EP3898968A1 - Nucleic acid encoded chemical libraries - Google Patents
Nucleic acid encoded chemical librariesInfo
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- EP3898968A1 EP3898968A1 EP19832693.6A EP19832693A EP3898968A1 EP 3898968 A1 EP3898968 A1 EP 3898968A1 EP 19832693 A EP19832693 A EP 19832693A EP 3898968 A1 EP3898968 A1 EP 3898968A1
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- European Patent Office
- Prior art keywords
- nucleic acid
- group
- strand
- chemical moieties
- strands
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
Definitions
- This invention relates to nucleic acid encoded chemical libraries, particular cyclised nucleic acid-encoded self-assembling chemical libraries (ESACs), and methods of production and screening thereof.
- ESACs cyclised nucleic acid-encoded self-assembling chemical libraries
- DECLs DNA-Encoded Chemical Libraries
- DECLs represent an innovative manner to discover new ligands toward target proteins of therapeutic interest.
- the members of nucleic acid encoded chemical libraries display pharmacophores made up of one or more chemical moieties (also called“building blocks”). These chemical libraries can be used to identify pharmacophores which are candidate binding agents or have improved characteristics, for example improved binding [R. M. Franzini, C. Randolph J. Med. Chem., 59, 6629-6644 (2016)].
- DECLs The basic concept of DECLs, i.e. the use of DNA as a tag for the unique identification of library members through sequencing, was proposed by Lerner and Brenner in 1992 [R. A. Lerner & S. Brenner Proc. Natl. Acad. Sci. USA, 89, 5381-5383 (1992)].
- the first examples of functional libraries were published in 2004 by the Neri group at ETH [S. Melkko et al Nat. Biotechnol., 22, 568-574 (2004)] and by the Liu group at Harvard University [Z. J. Gartner et al Science, 305, 1601-1605 (2004)].
- the first category displays an individual small molecule (it does not matter how complex it is) at the individual extremities of the double strand DNA-tag fragment, which serves as an amplifiable identification barcode, whereas in the second class two sets of small organic compounds are attached to the adjacent complementary DNA strands [R. M. Franzini, D. Neri, J. Scheuermann Acc. Chem. Res., 47, 1247-1255 (2014)].
- Most of the DECLs reported so far by both academia and industry were constructed by split-and-pool synthetic procedures [M. A. Clark et al. Nat. Chem. Biol. 5, 647-654 (2009)] aiming at drug-like molecules complying with Lipinski’s rule of five (R05) [R. M. Franzini & C. Randolph J. Med. Chem. 59, 6629-6644 (2016)].
- the first set of amino acid reactive moieties is singularly conjugated to short oligonucleotide and HPLC purified. All the conjugates are then pooled together in equimolar amounts and split into a number of aliquots equal to the number of amino acid reactive moieties in the second set. From this point on, it is not possible to singularly isolate the new conjugates. In order to ensure the quality and performance of the final library, it is necessary that the coupling reactions of each of the amino acid reactive moieties from the second set are high yielding and clean (there is no formation of side products). Therefore, it is not advisable to add amino acid reactive moieties with a peculiar reactivity to single-pharmacophore libraries.
- ESAC libraries In dual-pharmacophore DECLs, two building blocks are simultaneously connected to the extremities of complementary DNA strands, thus enabling the formation of combinatorial libraries by the self-assembly of oligonucleotide conjugates.
- Each member of the two sub-libraries (E1 and E2) forming the ESAC library is singularly synthesized and HPLC purified: this procedure allows the introduction in the ESAC libraries of compounds with low conjugation yields and formation of side products.
- the structure of ESAC libraries allows the members to explore a wide surface of the target protein, increasing the possibility to find binding pockets.
- ESAC libraries may facilitate the identification of synergistic amino acid reactive moieties, which recognize adjacent pockets on target proteins of interest [S. Melkko et al. Nat. Biotechnol., 22, 568-574 (2004)].
- the library structure, the construction procedure and the synergistic effect make the ESAC library suitable for the identification of new binding molecules which are able to interact with large surfaces of
- DNA encoded chemical libraries may preferentially yield binders for targets with defined pockets, such as kinases, proteases, or phosphatases
- targets with defined pockets
- targets such as kinases, proteases, or phosphatases
- the recognition of large surfaces of target proteins remains a challenge.
- millions of molecules have to be screened, in order to find a suitable candidate.
- the preparation of very large libraries of organic molecules and their purity are cumbersome.
- the complexity associated with the identification of specific binding molecule from a pool of candidates grows with the size of the chemical library to be screened. Summary
- BBs building blocks
- the present inventors have recognised that covalent linkage between the chemical moieties (also called building blocks (BBs)) on complementary strands of members of nucleic acid encoded chemical libraries facilitates the generation of large, high purity libraries of cyclised pharmacophores through a ring closure the chemical moieties (e.g. on the top) that may for example facilitate screening for molecules that bind to large surfaces of target proteins.
- BBs building blocks
- a first aspect of the invention provides a method of producing a nucleic acid encoded chemical library comprising;
- first conjugates comprising a first nucleic strand coupled to a first reactive group and a first set of one or more chemical moieties at a first end
- a second aspect of the invention provides a method of producing a member of a nucleic strand encoded chemical library comprising;
- first conjugate comprising a first nucleic strand coupled to a first reactive group and a first set of one or more chemical moieties
- a third aspect of the invention provides a nucleic acid encoded chemical library comprising;
- each member comprising;
- first and second nucleic strands are hybridised together to form double stranded molecules, and the first and second sets of chemical moieties are covalently linked to form cyclised pharmacophores coupled to the double stranded molecules.
- a suitable nucleic acid encoded chemical library may be produced by a method of the first aspect.
- Suitable members of the nucleic acid encoded chemical library may be produced by a method of the second aspect.
- one of the first and second nucleic acid strands of the first, second and third aspects may be coupled to one chemical moiety and the other of the first and second nucleic acid strands may be coupled to one chemical moiety (see for example Figure 1 A).
- one of the first and second nucleic acid strands of the first, second and third aspects may be coupled to two chemical moieties and the other of the first and second nucleic acid strands may be coupled to one chemical moiety (see for example Figures 1 B and 1 E).
- one of the first and second nucleic acid strands of the first, second and third aspects may be coupled to a first chemical moiety.
- a second chemical moiety may be attached to the first chemical moiety.
- a reactive group may be attached to the first or the second chemical moiety.
- the other of the first and second nucleic acid strands may be coupled to one chemical moiety which is attached to a reactive group (see for example Figures 1 B and 1 E).
- one of the first and second nucleic acid strands of the first, second and third aspects may be coupled to two chemical moieties and the other of the first and second nucleic acid strands may be coupled to two chemical moieties (see for example Figure 1 C).
- one of the first and second nucleic acid strands of the first, second and third aspects may be coupled to three chemical moieties and the other of the first and second nucleic acid strands may be coupled to one chemical moieties (see for example Figure 1 D).
- a fourth aspect of the invention provides a method of screening a nucleic acid encoded chemical library comprising;
- the populations of first and second conjugates and the libraries of the first to fourth aspects described above may display high purity.
- FIG 1 shows examples of different self-assembling chemical libraries (ESAC) embodiments.
- Figure 2 shows examples of synthetic and encoding strategies for dual-pharmacophore chemical libraries with ring closure on the top.
- Figure 3 shows some examples of chemical reactions/chemical compounds used for the ring closure of the complementary sub-population of nucleic acid conjugates.
- Figures 4A and 4B show a strategy for the generation of nucleic acid conjugates in which the extremities of the two nucleic acid strands (strand A and strand B) are coupled to chemical moieties and azide (strand A) and alkyne (strand B) reactive groups.
- Figure 4A shows the synthesis of an amino-modified d-spacer oligonucleotide azide conjugate (strand A).
- Figure 4B shows the synthesis of the amino-modified 48-mer oligonucleotide alkyne conjugate (strand B).
- Figure 5 shows an example of the production of a cyclic dual-pharmacophore chemical library by copper(l)- catalysed 1 , 2, 3-triazole-forming reaction, in which chemical moieties are coupled to the strands of the library members.
- An azide modified chemical moiety is coupled to a first nucleic acid strand (strand A).
- An alkyne modified chemical moiety is coupled to the second nucleic strand (strand B).
- Ring closure is performed between diverse populations of chemical moieties which are displayed at the extremities of the two DNA strands using a copper(l)-catalysed 1 , 2, 3-triazole-forming reaction.
- Figure 6 shows analytical UV traces (recording absorbance at 260 nm) of (A) nucleic acid strand A comprising the azide modified chemical moiety, (B) strand B comprising the alkyne modified chemical moiety, (C) strands A and B without ring closure reagents and (D) nucleic acid strands A and B with ring closure.
- Figure 7 shows the respective MS traces of (A) nucleic acid strand A comprising the azide modified chemical moiety, (B) strand B comprising the alkyne modified chemical moiety, (C) strands A and B without ring closure reagents and (D) nucleic acid strands A and B with ring closure.
- Figure 8 shows the synthesis scheme of (A) the 5’-amino-modified oligonucleotide azide acid conjugate (strand A1) by coupling the 2-azidoacetic acid onto the Elib2_Code1 oligonucleotide using s-NHS/EDC as coupling reagents in MOPS buffer, (B) the 3’-amino-modified, 5’-phosphorylated d-spacer oligonucleotide alkyne acid conjugate (strand B1) by coupling the 6-heptynoic acid onto the d-spacer using DMT-MM as coupling reagent in Borate buffer and (C) the ring closure of the nucleic acid strands A1 and B1 by 1 ,2,3- triazole formation reaction using copper(l) catalyst to obtain the Format 1.
- Figures 9 shows respectively the analytical LC-ESI-MS trace references of (A) nucleic acid strand A1 , (B) nucleic acid strand B1 , (C) the mixture of nucleic acid strands A1 and B1 without the ring closure reaction and (D) the nucleic acid strand A1 and strand B1 mixture after the reaction depicted on Figure 8C.
- Figure 10 shows (A) the synthesis scheme of the encoding step by the ligation of a custom oligonucleotide Elib4_Code2 onto the 5’-phosphorylated, 3’-amino-modified alkynyl acid strand B1 , synthesising the encoded oligonucleotide alkynyl conjugate strand B2 and (B) the synthesis scheme of DNA duplex Format 2 conjugate by the ring closure of nucleic acid strands A1 and B2 by 1 , 2, 3-triazole formation reaction using copper(l) catalyst to obtain Format 2.
- Figure 11 shows respectively the analytical LC-ESI-MS trace references of the (A) nucleic acid strand A1 ,
- FIG. 12 shows (A) the synthesis scheme of the Klenow fill-in of the nucleic acid strand A1 to nucleic acid strand A2, constructing the DNA duplex used for the ring closure reaction and (B) the synthesis scheme of the ring closure of the nucleic acid strands A2 and B2 by 1 , 2, 3-triazole formation reaction using copper(l) catalyst to obtain Format 3.
- Figures 13 shows respectively (A) the analytical LC-ESI-MS trace references of the nucleic acid strand B2, (B) the analytical LC-ESI-MS trace references of the duplex of nucleic acid strands A2 and B2 without the ring closure reaction, (C) the analytical LC-ESI-MS trace of the nucleic acid strands A2 and B2 duplex after the reaction depicted on Figure 12B.
- Figure 14 shows a denaturing electrophoresis gel of the ring closure formation by copper(l) catalysed azide alkyne cycloaddition in different formats and their corresponding references.
- the lanes are ordered as follows: (A) nucleic acid strand A1 , (B) nucleic acid strand B1 , (C) nucleic acid strands A1 and B1 reference mixture, (D) Ring closure formation of nucleic acid strand A1 and strand B1 (Format 1), (E) nucleic acid strand B2, (F) nucleic acid strands A1 and B2 reference mixture, (G) Ring closure formation of nucleic acid strand A1 and strand B2 (Format 2), (H) nucleic acid strands A2 and B2 reference mixture, (I) Ring closure formation of nucleic acid strand A2 and strand B2 (Format 3).
- Figure 15 shows (A) the synthesis scheme of the 5’-amino-modified oligonucleotide scaffold conjugates (nucleic acid strand C1) by coupling 14 tri-functional carboxylic acid scaffolds (first chemical moiety, BB1) onto custom Elib5_Code1 oligonucleotides using s-NHS/EDC as coupling reagents in TEA/HCI buffer.
