e) removing the shortened second at least partially double-stranded oligonucleotides;

f) providing a second oligonucleotide library comprising several subgroups of oligonucleotides and each subgroup comprising several members, whereby each member is a third at least partially double-stranded oligonucleotide comprising

fa) a recognition site, or part thereof, for a third type IIS restriction enzyme which cuts outside its recognition site;

fb) a single-stranded overhang, whereby the single-stranded overhang is different for the various subgroups of oligonucleotides of the second oligonucleotide library and whereby the single-stranded overhang of each one subgroup of the second oligonucleotide library is complementary to the single-stranded overhang of each of the first elongated at least partially double-stranded oligonucleotides; and

fc) a stretch of nucleotides which is different in the members of each subgroup of the second oligonucleotide library and corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library;

g) ligating the plurality of the first elongated at least partially double-stranded oligonucleotides with the members of the second oligonucleotide library, whereby those molecules are ligated at the overhangs of which are complementary to each other, whereupon second ligation products are formed;

h) cutting the second ligation products with the third type II S restriction enzyme in the nucleic acid sequence of the third at least partially double-stranded oligonucleotide, and providing for a plurality of second elongated at least partially double-stranded oligonucleotides and shortened third at least partially double-stranded oligonucleotides, whereby such plurality of second elongated at least partially double-stranded oligonucleotides consists of various subgroups which differ in their single-stranded overhangs;

i) removing the shortened third at least partially double-stranded oligonucleotides;

j) providing a third oligonucleotide library comprising several members, whereby each member is a fourth at least partially double-stranded oligonucleotide comprising

ja) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhangs of the second elongated at least partially double-stranded oligonucleotides; and

jb) a stretch, preferably adjacent to said overhang which is identical in the various members and provides for the third or first stretch of nucleotides of the elements of the nucleic acid library, whereby the members of the third oligonucleotide library differ in their single-stranded overhangs and the overhangs correspond to the second stretch, or part thereof, of the nucleic acid library; and

jc) a recognition site, or part thereof, for a fourth type IIS restriction enzyme which cuts outside its recognition site;

k) combining the plurality of second elongated at least partially double-stranded oligonucleotides and the third oligonucleotide library and ligating each molecule of the members of the third oligonucleotide library with one molecule of the plurality of the second elongated at least partially double-stranded oligonucleotides, whereby those molecules are ligated and the overhangs of which are complementary to each other, whereupon third ligation products are formed;

l) cutting the third ligation product with the fourth type IIS restriction enzyme in the nucleic acid sequence of the member of the third oligonucleotide library, providing for a plurality of third elongated at least partially double-stranded oligonucleotides and shortened fourth at least partially double-stranded oligonucleotides;

m) optionally removing the shortened fourth at least partially double-stranded oligonucleotides;

n) providing a fifth at least partially double-stranded oligonucleotide which has a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhang of the plurality of third elongated at least partially double-stranded oligonucleotides, whereby the fifth oligonucleotide comprises a recognition site, or part thereof, for a fifth type IIS restriction enzyme which cuts outside its recognition site;

o) combining the plurality of third elongated at least partially double-stranded oligonucleotides and the fourth oligonucleotide library and ligating each molecule of the plurality of the third elongated at least partially double-stranded oligonucleotides with one molecule of the fifth at least double-stranded oligonucleotide, whereupon a fourth ligation product is formed;

p) cutting the fourth ligation product with the fifth type IIS restriction enzyme in the nucleic acid sequence of the fifth at least partially double-stranded oligonucleotide, providing for a plurality of fourth elongated at least partially double-stranded oligonucleotides and the shortened fifth at least partially double-stranded oligonucleotides;

q) optionally removing the shortened fifth at least partially double-stranded oligonucleotides;

r) obtaining the nucleic acid library,

wherein the nucleic acid library is a library of nucleic acid molecules coding for a peptide, polypeptide, or protein;

whereby the peptide, polypeptide, or protein comprises one or several constant regions, and one or several variable regions;

wherein the one or several of the variable regions is/are encoded by the sequence of the second stretch of nucleotides of the elements of the nucleic acid library; and

wherein the second stretch of nucleotides of the elements of the nucleic acid library is the only difference between said elements.

16. The method according to claim 15 , further comprising the steps of:

r) repeating steps n) to q), whereby the fifth at least partially

double-stranded oligonucleotide of step n) is a further at least double-stranded oligonucleotide, the fourth ligation product in step o) is a further ligation product, the plurality of fourth elongated at least partially double-stranded oligonucleotides in step p) is a plurality of further elongated at least partially double-stranded oligonucleotides, and the shortened fifth at least partially double-stranded oligonucleotide of step p) is a shortened further at least partially double-stranded oligonucleotide.