- the resulting oligonucleotides conjugates were then Fmoc-deprotected to obtain the 14 tri-functional nucleic acid strands C1 that were pooled in equimolar quantity, giving the nucleic acid strands C1 Pool, (B) the synthesis scheme of the encoding step by the ligation of 293 custom 5’-phosphorylated oligonucleotides Elib5_Code2 onto the pool of 14 5’-amino-modified tri-functional carboxylic acid scaffolds (nucleic acid strand C1), giving 293 Elib5 sub-pools, (C) the synthesis scheme of the nucleic acid strands C2 sub-library by coupling 293 carboxylic acids (second chemical moiety, BB2) to the corresponding Elib5 sub-pools using DMT-MM as coupling reagent in MOPS buffer, yielding the final nucleic acid strands C2 sub-library.
- B the synthesis scheme of the encoding step by the ligation
- Figure 16 shows (A) the synthesis scheme of the nucleic acid strand D1 by coupling the 5-hexynoic acid onto the 3’-amino-modified, 5’-phosphorylated d-spacer oligonucleotide using DMT-MM as coupling reagent in Borate buffer and (B) the synthesis scheme of the encoding step by the ligation of a custom Elib6_Code3 oligonucleotide onto nucleic acid strand D1 , giving the encoded oligonucleotide alkynyl conjugate nucleic acid strand D2.
- Figure 17 shows the synthetic scheme of the ring closure of the nucleic acid strands C2 sub-library and nucleic acid strand D2 by 1 ,2,3-triazole cyclisation reaction using copper(l) catalyst to obtain the format 4.
- Figure 18 shows, respectively, (A) the analytical LC-ESI-MS trace references of the nucleic acid strands C2 sub-library, (B) the analytical LC-ESI-MS trace references of the nucleic acid strand D2, (C) the analytical LC-ESI-MS trace references of the mixture of the nucleic acid strands C2 sub-library and D2 without the ring closure reaction and (D) the analytical LC-ESI-MS trace of the nucleic acid strands C2 sub-library and nucleic acid strand D2 mixture after reaction depicted on Figure 17, to obtain Format 4.
- Figure 19 shows (A) the analytical LC-ESI-MS trace references of the mixture of nucleic acid strands C2 sub-library and D2 without the ring closure reaction at 80 °C (maximum afforded by the device), unlike all the previous figures, depicting the LC-ESI-MS traces at 60 °C and (B) the analytical LC-ESI-MS trace at 80 °C of the nucleic acid strands C2 sub-library and nucleic acid strand D2 mixture after reaction depicted on Figure 17 to obtains the Format 4.
- Figure 20 shows a denaturing electrophoresis gel of the ring closure formation of the format 4 by copper(l) catalysed azide alkyne cycloaddition and their corresponding references.
- the lanes are ordered as follows: (A) Pool of 4 ⁇ 02 Elib5 conjugates (nucleic acid strands C2 sub-library), (B) encoded alkyne conjugate (nucleic acid strand D2), (C) nucleic acid strands C2 sub-library and nucleic acid strand D2 mixture reference and (D) ring closure formation of C2 and D2 (Format 4).
- Figure 21 shows (A) the synthesis scheme of the Klenow fill-in of the nucleic acid strands C2 sub-library into nucleic acid strands C3 sub-library, constructing the DNA duplex used for the ring closure reaction and (B) the synthesis scheme of the ring closure of the nucleic acid strands C3 sub-library and nucleic acid strand D2 by 1 ,2,3-triazole formation reaction using copper(l) catalyst.
- Figure 22 shows (A) the analytical LC-ESI-MS trace references of the nucleic acid strand D2, (B) the analytical LC-ESI-MS trace references of the duplex of nucleic acid strands C3 sub-library and nucleic acid strand D2 without the ring closure reaction and (C) the analytical LC-ESI-MS trace of the nucleic acid strands C3 sub-library and nucleic acid strand D2 duplex after the reaction depicted in Figure 21 B (Format 5).
- Figure 23A shows the synthesis scheme of the Klenow fill-in of the strands C2 sub-library using a single custom YL_Code3 to yield the single-pharmacophore DNA duplex (Format 6A).
- Figures 23B, 23C and 23D show respectively the analytical LC-ESI-MS trace references of the strands C2 sub-library, the YL_Code3 and the single-pharmacophore DNA duplex (Format 6A). The analyses have been performed at 60 °C, except in the case of Figure 23D, to see the denaturation of the Format 6A at 80 °C.
- Figure 24 shows the synthesis scheme of the 5’-amino-modified oligonucleotide azide acid conjugate (strands F1) by coupling the acid alkynes onto the d-spacer oligonucleotide using DMT-MM as coupling reagent in Borate buffer ( Figure 24A), s-NHS/EDC as coupling reagents in MOPS buffer ( Figure 24B) and DMT-MM as coupling reagent in MOPS buffer ( Figure 24C).
- Figure 25A shows the synthesis scheme of the encoding step by the ligation of custom oligonucleotides Elib6_Code3#1-n onto 5’-phosphorylated, 3’-amino-modified alkynyl acid oligonucleotide conjugates (strands F1), giving strands F2.
- Figure 25B shows the synthesis scheme of the Klenow fill-in of the strands C2 sublibrary using the strands F2 sub-library to yield the dual-pharmacophore DNA duplex (Format 6B) composed of the strands F2 sub-library and the strands C3B sub-library.
- Figures 25C, 25D and 25E show respectively the analytical LC-ESI-MS trace references of the strands C2 sub-library, the strands F2 sub-library and the dual-pharmacophore DNA duplex (Format 6B).
- Figure 26A shows the synthetic scheme of the ring closure of strands C3B and strands F2 sub-libraries by 1 ,2,3-triazole formation reaction using copper(l) catalyst, yielding a cyclised dual-pharmacophore library (Format 6C).
- Figures 26B and 26C show respectively the analytical LC-ESI-MS trace references of the dualpharmacophore DNA duplex (Format 6B) and cyclised dual-pharmacophore library (Format 6C).
- Figure 27 shows the electrophoresis denaturing gel of the three different library formats (Formats 6A, 6B and 6C) and their corresponding references.
- the lanes are ordered as follow: (a) strands C2 sub-library, (b) strands F2 sub-library, (c) strands C2 and F2 sub-libraries mixture, (d) Ring closure of strands C2 and strands F2 sub-libraries (no Klenow fill-in), (e) strands C3B and strands F2 sub-libraries (Format 6B), (f) Ring closure of filled-in strands C3B and strands F2 sub-libraries (Format 6C), (g) strands C2 sub-library, (h) YL_Code3, (i) Klenow fill-in of strands C2 sub-library and YL_Code3 (Format 6A).
- Figures 28A and 28B show the DNA constructs built by the PCR1 of the single-pharmacophore (Format 6A) and the dual-pharmacophores (Formats 6B and 6C) respectively.
- Figure 29 shows the agarose gel of the PCR#2 pool, merging all formats (6A, 6B and 6C) before the gel extraction.
- Figure 30 shows the fingerprints of the Format 6A after sequencing of the affinity selections. Codes A, B and C on the figure correspond to Codes 1 , 2 and 3, respectively.
- Figure 30A shows the Format 6A library selection without CAIX, highlighting the non-specific binders.
- Figure 30B shows the Format 6A selection against CAIX.
- Figure 31 shows the fingerprints of the Format 6B after sequencing of the affinity selections. Codes A, B and C on the figure correspond to Codes 1 , 2 and 3, respectively.
- Figure 31 A shows the Format 6B library selection without CAIX, highlighting the non-specific binders.
- Figure 31 B shows the Format 6B selection against CAIX.
- Figure 32 shows the fingerprints of the Format 6C after sequencing of the affinity selections. Codes A, B and C on the figure correspond to Codes 1 , 2 and 3, respectively.
- Figure 32A shows the Format 6C library selection without CAIX, highlighting the non-specific binders.
- Figure 32B shows the Format 6C selection against CAIX.
- Figure 33A shows the most enriched compounds from the fingerprints of Format 6A.
- the fingerprint depicts two carboxylic acids, 6 and 53, as building block 2, described in Example 5.4.
- the building blocks 1 described in Example 5.2, do not show any preferential number.
- the single-pharmacophore setting displays preferential building blocks 2, keeping flexibility in respect of building blocks 1.
- Figure 33B shows the most enriched compounds from the fingerprints of Format 6B.
- the fingerprint depicts only the carboxylic acid 6 as building block 2, described in Example 5.4.
- the building blocks 1 described in Example 5.2, and building blocks 3, described in Example 8.2, do not show any preferential number.
- the dual-pharmacophore setting locks the carboxylic acid 6 as building block 2, inducing a rigidity of the structures.
- Figure 33C shows the most enriched compounds from the fingerprints of Format 6C.
- the fingerprint depicts only the carboxylic acid 6 as building block 2, described in Example 5.4, and the tri-functional scaffold 4 (the 4 th scaffold among the 14 described in the synthesis of the strands C2 sub-library in Example 5) as building block 1 , described in Example 5.2.
- the full set of acid alkynes, described in Example 8.2, is shown as building block 3.
- the cyclised dual-pharmacophore setting increases the rigidity by locking carboxylic acid 6 as building block 2 described in Example 5.4 and tri-functional scaffold 4 as building block 1 described in Example 5.2.
- Figure 34A shows the synthesis scheme of the 5’-amino-modified oligonucleotide scaffold conjugates (strand G1) by coupling tri-functional carboxylic acid scaffolds onto custom Elib5_Code1. The resulting
- FIG. 34B shows the synthesis scheme of the encoding step by the ligation of custom 5’-phosphorylated oligonucleotides Elib5_Code2 onto the pool of 5’-amino- modified tri-functional carboxylic acid scaffolds, yielding Elib5 sub-pools (Elib5-G sub-pools).
- Figure 34C shows the synthesis scheme of the strands G2 sub-library by coupling carboxylic acids to the corresponding Elib5 sub-pools, yielding the final strands G2 sub-library.
- Figure 35A shows the synthesis scheme of the strands H1 by coupling azide acids onto the 3’-amino- modified, 5’-phosphorylated d-spacer.
- Figure 35B shows the synthesis scheme of the encoding step by the ligation of a custom oligonucleotide Elib6_Code3 onto the strands H1 to obtain the strands H2 that were pooled in equimolar quantity, yielding the strands H2 sub-library.
- Figure 36A shows the synthesis scheme of the Klenow fill-in of the strand G2 to strand G3, yielding the DNA duplex used for the ring closure reaction.
- Figure 36B shows the synthesis scheme of the ring closure of the strands G3 and H2 by 1 , 2, 3-triazole formation reaction (Format 7).
- Figure 37A shows the synthesis scheme of the 5’-amino-modified oligonucleotide amino acid conjugates (strand 11) by coupling N-Fmoc protected amino acids onto custom Elib5_Code1. The resulting
- FIG. 37B shows the synthesis scheme of the encoding step by the ligation of custom 5’-phosphorylated oligonucleotides Elib5_Code2 onto the pool of 5’-amino- modified amino-conjugates, yielding Elib5 sub-pools (Elib5-I sub-pools).
- Figure 37C shows the synthesis scheme of the strands I2 sub-library by coupling acid azides to the corresponding Elib5 sub-pools, yielding the final strands I2 sub-library.
- Figure 38A shows the synthesis scheme of the strands F1 by coupling acid alkynes onto the 3’-amino- modified, 5’-phosphorylated d-spacer.
- Figure 38B shows the synthesis scheme of the encoding step by the ligation of a custom oligonucleotide Elib6_Code3 onto strands F1 , to obtain the strands F2 that were pooled in equimolar quantity, yielding the final strands F2 sub-library.
- Figure 39A shows the synthesis scheme of the Klenow fill-in of the strand 12 to strand 13, yielding the DNA duplex used for the ring closure reaction.
- Figure 39B shows the synthesis scheme of the ring closure of the strands 13 and F2 by 1 , 2, 3-triazole formation reaction (Format 8).
- Figure 40A shows the synthesis scheme of the 5’-amino-modified oligonucleotide amino acid conjugates (strand J1) by coupling N-Fmoc protected amino acids onto custom Elib5_Code1. The resulting
- Figure 40B shows the synthesis scheme of the encoding step by the ligation of custom 5’-phosphorylated oligonucleotides Elib5_Code2 onto the pool of 5’-amino- modified amino-conjugates, yielding Elib5 sub-pools (Elib5-J sub-pools).
- Figure 40C shows the synthesis scheme of the strands J2 sub-library by coupling acid alkynes to the corresponding Elib5 sub-pools, yielding the final strands J2 sub-library.
- Figure 41A shows the synthesis scheme of the Klenow fill-in of the strand J2 to strand J3, yielding the DNA duplex used for the ring closure reaction.
- Figure 41 B shows the synthesis scheme of the ring closure of the strands J3 and H2 by 1 ,2,3-triazole formation reaction (Format 9).
- Figure 42A shows the synthesis scheme of the 5’-amino-modified oligonucleotide acid ester conjugates (strand K1) by coupling acid esters onto custom Elib5_Code1. The resulting oligonucleotides conjugates were then hydrolysed to obtain free-acido strands K1 that were pooled in equimolar quantity, yielding the strands K1 Pool.
- Figure 42B shows the synthesis scheme of the encoding step by the ligation of custom 5’- phosphorylated oligonucleotides Elib5_Code2 onto the pool of 5’-amino-modified acido-conjugates, yielding Elib5 sub-pools (Elib5-K sub-pools).
- Figure 42C shows the synthesis scheme of the strands K2 sub-library by coupling amine azides to the corresponding Elib5 sub-pools, yielding the final strands K2 sub-library.
- Figure 43 shows the synthesis scheme of the Klenow fill-in of the strand K2 to strand K3, yielding the DNA duplex used for the ring closure reaction (upper panel) and the synthesis scheme of the ring closure of the strands K3 and F2 by 1 ,2,3-triazole formation reaction (Format 10; lower panel).
- Figure 44A shows the synthesis scheme of the 5’-amino-modified oligonucleotide acid ester conjugates (strand L1) by coupling acid esters onto custom Elib5_Code1. The resulting oligonucleotides conjugates were then hydrolysed to obtain free-acido strands L1 that were pooled in equimolar quantity, yielding the strands L1 Pool.
- Figure 44B shows the synthesis scheme of the encoding step by the ligation of custom 5’- phosphorylated oligonucleotides Elib5_Code2 onto the pool of 5’-amino-modified acido-conjugates, yielding Elib5 sub-pools (Elib5-L sub-pools).
- Figure 44C shows the synthesis scheme of the strands L2 sub-library by coupling amine alkynes to the corresponding Elib5 sub-pools, yielding the final strands L2 sub-library.
- Figure 45 shows the synthesis scheme of the Klenow fill-in of the strand L2 to strand L3, yielding the DNA duplex used for the ring closure reaction (upper panel) and the synthesis scheme of the ring closure of the strands L3 and H2 by 1 ,2,3-triazole formation reaction (Format 11 ; lower panel).
- Table 1 shows the conditions for copper (l)-catalysed 1 , 2, 3-triazole-forming reactions to achieve the ring closure in between diverse population of chemical moieties displayed at the extremities of the two DNA strands wherein an azide modified chemical moiety is coupled to a nucleic acid strand A and an alkyne modified chemical moiety is coupled to the second nucleic acid strand B.
- Table 2 shows the m/z results of a) nucleic acid strand A comprising the azide modified building block, b) nucleic acid strand B comprising the alkyne modified building block, and c) the ring closure example of nucleic acid strands A and B, and d) of the nucleic acid strands A and B without ring closure reagents.
- This invention relates to the production of a nucleic acid encoded chemical library through the hybridisation of a population of first conjugates comprising a first nucleic strand coupled to a first set of one or more chemical moieties, and a first reactive group to a population of second conjugates comprising a second nucleic strand coupled to a second set of one or more chemical moieties, and a second reactive group to produce double stranded molecules comprising first and second sets of chemical moieties.
- the first and second sets of chemical moieties may be located at an end of the double stranded molecules.
- the first and second reactive groups are then reacted to covalently link the first and second conjugates through ring closure, for example“on the top” of the sets of chemical moieties, and producing cyclised pharmacophores of high purity located at the end of the double stranded molecules that comprise the first and second sets of chemical moieties covalently linked together.
- the population of pharmacophores coupled to the double stranded molecules forms a nucleic acid-encoded chemical library.
- the methods described herein allow the generation of libraries of large pharmacophores that are both highly pure and highly diverse.
- nucleic acid encoded chemical library as described herein comprise a double-stranded nucleic acid molecule linked at an end to a pharmacophore that comprises a first set of chemical moieties coupled to the first nucleic acid strand and covalently linked to a second set of chemical moieties coupled to the second nucleic acid strand.
- one of the nucleic acid strands comprises coding sequences that encode the chemical moieties that constitute the pharmacophore displayed by the library member.
- a member of a nucleic acid encoded chemical library may be produced by a method comprising;
- first conjugate comprising a first nucleic strand coupled to a first reactive group and a first set of one or more chemical moieties
- the first and second conjugates may be pure (i.e. they may display high purity) and may be used to generate a pure library member.
- a nucleic acid-encoded library is a collection of library members, each of which displays a cyclised pharmacophore that is made up of one or more chemical moieties. The identity of the chemical moieties that constitute the pharmacophore is encoded into each library member through a nucleic acid strand that incorporates coding sequences that allow the identification of the chemical moieties in the pharmacophore. The members of the library display a diverse population of pharmacophores. This allows the screening of a large number of pharmacophores. For example, a nucleic acid-encoded library may comprise 10 6 or more different pharmacophores for screening.
- members of a nucleic acid-encoded chemical library may be formed from two nucleic acid strands (a first and a second strand) and two or more chemical moieties.
- the two or more chemical moieties may be composed of a first set of one or more chemical moieties coupled to a first nucleic strand and a second set of one or more chemical moieties coupled to a second nucleic strand.
- the first nucleic strand may be hybridised to the second nucleic acid strand to bring the first and second sets of chemical moieties into proximity.
- the first and second sets of chemical moieties are then covalently linked through a ring closure, for example on the top of the two strand extremities, to form the cyclised
- the first and second sets of chemical moieties are diverse.
- the population of first conjugates and the population of second conjugates may be sub-libraries which may be combined through hybridisation and covalent linkage as described herein to generate a nucleic acid-encoded library.
- the populations of first and second conjugates may be pure i.e. the populations may be essentially free of contaminant molecules, such as reactants and by-products. For example, 70% or more, 80% or more 90% or more, 95% or more, 99% or more or 99.5% or more of the molecular species in the populations may be conjugates as described herein. Purity may be determined by standard analytic techniques, such as HPLC, SDS-PAGE and MS. For example, purity may be expressed as the percentage of measured peak area of the first and second conjugates combined from the sum of peak area of all peaks, for example the sum of the peaks area of the first conjugate, the second conjugate and the contaminant molecule. The peak area may be measured under standard detection conditions, for example HPLC or MS conditions described elsewhere herein.
- the conjugates that form the library members described herein each comprise a nucleic strand, a set of one or chemical moieties and a reactive group.
- the set of chemical moieties may be coupled to an end of the nucleic acid strand.
- the reactive group may be coupled to the end of the nucleic acid strand or more preferably may be coupled to the set of chemical moieties.
- the first conjugate may thus comprise a first nucleic strand, a first set of one or more chemical moieties and a first reactive group.
- the second conjugate may comprise a second nucleic strand, a second set of one or more chemical moieties and a second reactive group.
- a nucleic acid strand is a polynucleotide chain (e.g.
- nucleic acid strands of the conjugates may be DNA, RNA or chimeric RNA/DNA.
- the nucleic acid strand(s) in the chemical libraries described herein are DNA.
- the first and second nucleic acid strands may comprise one or more complementary regions that comprise complementary nucleotide sequences.
- the first nucleic acid strand of the first conjugate may hybridise to the second nucleic acid strand of the second conjugate through the complementary regions in the two strands to form a double-stranded molecule.
- the first nucleic acid strand may comprise coding sequences that encode the chemical moieties in the first set.
- the second nucleic acid strand may comprise coding sequences that encode the chemical moieties in the second set.
- the first nucleic acid strand may further comprise coding sequences that encode the chemical moieties in the second set.
- the second nucleic acid strand may further comprise coding sequences that encode the chemical moieties in the first set.
- a coding sequence may encode one chemical moiety or more than one chemical moiety, for example two chemical moieties.
- Suitable methods for the incorporation of the coding sequences for the second set of chemical moieties into the first nucleic acid strand or the coding sequences for the first set of chemical moieties into the second nucleic acid strand are known in the art and include for example ligation or extension using a polymerase, as discussed below.
- a coding sequence can be any sequence of nucleic acid bases that is uniquely associated with a particular chemical moiety. This allows the identity of the chemical moiety to be determined by sequencing or otherwise‘reading’ the coding sequence.
- a coding sequence may encode one chemical moiety or more than one chemical moiety. Preferably a coding sequence encode one or two chemical moieties.
- coding sequences that are longer than necessary are that they provide the opportunity to differentiate codes by more than just a single nucleotide difference, which gives more confidence in the decoding process.
- a first chemical moiety from a population of 20 different moieties (20 compounds) may be encoded by 6 nucleotides
- a second chemical moiety from a population of 200 different moieties may be encoded by 8 nucleotides.
- the length of the coding sequence therefore depends on the number of chemical moieties to be encoded (i.e. the number of different chemical moieties in the library).
- a sequence of nucleotides and/or its complement may be used as a coding sequence to encode a chemical moiety.
- Suitable sequences for encoding chemical moieties in a library are well-known in the art. Examples of suitable coding sequences are shown in SEQ ID NOs: 1 , 3, 4, 6, 7, 9, 11 and 12.
- the second nucleic acid strand may not be complementary to the first nucleic acid strand when the strands are hybridised together in the double stranded nucleic acid molecule at positions where the coding sequences are located in the first nucleic acid strand.
- the second nucleic acid strand may comprise one or more spacer regions.
- the first nucleic acid strand may not be complementary to the second nucleic acid strand when the strands are hybridised together in the double stranded nucleic acid molecule at positions where the coding sequences are located in the second nucleic acid strand.
- the first nucleic acid strand may comprise one or more spacer regions.
- the spacer region is non-hybridisable and may be called a non-hybridisable spacer (also named d- spacer).
- the spacer region may be located in one of the first and second nucleic acid strands at a position that would otherwise hybridise with a coding sequence located in the other of the first and second nucleic acid strands in the double stranded nucleic acid molecule.
- regions in one of the first and second nucleic acid strands that are complementary to all of the coding sequences in the other of the first and second nucleic acid strands may be replaced by spacer regions.
- the non-hybridisable spacers may be located at positions in one of the first and second nucleic acid strands that correspond, when the first and second strands are hybridised together, to the positions of the coding sequences in the other of the first and second nucleic acid strands.
- a nucleic acid strand containing one or more spacer regions at positions corresponding to coding sequences may hybridise to nucleic acid strands containing different coding sequences. This may be useful in the production of diversity in self-assembling libraries.
- the spacer region (or d-spacer) is an abasic region that does not hybridise to nucleotide sequences and is not a template for a nucleic acid polymerase.
- Suitable spacer regions may comprise an abasic linker, such as an abasic phosphodiester backbone or a linker, such as an alkyl chain, polyethylene glycol or other oligomer that spans the spacer region.
- Suitable spacer regions may be obtained from commercial suppliers.
- An example of a spacer region or d-spacer sequence is shown in SEQ ID NO: 2
- nucleic acid strands comprising spacer regions, coding sequences and complementary regions are known in the art (see for example W02003/076943, W02009/077173, WO2015/091207; and W. Decurtins et al. Nat. Protoc. 11, 764-780 (2016); M. Wichert et al Nat. Chem., 7, 241-249 (2015)).
- a nucleic acid strand in a conjugate described herein may be coupled to a set of one or more chemical moieties.
- the first and second sets of chemical moieties may be coupled to the first and second nucleic acid strands directly or via a linker.
- the first set of chemical moieties may be coupled to one of the 5’ and 3’ ends of the first nucleic acid strand in the first conjugate and the second set of chemical moieties may be coupled to the other of the 5’ and 3’ ends of the second nucleic acid strand in the second conjugate.
- the two sets of chemical moieties may be located at the same end of the double stranded molecule following hybridisation of the first and second nucleic acid strands. This facilitates the reaction of the first and second reactive groups to covalently link the sets of chemical moieties through a ring closure and form the cyclised pharmacophore.
- the first and/or second sets of chemical moieties may be diverse.
- the first and/or second conjugates that form the library members may themselves be members of a sub-library, each nucleic acid strand in the population of first and/or second conjugates being coupled to a different combination of chemical moieties.