17. The method according to claim 16 , whereby after ligation step o) the further ligation product is cleaved in the nucleic acid sequence of the further elongated at least partially double-stranded oligonucleotide.

18. A method for the manufacture of a nucleic acid molecule library comprising a plurality of elements, whereby the elements comprise at least one constant stretch and at least one variable stretch and whereby the at least one constant stretch comprises a nucleotide sequence which is the same in all of the elements, and the at least one variable stretch comprises a nucleotide sequence which is different in all of the elements, the method comprises the following steps:

a) providing a first oligonucleotide, whereby the first oligonucleotide is either an at least partially double-stranded oligonucleotide having a single-stranded overhang and comprising a recognition site for a first type IIS restriction enzyme which cuts outside its recognition sequence, or a nucleic acid library comprising a plurality of elements, whereby the nucleic acid library is the nucleic acid library according to claim 1 or 4 and the elements of the nucleic acid library have a single-stranded overhang, and the elements of the nucleic acid library comprise a recognition site for a first type IIS restriction enzyme which cuts outside its recognition sequence;

b) providing a second oligonucleotide, whereby such second oligonucleotide is a nucleic acid library comprising a plurality of elements, whereby the nucleic acid library is the nucleic acid library according to claim 1 or 4 and the elements of the nucleic acid library have a single-stranded overhang which is complementary to the single-stranded overhang of the oligonucleotide provided in step a), the elements of the nucleic acid library comprise a recognition site for a second type IIS restriction enzyme which cuts outside its recognition sequence, whereby the second type IIS restriction enzyme of the elements of the nucleic acid library is different from the first type IIS restriction enzyme of the oligonucleotide provided in step a);

c) combining the oligonucleotides of step a) and step b) and ligating one molecule of the oligonucleotide of step a) with each one molecule of each one element of the nucleic acid library of step b) via their single-stranded overhangs under the proviso that the oligonucleotide of step a) is different from the nucleic acid library according to claim 1 or 4 ;

or

combining the oligonucleotides of step a) and step b) and ligating each one molecule of each one element of the nucleic acid library of step a) with each one molecule of each one element of the nucleic acid library of step b) via their single-stranded overhangs under the proviso that the oligonucleotide of step a) is a nucleic acid library according to claim 1 or 4 ;

d) optionally removing non-reacted reactants and enzymes;

e) cleaving the ligation product of step c) with a type IIS restriction enzyme which cuts outside its recognition sequence, whereby the cleavage occurs in the oligonucleotide of step a) or step b) providing for a library of extended nucleic acid molecules; and

f) optionally separating the extended nucleic acid molecules from the reaction mixture;

g) obtaining the nucleic acid library.

19. The method according to claim 18 , whereby the step a) to f) are repeated under the provisos that the oligonucleotide of step a) is a third oligonucleotide which is the library of extended nucleic acid molecules provided in step e), and the oligonucleotide of step b) are fourth oligonucleotides which are either a library according to claim 1 or 4 , or fourth at least partially double-stranded oligonucleotides having a single-stranded overhang and comprising a recognition site for a fourth type IIS restriction enzyme which cuts outside its recognition sequence, whereby the third and the fourth oligonucleotides each comprise a single-stranded overhang and whereby both overhangs are complementary to each other; when combining the oligonucleotides of step a) and step b) and ligating one molecule of each one element of the nucleic acid library of step a) with one molecule of the oligonucleotide or each one molecule of each one element of the nucleic acid library of step b) via their single-stranded overhangs, the oligonucleotide of step a) is different from the oligonucleotide or library of extended nucleic acid molecules of step b); or when combining the oligonucleotides of step a) and step b) and ligating each one molecule of each one element of the nucleic acid library of step a) with one molecule of the oligonucleotide or each one molecule of each one element of the nucleic acid library of step b) via their single-stranded overhangs, the oligonucleotide of step a) is a library of extended nucleic acid molecules; and wherein in step e) a further library of extended nucleic acid molecules is obtained.

20. The method according to claim 18 , wherein the variable stretch is provided by the second stretch of the elements of the nucleic acid library as described in claim 1 .

21. The method according to claim 18 , wherein the constant stretch or part thereof is provided by the first stretch and/or the third stretch of the elements of the nucleic acid library as described in claim 1 .