- a sub-library of conjugates may comprise different sets of chemical moieties coupled to nucleic acid strands.
- the conjugates in a sub-library may assemble through hybridisation of the first and second nucleic acid strands with conjugates from the same sub-library or a different sub-library, for example a sub-library of conjugates comprising a different number of chemical moieties, to produce a double-stranded library.
- nucleic acid strands coupled to a set containing a single chemical moiety may assemble with nucleic acid strands coupled to a set containing two chemical moieties, thereby presenting, following reaction of the reactive groups, a pharmacophore consisting of three chemical moieties and a covalent linkage.
- nucleic acid strands coupled to a first set of two or more chemical moieties may assemble with nucleic acid strands coupled to a second set of two or more chemical moieties, thereby presenting, following reaction of the reactive groups, a cyclised pharmacophore consisting of four or more chemical moieties and a covalent linkage.
- a set of chemical moieties may comprise 1 , 2, 3, 4, 5 or more chemical moieties.
- each of the first and second sets of chemical moieties may comprise 1 , 2, 3 or more chemical moieties.
- the reaction of the first and second reactive groups to form a covalent linkage generates a cyclised pharmacophore through a ring closure, for example on the top of the two strand extremities.
- the cyclised pharmacophore may comprise the first and second sets of chemical moieties and the covalent linkage.
- the covalent linkage in between the two conjugates may be part of the pharmacophore or may be part of a branched chemical space between the nucleic acid strand and the pharmacophore.
- the covalent linkage provides one of a pair of ring closures that generate a cyclised population of library members (e.g. one of a top or bottom ring closure, preferably with a ring closure on the top).
- the other of the pair of ring closures (e.g. the other of the top and bottom closures) is provided by the hybridisation of the first and second nucleic acid strands to form a double stranded molecule.
- the presence and identity of the covalent linkage may affect the binding properties of the pharmacophore, and may for example introduce of lipophilic or hydrophilic chemical spaces, and/or additional functional groups into the pharmacophore.
- a nucleic acid-encoded library as described herein may include members with different covalent linkages. This may be useful for example in increasing the diversity contained in the library.
- a pharmacophore is an assembly of molecular features or elements which is capable of specifically interacting with a target. Different combinations of chemical moieties produce different pharmacophores which are displayed by different members of the library. In addition, different covalent linkages may also produce different pharmacophores which may be combined following production of a library as described herein.
- the pharmacophore may be formed from the first set of chemical moieties on the first nucleic acid strand, the second set of chemical moieties on the second nucleic acid strand and the covalent linkage between the two sets of moieties.
- the chemical moieties within a set on the same nucleic acid strand will be covalently bonded together and the different sets of chemical moieties on different nucleic acid strands will be brought into proximity by the assembly of the nucleic acid strands, followed by ring closure with a covalent linkage through reaction of the reactive groups.
- the reaction of the reactive groups covalently links the sets of chemical moieties to form a cyclised pharmacophore following hybridisation of the first and second strands, to form the library member.
- a chemical library member may display a pharmacophore which comprises or consists of any of 2, 3, 4, 5 or more chemical moieties.
- the chemical moieties may be covalently linked together, for example with a covalent connection at an end of the first and second conjugates (e.g.“on the top” of the two conjugates).
- Both nucleic acid strands may be coupled to one or more chemical moieties in the pharmacophore.
- a first strand may be coupled to the first set of chemical moieties and the second strand may be coupled to the second set of chemical moieties and the chemical moieties in the sets may be covalently linked by reaction of the reactive groups.
- the covalent linkage itself may form part of the pharmacophore.
- a pharmacophore presented on a double stranded nucleic acid molecule may form a closed structure through the covalent linkage and the non-covalent hybridisation of the nucleic acid strands and may be described as cyclic.
- the total molecular weight of the chemical moieties in the cyclised pharmacophore may be less than 3kD, preferably less than 1 kD, more preferably less than 500Da.
- Suitable chemical moieties include small organic molecules, amino acid residues or other amino- containing moieties (optionally with appropriate amino protection); and peptides or globular proteins (including antibody domains).
- a chemical moiety may have a molecular weight of 300 Da or less, for example about 100 to 300 Da.
- Populations of chemical moieties for use in the generation of libraries are well-known in the art (see for example W. Decurtins et al. Nat. Protoc. 11, 764-780 (2016); M. Wichert et al Nat. Chem., 7, 241-249 (2015) ; Mannocci et al., PNAS 105, 17670-17675 (2008); Mannocci et al., Bioconj. Chem.
- a set of chemical moieties may be covalently coupled to the nucleic acid strand directly or indirectly, for example via a linker.
- Suitable linkers such as alkyl chains, are well known in the art.
- the chemical moieties may be coupled directly using conventional synthetic chemistries, for example amide or other conventional linkages.
- Chemical moieties may be coupled to a nucleic acid strand via other chemical moieties.
- each of the chemical moieties within a set may be covalently bonded to other chemical moieties and one of the chemical moieties may be coupled to the nucleic acid strand.
- Suitable methods for covalently bonding chemical moieties and coupling chemical moieties to nucleic acid strands are well known in the art (see for example W. Decurtins et al. Nat. Protoc. 11, 764-780 (2016); M. Wichert et al. Nat. Chem., 7, 241- 249 (2015)).
- the first and second sets of chemical moieties may be coupled to the nucleic acid strands such that they are located at the same end of the double-stranded molecule formed by hybridisation of the strands.
- the first set of chemical moieties may be coupled to the 5’ end of the first nucleic acid strand and the second set of chemical moieties may be coupled to the 3’ end of the second nucleic acid strand; or the first set of chemical moieties may be coupled to the 3’ end of the first nucleic acid strand and the second set of chemical moieties may be coupled to the 5’ end of the second nucleic acid strand.
- Hybridisation establishes non-covalent sequence-specific base-pairing between the complementary regions of the first and second nucleic acid strands and brings the set of chemical moieties attached to the strands into proximity.
- the complementary regions of the strands will anneal together such that the strands form a double stranded molecule with the sets of chemical moieties at one end.
- Suitable hybridisation conditions are well-known in the art. Typical hybridisation temperatures for the sequence specific annealing of two polynucleotide strands may be between 4°C and 70°C.
- the first nucleic acid strands of a sub-library of first conjugates may be coupled to a first diverse set of one or more chemical moieties and the second nucleic acid strands of a sub-library of second conjugates may be coupled to a second diverse set of one or more chemical moieties.
- the first and second diverse sets may be the same or different.
- pharmacophores may be generated from the different combinations of the sets of chemical moieties coupled to the nucleic acid strands. This increases the number of different pharmacophores in the library.
- the coding sequences in the second nucleic acid strand may be incorporated into the first nucleic acid strand. This allows the first strand to contain coding sequences for all of the chemical moieties in the first and second sets. The chemical moieties displayed by the library member can thus be identified by sequencing the first nucleic acid strand.
- the hybridisation of the first and second nucleic acid strands may leave a single-stranded overhanging region of the second nucleic acid strand.
- the single-stranded region of the second nucleic acid strand may comprise coding sequences encoding the set of chemical moieties attached to the second strand.
- a method may comprise;
- the first nucleic acid strand incorporates the complement of the coding sequences in the second nucleic acid strand.
- the coding sequences in the first nucleic acid strand may be incorporated into the second nucleic acid strand. This allows the second strand to contain coding sequences for all of the chemical moieties in the first and second sets.
- the chemical moieties displayed by the library member can thus be identified by sequencing the second nucleic acid strand.
- the hybridisation of the first and second nucleic acid strands may leave a single-stranded overhanging region of the first nucleic acid strand.
- the single-stranded region of the first nucleic acid strand may comprise coding sequences encoding the set of chemical moieties attached to the first strand.
- a method may comprise;
- the second nucleic acid strand incorporates the complement of the coding sequences in the first nucleic acid strand.
- Suitable techniques for 5’ to 3’ extension of nucleic acid strands along a template nucleic acid strand are well known in the art.
- the second nucleic acid strand may be extended by addition of nucleotides for polymerisation (normally in excess), preferably deoxynucleotides (dNTPs), and a polymerase (e.g. Taq or Klenow polymerase) in a suitable buffer, incubated at a suitable temperature (e.g. 37°C for Klenow polymerase or 65°C or 72°C for Taq).
- the coding sequences in the second nucleic acid strand may be incorporated into the first nucleic acid strand by ligation. This allows the first strand to contain coding sequences for all of the chemical moieties in the first and second sets.
- the chemical moieties displayed by the library member can thus be identified by sequencing the first nucleic acid strand.
- the hybridisation of the first and second nucleic acid strands may leave a single-stranded overhanging region of the second nucleic acid strand.
- the single- stranded region of the second nucleic acid strand may comprise coding sequences encoding the set of chemical moieties attached to the second strand.
- a method may comprise;
- a coding oligonucleotide comprising the complement of the coding sequences in the second nucleic acid strand
- the coding sequences in the first nucleic acid strand may be incorporated into the second nucleic acid strand by ligation. This allows the second nucleic acid strand to contain coding sequences for all of the chemical moieties in the first and second sets.
- the chemical moieties displayed by the library member can thus be identified by sequencing the second nucleic acid strand.
- the hybridisation of the first and second nucleic acid strands may leave a single-stranded overhanging region of the first nucleic acid strand.
- the single-stranded region of the first nucleic acid strand may comprise coding sequences encoding the set of chemical moieties attached to the first strand.
- a method may comprise;
- a coding oligonucleotide comprising the complement of the coding sequences in the first nucleic acid strand
- the coding oligonucleotide may be ligated to the first or second nucleic acid strand by any convenient technique.
- an adaptor oligonucleotide may be used.
- the nucleic acid strand may comprise a proximal end that is coupled to the chemical moiety, for example the 5' end, and a distal end to which the coding sequence is added, for example the 3' end.
- the nucleic acid strand may further comprise an annealing region which hybridises with an adaptor oligonucleotide. The annealing region may be located adjacent the distal end of the nucleic acid strand to facilitate ligation of the coding oligonucleotide.
- An adaptor oligonucleotide may serve as a template to facilitate the ligation of the first or second nucleic acid strand and the coding oligonucleotide.
- a single adaptor oligonucleotide may facilitate the ligation of multiple nucleic acid strands and coding oligonucleotides.
- a set consisting of 1 , 2, 3, 4, 5 or more adaptor oligonucleotides may be used to facilitate ligation of all of the nucleic acid strands in the sub-library to coding oligonucleotides.
- the sequence of the adaptor oligonucleotide is the same regardless of the chemical moiety(s) coupled to the nucleic acid strand i.e. only 1 adaptor oligonucleotide is used. This reduces the total number of oligonucleotides required to generate the nucleic acid encoded chemical library.
- the adaptor oligonucleotide hybridises with the nucleic acid strand and the coding oligonucleotide and brings the ends of the nucleic acid strand and coding sequence into association within a double-stranded trimeric complex, such that they can be ligated together by a ligase.
- the adaptor oligonucleotide may bring into association the 3' end of the nucleic acid strand to the 5' end of the coding oligonucleotide or the 5' end of the nucleic acid strand to the 3' end of the coding oligonucleotide.
- Suitable hybridisation conditions for the hybridisation of polynucleotides are well-known in the art and include for example a temperature of between 0°C and 70°C. Suitable ligation conditions are well-known in the art.
- the adaptor oligonucleotide may be DNA, RNA or chimeric (i.e. containing both deoxyribonucleotides and ribonucleotides).
- a suitable adaptor oligonucleotide may, for example be 10 to 35 bases, preferably 14 to 30 bases in length. Examples of suitable adaptor oligonucleotide are shown in SEQ ID NOs: 5, 8 and 10.
- Suitable adaptor oligonucleotides may be synthesized using appropriate techniques.
- the adaptor may remain hybridised to the first nucleic acid strand, for example within a nucleic acid spacer strand, and may form part of the library member that is produced.
- the adaptor may be removable or removed by purification following the ligation step.
- adaptor may be separated under denaturing conditions on the basis of their small size relative to the nucleic acid strand incorporating the identifier oligonucleotide. More preferably, the adaptor oligonucleotide may be cleavable.
- the adaptor oligonucleotide may be cleaved enzymatically, for example using RNAase, or chemically, for example by base hydrolysis (typically, exposure to pH>12 at room temperature or greater).
- the nucleic acid strand may be purified following removal of the adaptor for example to remove fragments of a cleaved or degraded adaptor. Suitable purification methods are well known in the art.
- the first and second conjugates are covalently linked in the libraries described herein by reaction of the first reactive group on the first conjugate with the second reactive group on the second conjugate.
- the reactive groups may be located at the same end of the nucleic strands as the sets of chemical moieties.
- the sets of chemical moieties comprise the reactive groups.
- the first set of one or more chemical moieties may comprise the first reactive group and/or the second set of one or more chemical moieties may comprise the second reactive group.
- one or both of the first and second sets may comprise a chemical moiety that is modified to further comprise a reactive group (i.e. a reactive-group modified chemical moiety).
- one or both of the reactive groups may be directly linked to the nucleic acid strands.
- the reactive groups may be linked to the nucleic acid strands or sets of chemical moieties directly or through a linker, such as an alkyl- or polyethylene glycol- chain.
- the first and second reactive groups of the first and second conjugates may be any pair of chemical groups that react together to form a covalent linkage.
- Any convenient chemistry may be employed to react the reactive groups, including for example, a click reaction, such as azide alkyne cycloaddition or alkene hydrothiolation (see respectively for example Liu et al. Beilstein J. Org. Chem. (2016), 14, 2404-2410 and Chalker et al. Chem. Asian J. (2009), 4, 630-640) or 1 ,3- dipolar cycloaddition, nitrone-olefin cycloaddition, Diels Alder reaction (see for example, Buller et al.
- a click reaction such as azide alkyne cycloaddition or alkene hydrothiolation
- 1 ,3- dipolar cycloaddition, nitrone-olefin cycloaddition, Diels Alder reaction see for example, Buller et al.
- Suitable reactive groups may be selected from carboxyl, sulfonyl halide, acyl halide, aryl halide, isocyanate, isothiocyanate, carbonyl, alkyl halide, alkenyl, boronyl, amino, azido, alkynyl and thiol groups.
- covalent linkage may be achieved through click chemistry.
- Suitable click chemistry reactions includel ,3-dipolar cycloaddition, for example azide alkyne cycloaddition (CuAAC), such as copper(l)-catalysed CuAAC.
- CuAAC azide alkyne cycloaddition
- one of the first and second reactive groups may comprise an azido group and the other of the first and second reactive groups may comprise an alkynyl (CoC) group.
- a first reactive group comprising one of an alkynyl or an azido group may react with a second reactive group comprising the other of the alkynyl or the azido group to form covalent linkage via a 1 ,2,3-triazole moiety.
- the first and second conjugates may be admixed in aqueous buffer in the presence of copper salt, such as CuSC> 4 , and, if necessary, reducing agent, at a suitable temperature to effect the azide alkyne cycloaddition of the first and second reactive groups.
- copper salt such as CuSC> 4
- reducing agent at a suitable temperature to effect the azide alkyne cycloaddition of the first and second reactive groups.
- Suitable click chemistry reactions include alkene hydrothiolation (thiol-ene reaction).
- one of the first and second reactive groups may comprise an alkenyl group and the other of the first and second reactive groups may comprise a thiol group.
- a first reactive group comprising one of an alkenyl group and a thiol group may react with a second reactive group comprising the other of the alkenyl or thiol group to form covalent linkage [see for example Chalker et al. Chem. Asian J. (2009), 4, 630-640]
- Covalent linkage may be achieved through 1 , 3-dipolar cycloaddition.
- one of the first and second reactive groups may comprise a diazoalkane group and the other of the first and second reactive groups may comprise a vinyl group.
- a first reactive group comprising one of a diazoalkane group and a vinyl group may react with a second reactive group comprising the other of the diazoalkane group or vinyl group, to form a covalent linkage.
- Covalent linkage may be achieved through nitrone-olefin cycloaddition.
- one of the first and second reactive groups may comprise a nitrone group and the other of the first and second reactive groups may comprise an alkenyl or alkynyl group.
- a first reactive group comprising one of a nitrone group and an alkenyl/alkynyl group may react with a second reactive group comprising the other of the nitrone group and alkenyl/alkynyl group to form a covalent linkage
- Covalent linkage may be achieved through a sulfhydryl/maleimide reaction.
- a first reactive group comprising one of a sulfhydryl or maleimide group may react with a second reactive group comprising the other of the sulfhydryl or maleimide group to form a 3-thiosuccinimidyl ether linkage [see for example Chalker et al. Chem. Asian J. (2009), 4, 630-640]
- Covalent linkage may be achieved through a Diels-Alder Cycloaddition reaction.
- a first reactive group comprising one of a dienyl or imine group and an alkenyl group may react with a second reactive group comprising the other of the dienyl or imine group and the alkenyl group to form a substituted cyclohexene linkage.
- the first and second conjugates may be admixed in aqueous buffer in the presence, if necessary, of a suitable condensing agent at a suitable temperature to effect the cycloaddition reaction [see for example, Buller et al. Bioorganic & Medicinal Chemistry Letters (2008), 18, 5926-5931 ]
- Covalent linkage may be achieved through an amination reaction.
- a first reactive group comprising one of an amine group and an carbonyl group or activated version thereof (e.g. ester, acid anhydride, acid halide or activated ester such as N-hydroxysuccinimide ester) may react with a second reactive group comprising the other of the amine group or the carbonyl group or activated version thereof to form an amide linkage.
- a solution of one of the first and second conjugates may be prepared in DMSO and it is added to the other of the first and second conjugates in aqueous buffer in the presence, if necessary, of a suitable condensing agent and shacked at a suitable temperature [see for example Y. Li et al. ACS Comb. Sci. (2016) 18, 438-444]
- Covalent linkage may be achieved through reductive amination/reductive alkylation.
- one of the first and second reactive groups may comprise a carbonyl group, such as an aldehyde group, and the other of the first and second reactive groups may comprise an amino group.
- a first reactive group comprising one of a carbonyl group and an amino group may react with a second reactive group comprising the other of the carbonyl group and amino group to form a covalent linkage
- Covalent linkage may be achieved through Suzuki cross-coupling.
- a first reactive group comprising one of a boronyl group and a halide group, such as I, OTf, Br or Cl, may react with a second reactive group comprising the other of the boronyl or halide group, to form a covalent linkage [see for example Li J. Y. et al. Bioconjugate Chem. (2016) 29 (1 1), 3841 -3846]
- Covalent linkage may be achieved through disulfide formation.
- first and second reactive groups comprising thiol groups may react together to form a disulfide linkage.
- Covalent linkage may be achieved through cyclic sulfide formation.
- a first reactive group comprising one of an alkenyl group and a disulfide group
- a second reactive group comprising the other of the alkenyl group and disulfide group to form a cyclic sulfide linkage.
- Covalent linkage may be achieved through ether bond formation.
- a first reactive group comprising one of a hydroxyl group and a halide group may react with a second reactive group comprising the other of the hydroxyl group and a halide group to form an ether linkage.
- Covalent linkage may be achieved through urea/thiourea formation.
- a first reactive group comprising one of an amino group and an isothiocyanate or isocyanate group may react with a second reactive group comprising the other of the amine group and isothiocyanate/isocyanate group to form a urea or thiourea linkage.
- a solution of one of the first and second conjugates may be prepared in DMSO or CFhCN and it is added to the other of the first and second conjugates in aqueous buffer and shacked at a suitable temperature.
- Covalent linkage may be achieved through a photo-redox decarboxylative reaction.
- a first reactive group comprising one of a carboxylic group and a vinyl group may react with a second reactive group comprising the other of the carboxylic group and vinyl group to produce a carbon-carbon linkage.
- Covalent linkage may be achieved through a metathesis reaction.
- first and second reactive groups comprising alkenyl groups may react together to form a carbon-carbon linkage.
- Covalent linkage may be achieved through an Eglinton or Hay reaction.
- first and second reactive groups comprising alkynyl groups may react together to form a carbon-carbon linkage.
- Covalent linkage may be achieved through sulfonylation reaction.
- a first reactive group comprising one of a sulfonyl halide group and an amino group may react with a second reactive group comprising the other of the sulfonyl halide or amino group, to form a sulfonamide linkage.
- Covalent linkage may be achieved through an alkylation reaction.
- a first reactive group comprising one of an amino group and an alkyl halide group may react with a second reactive group comprising the other of the amino and alkyl halide group to form an amino linkage.
- Covalent linkage may be achieved through carbamate formation.
- a first reactive group comprising one of an amino group and a carbonyldiimidazole group may react with a second reactive group comprising the other of the amino and carbonyldiimidazole group to form a carbamate linkage.
- Covalent linkage may be achieved through an acylation reaction.
- a first reactive group comprising one of an amino group and an acyl halide group may react with a second reactive group comprising the other of the amino and acyl halide group to form an amino linkage.
- Covalent linkage may be achieved through a Michael reaction.
- a first reactive group comprising one of an amino group and an acrylamide group may react with a second reactive group comprising the other of the amino and acrylamide group to form an amino linkage.
- Covalent linkage may be achieved through a Michael addition.
- a first reactive group comprising one of an a,b-unsaturated carbonyl group and an alkenyl group may react with a second reactive group comprising the other of the a,b-unsaturated carbonyl group and alkenyl group to form an amino linkage.
- Covalent linkage may be achieved through alkene-alkyne oxidative coupling.
- a first reactive group comprising one of an alkenyl group and an alkynyl group may react with a second reactive group comprising the other of the alkenyl group and alkynyl group to form a carbon-carbon linkage.
- Covalent linkage may be achieved through a Heck reaction.
- a first reactive group comprising one of an alkenyl group and a halide group may react with a second reactive group comprising the other of the alkenyl group and halide group to form a carbon-carbon linkage.
- Covalent linkage may be achieved through a Sonogashira reaction.
- a first reactive group comprising one of an alkynyl group and a halide group may react with a second reactive group comprising the other of the alkynyl group and halide group to form a carbon-carbon linkage.
- the library may be isolated and/or purified. For example, after the covalent linkage of the first and second reactive
- the reaction crude may be analysed, for example by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), in order to confirm the formation of the desired products.
- Library members comprising the covalent linkage may be isolated from the reaction mixture, for example by ethanol precipitation, high performance liquid chromatography (HPLC) purification and lyophilisation of the pure fraction.
- HPLC high performance liquid chromatography
- the library may be combined with one or more additional libraries to produce an expanded library.
- the library or expanded library may be stored or used in screening applications.
- a nucleic acid encoded library may be screened for binding to a target molecule. Methods of screening using nucleic acid encoded libraries are described in more detail below.
- a nucleic acid encoded chemical library contains members that together display a diverse population of pharmacophores. As described above, the nucleic acid strands hybridise to form a duplex nucleic acid molecule which is coupled to the sets of one or more chemical moieties attached to each strand.
- the nucleic acid strands may self-assemble through the hybridisation of complementary regions in each strand to form a double-stranded or partially double stranded nucleic acid molecule.
- the first and second sets of chemical moieties coupled to the strands are then covalently connected through the reactive groups together to form a cyclised pharmacophore (i.e. both the nucleic acid strands and optionally the covalent linkage contribute to the pharmacophore) producing a population of library members comprising cyclised pharmacophores .
- the population may display high purity.
- a nucleic acid encoded chemical library may comprise;
- each member comprising;
- first and second nucleic strands are hybridised together to form double stranded molecules, and the first and second sets of chemical moieties are covalently linked to form cyclised pharmacophores coupled to an end of the double stranded molecules, forming a chemical library.
- the library may display high purity i.e. the library may be essentially free of containment molecules, such as reactants and by-products, including unreacted first and second conjugates.
- containment molecules such as reactants and by-products, including unreacted first and second conjugates.
- 70% or more, 80% or more 90% or more, 95% or more, 99% or more or 99.5% or more of the molecular species in the populations may be library members as described herein.
- Purity may be determined by standard analytic techniques, such as HPLC, SDS-PAGE and LC-MS.
- purity may be expressed as the percentage of measured peak area of the first and second conjugates combined from the sum of peak area of all peaks, for example the sum of the peaks area of the first conjugate, the second conjugate and the containment molecule.
- the peak area may be measured under standard detection conditions, for example HPLC or MS conditions described elsewhere herein.
- Libraries produced by the methods described above may comprise 1000 or more, 10000 or more, 100000 or more or 1000000 or more different library members each different member displaying a different pharmacophore formed from a different combination of chemical moieties.
- a library produced by the methods described above may comprise 10 3 to 10 9 library members.
- the first and second sets of chemical moieties are diverse. Each pharmacophore in the library is formed from the covalent linkage of the first and second diverse sets of chemical moieties that are coupled to the first and second nucleic acid strands of the library members.
- the first nucleic strand may comprise coding sequences that identify the chemical moieties in the pharmacophore attached to it.
- Suitable nucleic acid encoded chemical libraries may be produced by the method described herein.
- members may include nucleic acid strands which are coupled to the same number and type of chemical moiety, but which are linked in a different order to each nucleic acid strand.
- nucleic acid strands may include the moieties linked in the order A-B, where A is distal to the nucleic acid strand and B is proximal to the nucleic acid strand, while others may contain the same two chemical moieties linked in the order B-A where B is distal to the nucleic acid strand and A is proximal to the nucleic acid strand. Assembly of each of these strands individually with a partner strand coupled to a single moiety ‘C’ will produce two library members having pharmacophores with different structures, even though they are composed of the same chemical moieties.
- members which include three chemical moieties A’, B’ and C’, where members may include the moieties linked as A’-B’-C’, A’-C’-B’, B’-A’-C’, B’-C’-A’, C’-A’-B’ and/or C’-B’-A’ (ordered as proximal-middle-distal with respect to the nucleic acid strand in each case).
- Other arrangements of chemical moieties are possible, for example A’ and B’ may both be linked to C’ but not to each other, or all of A’, B’ and C’ may form a covalently linked compound.
- the number of different members represents the complexity of a library and is defined by number of different chemical moieties, the number of chemical moieties in each pharmacophore, the number of different covalent linkages and therefore the number of different pharmacophores in the library.
- the number of different pharmacophores of any particular library can be determined by multiplying the number of different types of chemical moieties and the number of different types of chemical linkage together. For example, if each library member has two chemical moieties in the pharmacophore, and there are twenty types of each chemical moiety and one type of covalent linkage, then the resulting library has 400 members. If, for example, there are three chemical moieties in the pharmacophore, each of which has twenty variants, and one type of covalent linkage then the resulting library has 8000 members.
- each chemical moiety is present in the library in approximately equimolar amounts.
- the members of a nucleic acid encoded chemical library may be linked to a solid support such as a bead, array or other substrate surface.
- the library members can be free in solution.
- Nucleic acid encoded chemical libraries produced as described herein may be used in a variety of screening methods.
- the library can be used in a method for identifying a pharmacophore that participates in a preselected binding interaction with a biological macromolecule.
- a nucleic acid encoded chemical library generated according to the methods described herein provides a repertoire of chemical diversity in which each chemical moiety is linked to a coding sequence that facilitates identification of the chemical moiety.
- the library may be used to screen for pharmacophores with particular properties, e.g. pharmacophores that bind a target molecule e.g. a protein.
- Nucleic acid-encoded libraries may in particular be useful in the identification of pharmacophores which are candidates for binding to a target of interest, such as a protein, or which have improved characteristics compared to previously known pharmacophores, such as improved binding affinity to a target of interest.
- Suitable targets for nucleic acid-encoded libraries of pharmacophores are well known in the art.
- a method of screening a nucleic acid encoded chemical library may comprise contacting a nucleic acid encoded chemical library produced by a method described herein with a target molecule and
- the chemical library may be contacted with the target under binding conditions (i.e. in a binding reaction admixture) for a time period sufficient for the target molecule to interact with the library and bind to at least one member thereof.
- Suitable binding conditions are generally compatible with the known natural binding function of the target molecule.
- Compatible conditions include buffer, pH and temperature conditions that maintain the biological activity of the target molecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction.
- those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the target molecule of interest.
- the preferred binding conditions would be conditions suitable for the antibody to immunoreact with its immunogen, or a known
- the binding conditions would be those compatible with measuring receptor ligand interactions.
- a time period sufficient for the target molecule to bind to at least one member of the library is typically that length of time required for the target molecule to interact with its normal binding partner under conditions compatible with interaction.
- the time periods can vary depending on the target molecule and its respective concentration, admixing times are typically for at least a few minutes, and usually not longer than several hours, although nothing is to preclude using longer admixing times for binding to occur.
- Binding between a library member and the target molecule may result in the formation of a binding reaction complex, which is a stable product of the interaction between a target molecule and the pharmacophore of the library member as described herein.
- the product is referred to as a stable product in that the interaction is maintained over sufficient time that the complex can be isolated from the rest of the members of the library without the complex becoming significantly disassociated.
- the admixture of a library and the target molecule may be a heterogeneous or homogeneous admixture.
- the members of the library may be in the solid phase with the target molecule present in the liquid phase.
- the target molecule may be in the solid phase with the members of the library present in the liquid phase.
- both the library members and the target molecule may both be in the liquid phase.
- the target molecule may be any molecule which the pharmacophore is a candidate for interacting with.
- the target molecule may be a biological target molecule as described herein or any other molecule of interest. Suitable target molecules include biological targets, for example biological macromolecules, such as proteins.
- the target may be a receptor, enzyme, for example a kinase, protease, or phosphatase, an antigen or an oligosaccharide.
- the interaction with the target is generally through specific binding of all or part of the pharmacophore with the target.
- some or all of the chemical moieties, or parts of the chemical moieties which form the pharmacophore may specifically bind to the target.
- the binding between the pharmacophore and target may occur through intermolecular forces such as ionic bonds, hydrogen bonds and van der Waals forces, which are generally reversible.
- the binding may occur through covalent bonding, which is generally irreversible, although this is generally rare in biological systems.
- the one or more selected members may bind to a large surface of a target protein.
- the one or more selected members may for example inhibit protein-protein interactions (PPIs) of the target protein.
- PPIs protein-protein interactions
- the one or more selected library members may be isolated and/or purified. Any suitable separation technique selective for library members bound to the target molecule may be employed to isolate the one or more selected library members from the binding reaction admixture.
- a variety of separation techniques may be employed.
- a target which is a biological macromolecule may be provided in admixture in the form of a solid phase reagent, i.e. , affixed to a solid support, and thus can readily be separated from the liquid phase, thereby removing the majority of library members. Separation of the solid phase from the binding reaction admixture can optionally be accompanied by washes of the solid support to rinse library members having lower binding affinities off the solid support.
- a secondary binding means specific for the target molecule can be used to separate the macromolecule from the binding reaction admixture.
- an immobilised antibody immunospecific for the target molecule may be provided as a solid phase- affixed antibody to the binding reaction admixture after the binding reaction complex is formed. The immobilised antibody immunoreacts with the target molecule present in the binding reaction admixture to form an antibody- target molecule immunoreaction complex. Thereafter, by separation of the solid phase from the binding reaction admixture, the immunoreaction complex, and therefore any binding reaction complex, may be separated from the admixture to form isolated library member.
- a binding member can be operatively linked to the target molecule to facilitate its retrieval from the binding reaction admixture.
- exemplary binding members include the following high affinity pairs: biotin- avidin, protein A-Fc receptor, ferritin-magnetic beads.
- the target molecule is operatively linked (conjugated) to biotin, protein A, ferritin or other binding member, and the binding reaction complex is isolated by the use of the corresponding binding partner in the solid phase, e.g., solid-phase avidin, solid- phase Fc receptor or solid phase magnetic beads.
- solid supports on which to operatively link proteinaceous molecules is generally well known in the art.
- Useful solid support matrices are well known in the art and include cross-linked dextran such as that available under the tradename SEPHADEXTM from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose, borosilicate, polystyrene or latex beads about 1 micron to about 5 millimetres in diameter, polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose or nylon-based webs such as sheets, strips, paddles, plates microtiter plate wells and the like insoluble matrices
- the cyclised pharmacophore formed by the covalently linked chemical moieties of the one or more selected members may, for example, be a ligand, substrate, inhibitor or activator or may be useful in the development of any one of these.
- the cyclised pharmacophore may be an agonist or antagonist or a candidate agonist or antagonist or may be used as a model or lead in the development of such an agonist or antagonist.
- the identity of the pharmacophore that is displayed by a selected library member may be determined by decoding the coding sequences that are incorporated into the first nucleic acid strand of the selected library member.
- the first nucleic acid strands of the one or more selected library members may be amplified to produce amplification products and the amplification products may be sequenced to determine the coding sequences in the first nucleic acid strands.
- the identity of the first and second sets of chemical moieties in the one or more selected library members may be determined from the coding sequences in the first nucleic acid strands.
- a preferred method for decoding the coding sequences in the first nucleic acid strand is the use of high throughput sequencing methods (NGS sequencing), such as the lllumina HTDS (lllumina high-throughput sequencing) or the 454-Roche Genome Sequencer system.
- NGS sequencing high throughput sequencing methods
- PCR products have to contain suitable adaptor sequences at their extremities (called adaptor sequence A and B), which can be either added after a PCR reaction by ligation, or they can be incorporated in the PCR reactions, if the PCR primers contain on their 5’-ends sequences corresponding to an adaptor region.
- the next step of a particular sequencing process is the annealing of PCR amplicons on nucleic acid Capture Beads, emulsification of beads and PCR reagents in water-in-oil microreactors, and clonal emPCR amplification inside these microreactors.
- the Capture beads After breaking of the emulsion, the Capture beads are mixed with Enzyme Beads, and loaded on a PicoTiterPlate. Pyrosequencing allows the recording of individual sequences for each nucleic acid species displayed at Capture Beads, trapped in the wells of PicoTiterPlates.
- nucleic acid encoded chemical library produced as described herein is for lead optimization.
- Lead optimization may involve combining a known pharmacophore, formed from one or more chemical moieties with one or more further chemical moieties, as described herein with the aim of improving the characteristics of the known pharmacophore, for example the binding affinity.
- nucleic acid strands from first and second sub-libraries of conjugates may be hybridised and the sets of chemical moieties covalently linked to form a library.
- the first sub-library may comprise library members which are coupled to the known pharmacophore and the second sub-library comprises members coupled to one or more candidate chemical moieties.
- the second sub-library generally comprises a variety of different chemical moieties, because this increases the variety of structure in the pharmacophores of the assembled library members.
- the identities of the chemical moieties in the resultant covalently linked cyclised pharmacophore are encoded into the library member using the methods known in the art and described elsewhere herein.
- Example 1 The copperdj-catalvsed 1.2.3-triazole-forminq reaction ("top" ring closurej between azide- modified nucleic acid strand A and alkvne-modified nucleic acid strand B.
- Custom oligonucleotides were lyophilized and further purified by EtOH precipitation and re-dissolved in H2O. The final concentration was determined by UV absorbance measurement at 260 nm using a NanoDrop 2000 instrument.
- Amino-modified 48-mer oligonucleotide (SEQ ID NO: 1)
- oligonucleotide conjugates was performed on an Agilent 1200 Series with a C18-Xterra ⁇ Prep RP column (112 A, 5 pm, 10 x 150 mm) using a gradient of eluent A (TEAA 100 mM) and eluent B.
- the colloidal solution was left at -20 °C for 72 h and then centrifuged at 4 °C for 30 min at 15000 rpm. The resulting supernatant was discarded and the pellet was dried using a SpeedVac.
- the crude mixture was purified by RP-HPLC on a C18-Xterra ⁇ Prep RP column (112 A, 5 pm,
- alkynyl conjugate B 5-hexynoic acid (2.5 pL, 200 mM in DMSO) and DMT-MM (1.25 pL, 400 mM in H2O) were incubated for 2 h at 25 °C.
- Amino-modified 48-mer oligonucleotide (SEQ ID NO: 2) (10 nmol) in Borate Buffer (13.2 pL, 250 mM, pH 9.4) was then added to the mixture and the reaction was stirred for 2 h at 25 °C.
- 10% (v/v) of 5 M NaCI was added, followed by 2.5-3 volumes of cold absolute EtOH.
- the colloidal solution was left at -20 °C for 72 h and then centrifuged at 4 °C for 30 min at 15000 rpm. The resulting supernatant was discarded and the pellet was dried using a SpeedVac.
- the crude mixture was purified by RP-HPLC on a C18-Xterra ⁇ Prep RP column (112 A, 5 pm, 10 x 150 mm) using a gradient of eluent A (TEAA 100 mM) and eluent B (TEAA 100 mM in 80% ACN).
- the fractions containing the product were combined and lyophilised to obtain the alkyne product (B), as determined by measuring the UV absorbance at 260 nm of a water solution on a Thermofisher Nanodrop 2000.
- LC-ESI-MS 15088.73 m/z, found: 15088.01 m/z.
- Example 2 Construction of a cvclised dual-pharmacophore oligonucleotide conjugate composed of an azido acid DNA-encoded compound and an alkynyl acid attached on a complementary DNA strand (Format 1 , Figures 8 and 9)
- the synthetic oligonucleotides used for the construction of the oligonucleotide conjugates are shown below.
- the synthetic oligonucleotides were stored as 100 mM stock solutions at -20 °C.
- the oligonucleotide conjugates were precipitated by adding 10% v/v 5M NaCI and 2.5-3 volumes of cold absolute EtOH.
- the oligonucleotide conjugates were stored at -20 °C for at least 2 h before centrifugation (1 h, 15 ⁇ 00 rpm, 4 °C). Immediately after the centrifugation, the supernatants were carefully discarded, and the remaining pellets were vacuum dried.
- the oligonucleotide quantities were estimated by UV absorbance using a NanodropTM 2000/2000c spectrophotometer. 1 mI of the oligonucleotide conjugate solution was released directly onto the optical measurement surface. The absorbance at 260 nm was extracted from the computed data to calculate the amount of oligonucleotide, knowing the absorption coefficient ( e ) of the corresponding DNA sequence.
- a tandem-quadrupole mass spectrometer (Agilent 6100 Series Single Quadrupole MS) with electrospray ionization (ESI) source was used for mass detection and analysis. Mass spectrometric analyses were performed in negative ion-mode. ESI interface parameters were set as follows: dissolution temperature (200 °C), source temperature (110 °C), capillary voltage (3.0 kV), cone voltage (40 V), scan time (0.5 s), inter-scan delay time (0.1 s).
- the cyclisation reaction was analysed on native polyacrylamide 20% TBE gels (1 .0 mm, 15 wells) and on denaturing polyacrylamide 15% TBE-Urea gels (1.0 mm, 15 wells). A current of 120 mA with a voltage of 200 V was applied for 1 h on the electrophoresis box. The gels were then stained with SYBR Green I during 30 min and analysed by UV excitation (Figure 14).
- a cyclised dual-pharmacophore double-stranded oligonucleotide construct (Format 1) was generated from two hybridised single-stranded oligonucleotide conjugates (strand A1 and strand B1) by copper(l)-catalysed azide alkyne cycloaddition, as shown in Figure 8C.
- LC-ESI-MS was used to analyse the reactants and products. Single peaks were observed for strand A1 comprising the azide modified building block ( Figure 9A) and strand B1 comprising the alkyne modified building block ( Figure 9B) in the LC-ESI-MS trace.
- Example 3 Construction of a cyclised dual-pharmacophore oligonucleotide conjugate composed of an DNA- encoded azido compound and an alkvnyl acid encoded by a complementary DNA strand ( Figures 10 and 11)
- the synthetic oligonucleotides used for the encoding ligation step are shown below.
- the synthetic oligonucleotides were stored as 100 pM stock solutions at -20 °C.
- strand B1 For the synthesis of strand B1 , as described in Example 2.2, 50 pi of a solution of 1 mM of d-spacer oligonucleotide (SEQ ID NO: 2) in 250 mM Borate, 12.5 pi of 200 mM solution of 6-heptynoic acid, and 6.25 pi of a 400 mM solution of DMT-MM were stirred for 2 h at 25 °C.
- SEQ ID NO: 2 d-spacer oligonucleotide
- DNA-Compounds (strands A1 and B1) were precipitated with cold absolute EtOH and re-dissolved in H2O before Nanodrop measurement and LC-ESI-MS analysis, as described in Examples 2.4 and 2.5 respectively, yielding, the azido acid oligonucleotide conjugate (strand A1) and the alkynyl acid
- oligonucleotide conjugate (strand B1). 3.2 Encoding by ligation [oligonucleotide alkynyl conjugate strand B2, Figure 10A]
- TEAA triethylammonium acetate
- Buffer B 100 mM TEAA in 80% MeCN/20% H2O
- T 25-30 °C
- p 100-170 bar
- Abs 260 nm
- the Elib4_aT RNA adaptor came out at 6-6.5 min, the strand B1 between 8-9 min followed by the Elib4_Code2 oligonucleotide at 9-11 min.
- oligonucleotide alkynyl conjugate strand B2 showed a retention time of 12.5 min with a peak ranging between 11.5 and 13.5 min. The purity of the fractions was verified by LC-ESI-MS analysis as described in Example 2.5 and the recovered amount estimated by Nanodrop measurement after lyophilization as described in the Example 2.4.
- the DNA-Compound was diluted in H2O for Nanodrop Measurement and LC-ESI-MS analysis, as described in Examples 2.4 and 2.5, respectively, ( Figure 11) and polyacrylamide gel electrophoresis as described in Example 2.7 ( Figure 14).
- strand A1 and strand B2 Two single-stranded DNA conjugates (strand A1 and strand B2) were synthesized.
- the oligonucleotide section of strand B1 was extended by ligation to yield the encoded oligonucleotide alkynyl conjugate strand B2, as shown in Figure 10A.
- a cyclised dual-pharmacophore double-stranded oligonucleotide construct (Format 2) was generated from two hybridised single-stranded oligonucleotide conjugates (strand A1 and strand B2) by copper(l)-catalysed azide alkyne cycloaddition ( Figure 10B).
- LC-ESI-MS was used to analyse the reactants and products. Single peaks were observed for strand A1 comprising the azide modified building block ( Figure 1 1 A) and strand B2 comprising the alkyne modified building block ( Figure 11 B) in the LC-ESI-MS trace.
- Example 4 Construction of a third cvclised dual-pharmacophore DNA duplex composed of a DNA-encoded azido compound and an alkvnyl acid encoded by a complementary DNA strand (Format 3, Figures 1A, 12 to 14)
- Strand B2 was obtained from strand B1 followed extended by a ligation step ( Figure 10A).
- strand B1 50 pi of a solution of 1 mM of d-spacer oligonucleotide (SEQ ID NO: 2) in 250 mM Borate, 12.5 pi of 200 mM solution of 6-heptynoic acid, and 6.25 pi of a 400 mM solution of DMT-MM were stirred for 2 h at 25 °C.
- the DNA-Compound was precipitated with cold absolute EtOH and re-dissolved in H2O before Nanodrop measurement and LC-ESI-MS analysis, as described in Examples 2.4 and 2.5 respectively, to obtain the oligonucleotide alkynyl conjugate (strand B1).
- the strand B1 was encoded by ligation: 4.6 pi of 434 pM oligonucleotide alkynyl conjugate strand B1 (2 nmol), 62.5 pi of 48.3 pM coding oligonucleotide Elib4_Code2 (SEQ ID NO: 4) (3 nmol), 106,7 pi of 30 pM RNA adaptor oligonucleotide (SEQ ID NO: 5) were vacuum-dried and re-dissolved in 89.5 pi H2O and 10 pi of 10x ligase buffer. The mixture was heated up to 90 °C for 2 min and passively cooled down to 25 °C (hybridisation). Afterwards, 0.5 pi T4 ligase was added.
- Ligation was performed for 16 h at 16 °C.
- the ligase was inactivated for 10 min at 65 °C.
- the DNA- compound was purified by HPLC, as described in Example 3.3, and quantify by Nanodrop measurement, as described in Example 2.4, and LC-ESI-MS analysed, as described in Example 2.5, to obtain the encoded oligonucleotide alkynyl conjugate (strand B2).
- the reaction solution was cleaned up by cartridge purification, giving the DNA duplex composed of strand B2 and its complementary extended oligonucleotide (strand A2).
- the completion of the reaction was estimated by electrophoresis with QIAxce/ Advanced Instrument (see below the clean-up purification and the automated procedure of gel electrophoresis).
- the DNA duplex built up by the Klenow fill-in reaction was separated from the remaining DNA single strands and enzyme debris.
- a Spin Column for DNA (silica membrane, 800 pi loading capacity, QIA Quick ® PCR Purification kit) was used to bind the DNA oligonucleotides containing at least 100 base pairs (bp) to the silica membrane by mixing the Klenow reaction mixture as 1 volume to 5 volumes of binding buffer
- the spin column was centrifuged (25 °C, 13 ⁇ 00 rpm, 1 min) to filter the solution. After discarding of the recovered liquid, 750 pi of the Washing buffer (furnished with the PCR purification kit) was added on the spin column before another centrifugation (25 °C, 13 ⁇ 00 rpm, 1 min). After the discarding, 50 pi of H2O were added to the spin column. The liquid was left diffusing during 1 min before a last centrifugation (25 °C, 13 ⁇ 00 rpm, 90 s), recovering the cleaned up eluted DNA duplex.
- the Klenow fill-in reaction was analysed by electrophoresis automated by QIAxcel Advanced Instrument.
- the samples were loaded into the appropriate analysis tubes (row of 12 tubes).
- the unused tubes were filled with Dilution buffer (included with the QIAxcel Advanced Instrument or provided by QIAGEN).
- the resulting gel was computed by the QIAGEN software, before LC-ESI-MS analysis and Nanodrop measurement.
- the DNA-Compound was diluted in H2O for Nanodrop measurement and LC-ESI-MS analysis, as described in Examples 2.4 and 2.5 respectively ( Figure 13) and polyacrylamide gel electrophoresis as described in Example 2.7.
- Two single-stranded DNA conjugates (strand A2 and strand B2) were synthesized.
- the oligonucleotide section of strand A1 were extended by Klenow Fill-in polymerisation to yield the strand A2 ( Figure 12A).
- a cyclised dual-pharmacophore double-stranded oligonucleotide construct (Format 3) was generated from two hybridised single-stranded oligonucleotide conjugates (strand A2 and strand B2) by copper(l)-catalysed azide alkyne cycloaddition ( Figure 12B).
- Example 5 Construction of a sub-library of oligonucleotide-compound conjugates displaying two chemical Building Blocks (2BB) using 5'-aminomodified oligonucleotides (Figure 15)
- BB1 Building Block 1
- the oligonucleotides were stored as 100 pM stock solutions at -20 °C.
- the sequences of the oligonucleotides were stored as 100 pM stock solutions at -20 °C.
- oligonucleotides used for the encoding ligation step are shown below.
- Each BB has been tagged with a custom oligonucleotide, carrying an encoding region, with unique nucleotides defined as X, and a non-coding region.
- the pool of strands C1 was aliquoted in 293 reaction vessels before the splint ligation of the second code (Elib5_Code2, SEQ ID NO: 7), each of them containing distinct encoded sequences X, yielding 293 Elib5 sub-pools (Figure 15 B).
- the 293 Elib5 sub-pools were coupled with 293 carboxylic acids referred as building blocks 2 (BB2), yielding the strands C2.
- the strands C2 were mixed, purified by HPLC yielding the strands C2 sub-library ( Figure 15C).
- the strands C1 Pool was then split in 10 tubes followed by drying under vacuum. The removal of the Fmoc group was performed by addition of 5 pi triethylamine to solutions of 4 mM encoded compounds and stirring for 6 h at 37 °C. The DNA-Compounds were ethanol precipitated and vacuum-dried and the amounts were estimated by Nanodrop as described in Example 2.4, yielding the strands C1 Pool.
- the ligated oligonucleotide-compound conjugates carrying the BB1 and BB2 chemical moieties were precipitated and vacuum-dried as described above. All the ligated oligonucleotides-compound conjugates were then mixed together and vacuum-dried as described above, giving the sub-library pool (strands C2 sub-library). Purification of the strands C2 sub-library was performed by HPLC as described in Example 3.3 to generate the desired sub-library.
- a 4 ⁇ 02 strands C2 sub-library was synthesized from 14 tri-functional carboxylic acid scaffolds as chemical moiety BB1 and 293 carboxylic acids as chemical moiety BB2, assembled in combinatorial fashion.
- Example 6 Construction of a cvclised dual-pharmacophore oligonucleotide library composed of a trifunctional DNA-encoded azido sub-library (strands C2 sub-library) and an alkvnyl acid encoded by a complementary DNA strand (strand D2) (Format 4, Figures 16 to 19)
- the sequences of the synthetic oligonucleotides used for the encoding ligation step are shown below.
- the synthetic oligonucleotides were stored as 100 pM stock solutions at -20 °C.
- the ligase was inactivated for 10 min at 65 °C.
- the DNA-compound was purified by HPLC, as described in Example 3.3, quantified by Nanodrop, as described in Example 2.4, LC-ESI-MS analysed, as described in Example 2.5, to obtain the encoded oligonucleotide alkynyl conjugate (strand D2).
- a cyclised dual-pharmacophore double-stranded library was generated from a single-stranded azido-sub- library (strands C2 pool) and a single-stranded alkyne conjugate (strand D2) by copper (l)-catalysed azide alkyne cycloaddition.
- Example 7 Construction of a cyclised dual-pharmacophore oligonucleotide final library format composed of a filled-in tri-functional DNA-encoded azido sub-library (strands C3 sub-library) and an alkvnyl acid encoded by a complementary DNA strand (strand D2) (Format 5, Figures 1 E, 21 and 22)
- strands C2 sub-library tri-functional DNA-encoded azido sub-library (strands C2 sub-library), as described in Example 5, and an encoded oligonucleotide alkynyl conjugate (strand D2), as described in Example 6, 1.7 mI of 58.3 mM of strands C2 sub-library (100 pmol in H2O), 2.0 mI of 49 mM of strand D2 (100 pmol), 10 mI of NEB 2 10x Klenow buffer and 80.2 mI H2O were mixed, heated up at 90 °C and passively cooled down to 25 °C (hybridisation).
- the DNA-compound was diluted in H2O for Nanodrop measurement, LC-ESI-MS analysis and polyacrylamide gel electrophoresis, as described in Examples 2.4, 2.5 and 2.7 respectively.
- a cyclised dual-pharmacophore double-stranded library was generated from a single-stranded azido-sub-library extended by Klenow Fill-in (strands C3 sub-library) and a single-stranded alkyne conjugate (strand D2) by copper(l)-catalysed azide alkyne cycloaddition, as shown in Figure 21.
- Example 8 Construction of a cyclised dual-pharmacophore oligonucleotide library format composed of a trifunctional DNA-encoded azide scaffold sub-library (strands C3 sub-library) and an alkvnyl encoded by a complementary DNA sub-library [strands F2 sub-library ( Figures 23 to 33)1.
- the sequences of the synthetic oligonucleotides used for the encoding ligation step are shown below.
- the synthetic oligonucleotides were stored as 100 pM stock solutions at -20 °C.
- the strands F1 were individually purified by HPLC, as described in Example 3.3, vacuum-dried and redissolved in H2O before LC-ESI-MS analysis, as described in Example 2.5, and Nanodrop measurement, as described in Example 2.4.
- the ligase was inactivated for 10 min at 65 °C.
- the DNA-compound was purified by HPLC, as described in Example 3.3, quantified by Nanodrop, as described in Example 2.4, LC-ESI-MS analysed, as described in Example 2.5, to obtain encoded oligonucleotide alkynyl conjugates (strand F2).
- Equimolar amounts of strands F2 were mixed together to generate the desired strands F2 sub-library, as described in Example 5.1.
- reaction solution was cleaned up by cartridge purification, giving the dual-pharmacophore DNA duplex (Format 6B) composed of strands F2 sub-library and strands C3B sub-library.
- Form 6B dual-pharmacophore DNA duplex
- the completion of the reaction was estimated by electrophoresis with QIAxcel Advanced Instrument and then cleaned up by cartridge purification, both procedures being described in Examples 4.3 and 4.4.
- Affinity selections were performed using a Thermo Scientific KingFisher magnetic particles processor.
- the DNA-encoded chemical library (6.8 nM of Format 6A or 68 nM of Formats 6B and C in PBST) for 1 h with continuous gentle mixing.
- CAIX-coated beads were washed three times with 200 mI PBST (300) and subsequently incubated with 100 mI of the DNA-encoded chemical library (6.8 nM of Format 6A or 68 nM of Formats 6B and C in PBST (300)) for 1 h with continuous gentle mixing. After removing unbound library members by washing with 200 mI PBST (300) for five times, beads carrying bound library members were re-suspended in 100 mI Tris buffer
- the PCR1 is first performed in order to encode the affinity selection experiments.
- the mixtures were incubated first to 98 °C for 1 min and subsequently heat to 98 °C for 10 s (denaturation of the Template DNA duplex) and cooled down to 72 °C for 15 s (annealing of the Primers and elongation of the DNA strands).
- the PCR2 is then performed in order to allow the sequencing of the DNA.
- a mixture composed of 25 mI of Phusion MasterMix, 3 m1 10 mM of lllumina Primer 2a, 3 mI 10 mM of lllumina Primer 2b and 14 mI H2O were added 5 mI PCR#1 .
- the mixtures were incubated first to 98 °C for 3 min and subsequently heat to 98 °C for 45 s (denaturation of the Template DNA duplex), cooled down to 72 °C for 45 s (annealing of the Primers) and heated up to 72 °C for 45 s (elongation of the DNA strands).
- the PCR#2 pool was mixed with loading dye and split into four different wells.
- the DNA migration was performed by applying a voltage of 110 V and an open current (about 70 mA) during 27 min.
- the migration was analysed by a brief UV excitation and the PCR#2 bonds were excised from the gel.
- the DNA-compounds were then precipitated by adding 10 mI of 5 M NaCI and 300 mI of absolute ethanol before overnight storage at -20 °C. After 1 h of centrifugation at 14 ⁇ 00 rpm, the supernatant was carefully removed and the pellet re-dissolved in 500 mI of cold 80% EtOH before an additional centrifugation at 14 ⁇ 00 rpm for 30 min. The supernatant was carefully removed and the pellet was air dried for 1 h. The DNA compounds were re-dissolved in 100 mI. A small quantity was used for a last analysis by agarose gel, as described in Example 8.8. The sequencing sample was normalized at 14 ng.pl -1 in 55 mI.
- the lllumina HTDS consists in the capture of the different DNA nucleotides on the surface of the reaction flow cell, which presents complementary sequences to lllumina Primers introduced during the PCR2, as described in Example 8.7. These oligonucleotides were amplified until the formation of several“clusters”
- a single-pharmacophore double-stranded library (Format 6A) has been generated from a single-stranded azido-sub-library (strands C2 sub-library) and its complementary oligonucleotide built by Klenow fill-in.
- a dual-pharmacophore double-stranded library (Format 6B) has been generated from a single- stranded azido-sub-library (strands C2 sub-library) and a single-stranded alkynyl sub-library (strands F2 sublibrary).
- a cyclised dual-pharmacophore double-stranded library (Format 6C) has been generated from a single-stranded azido-sub-library (strands C3B sub-library) and a single-stranded alkynyl sub-library (strands F2 sub-library) by copper (l)-catalysed azide alkyne cycloaddition.
- These three libraries have been screened against CAIX as a protein target of interest to provide sets of binders specific for each library.
- the structures of the binders showed indeed an increase of the rigidity with the cyclised dual-pharmacophore settings.
- Example 9 Construction of a cvclised dual-pharmacophore oligonucleotide library format composed of a trifunctional DNA-encoded alkvnyl scaffold sub-library (strands G3 sub-library ' ) and an azido encoded by a complementary DNA sub-library [strands H2 sub-library ( Figures 34 to 3611
- Format 7 comprises a tri-functional scaffold (carboxylic acid, /V-Fmoc protected amine and alkyne moiety) as first building block (BB1), encoded by Elib5_Code1 (SEQ ID NO: 6), a carboxylic acid as second building block (BB2), encoded by Elib5_Code2 (SEQ ID NO: 7) providing a first population of nucleic acid conjugates (strands G2 sub-library, Figure 34), and an acid azide as third building block (BB3), encoded by
- Elib6_Code3#1 -n (SEQ ID NO: 12; where, n is the total number of BB3) providing a second population of nucleic acid conjugates (strands H2 sub-library, Figure 35).
- the double-stranded molecules (strands G3 sublibrary and strands H2 sub-library) are then covalently linked by 1 ,2,3-triazole formation reaction between the azide (strands H2 sub-library), and the alkyne (strands G3 sub-library, Figure 36A), yielding the final cyclised double-stranded population of molecules forming a chemical library (Format 7, Figure 36B).
- Example 10 Construction of a cvclised dual-pharmacophore oligonucleotide library format composed of an azido DNA-encoded sub-library (strands I3 sub-library) and an alkvnyl encoded by a complementary DNA sub-library [strands F2 sub-library ( Figures 37 to 39)1
- Format 8 comprises a /V-Fmoc amino acid as first building block (BB1), encoded by Elib5_Code1 (SEQ ID NO: 6), an acid azide as second building block (BB2), encoded by Elib5_Code2 (SEQ ID NO: 7) providing a first population of nucleic acid conjugates (strands I2 sub-library, Figure 37), and an acid alkyne as third building block (BB3), encoded by Elib6_Code3#1 -n (SEQ ID NO: 12; wherein n is the total number of BB3) providing a second population of nucleic acid conjugates (strands F2 sub-library, Figure 38).
- the double- stranded molecules (strands I3 sub-library and strands F2 sub-library) are then covalently linked by 1 ,2,3- triazole formation reaction between the azide (strands I3 sub-library, Figure 39A), and the alkyne (strands F2 sub-library), yielding the final cyclised double-stranded population of molecules forming a chemical library (Format 8, Figure 39B).
- Example 1 1 Construction of a cvclised dual-pharmacophore oligonucleotide library format composed of an alkvnyl DNA-encoded sub-library (strands J3 sub-library) and an azido encoded by a complementary DNA sub-library [strands H2 sub-library ( Figures 40 and 41)1
- the Format 9 comprises a /V-Fmoc amino acid as first building block (BB1), encoded by Elib5_Code1 (SEQ ID NO: 6), an acid alkyne as second building block (BB2), encoded by Elib5_Code2 (SEQ ID NO: 7) providing a first population of nucleic acid conjugates (strands J2 sub-library, Figure 40), and an acid azide as third building block (BB3), encoded by Elib6_Code3#1 -n (SEQ ID NO: 12) providing a second population of nucleic acid conjugates (strands H2 sub-library, as described in Example 9 and Figure 35).
- the double- stranded molecules (strands J3 sub-library and strands H2 sub-library) are then covalently linked by 1 ,2,3- triazole formation reaction between the alkyne (strands J3 sub-library, Figure 41A), and the azide (strands H2 sub-library), yielding the final cyclised double-stranded population of molecules forming a chemical library (Format 9, Figure 41 B).
- Example 12 Construction of a cvclised dual-pharmacophore oligonucleotide library format composed of an azido DNA-encoded sub-library (strands K3 sub-library) and an alkvnyl encoded by a complementary DNA sub-library [strands F2 sub-library ( Figures 42 and 43)1
- the Format 10 comprises an acid ester as first building block (BB1), encoded by Elib5_Code1 (SEQ ID NO: 6), an amine azide as second building block (BB2), encoded by Elib5_Code2 (SEQ ID NO: 7) providing a first population of nucleic acid conjugates (strands K2 sub-library, Figures 42), and an acid alkyne as third building block (BB3), encoded by Elib6_Code3#1-n (SEQ ID NO: 12) providing a second population of nucleic acid conjugates (strands F2 sub-library, as described in Example 10, Figures 38).
- BB1 first building block
- BB2
- the double- stranded molecules (strands K3 sub-library and strands F2 sub-library) are then covalently linked by 1 ,2,3- triazole formation reaction between the azide (strands K3 sub-library, Figures 43 upper panel), and the alkyne (strands F2 sub-library), yielding the final cyclised double-stranded population of molecules forming a chemical library (Format 10, Figures 43 lower panel).
- Example 13 Construction of a cyclised dual-pharmacophore oligonucleotide library format composed of an alkvnyl DNA-encoded sub-library (strands L3 sub-library) and an azido encoded by a complementary DNA sub-library [strands H2 sub-library ( Figures 44 and 45)1
- the Format 11 comprises an acid ester as first building block (BB1), encoded by Elib5_Code1 (SEQ ID NO: 6), an amine alkyne as second building block (BB2), encoded by Elib5_Code2 (SEQ ID NO: 7) providing a first population of nucleic acid conjugates (strands L2 sub-library, Figures 44) and an acid azide as third building block (BB3), encoded by Elib6_Code3#1-n (SEQ ID NO: 12) providing a second population of nucleic acid conjugates (strands H2 sub-library, as described in Example 9 and Figures 35).
- the double- stranded molecules (strands L3 sub-library and strands H2 sub-library) are then covalently linked by 1 ,2,3- triazole formation reaction between the alkyne (strands L3 sub-library, Figures 45 upper panel), and the azide (strands H2 sub-library), yielding the final cyclised double-stranded population of molecules forming a chemical library (Format 11 , Figures 45 lower panel).
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