# US 10,865,409 B2 · Methods for tagging DNA-encoded libraries

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- Number: US 10,865,409 B2
- Document ID: US10865409B2
- Status: Granted
- Kind: B2
- Application number: 14/343,306
- Filed: 2012-09-07
- Published: 2014-10-23
- Granted: 2020-12-15
- Earliest claimed: 2011-09-07
- Assignees: X-Chem, Inc.
- Applicants: Anthony D. Keefe; Richard W. Wagner; Alexander Litovchick; Matthew Clark; John W. Cuozzo; Ying Zhang; Paolo A. Centrella; Christopher D. Hupp
- Inventors: Anthony D. Keefe; Richard W. Wagner; Alexander Litovchick; Matthew Clark; John W. Cuozzo; Ying Zhang; Paolo A. Centrella; Christopher D. Hupp
- Web page: https://patentgrep.com/patent/US10865409B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1068: {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}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C40B 50/16: involving encoding steps
- C07B 2200/11: Compounds covalently bound to a solid support
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C07B: GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR (preparation of carboxylic acid esters by telomerisation C07C67/47; telomerisation C08F)

## Abstract

The present invention relates to oligonucleotide-encoded libraries and methods of tagging such libraries. In particular, the methods and oligonucleotides can include one or more 2′-substituted nucleotides, such as 2′-O-methyl or 2′-fluoro nucleotides, and other conditions or reagents to enhance enzyme ligation or one or more chemical functionalities to support chemical ligation.

## Claims

### Claim 1

A method of tagging a first library comprising an oligonucleotide-encoded chemical entity, said method comprising:
(i) providing a single-stranded oligonucleotide headpiece having a first functional group and a second functional group, wherein said headpiece comprises at least one terminal 2′-substituted nucleotide comprising said second functional group, wherein said at least one terminal 2′-substituted nucleotide is at the 5′-terminus and/or the 3′-terminus of said headpiece;
(ii) binding said first functional group of said headpiece to a first component of said chemical entity, wherein said headpiece is directly connected to said first component or said headpiece is indirectly connected to said first component by a bifunctional linker; and
(iii) ligating said second functional group of said headpiece to a single-stranded oligonucleotide first building block tag to form a complex, wherein said first building block tag comprises at least one terminal 2′-substituted nucleotide at the 5′- and/or the 3′-terminus and said ligating forms a covalent bond between a 2′-substituted nucleotide on the headpiece and a 2′-substituted nucleotide on the first building block tag, and wherein the ligating is carried out using enzymatic ligation;
wherein said steps (ii) and (iii) can be performed in any order; and
wherein said first building block tag encodes for the binding reaction of said step (ii), thereby providing a tagged library.

### Claim 2 (depends on 1)

The method of claim 1, wherein said first building block tag further comprises at least one 2′-substituted nucleotide at an internal position.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein each 2′-substituted nucleotide is, independently, 2′-O-methyl guanine, 2′-O-methyl uracil, 2′-O-methyl adenosine, 2′-O-methyl thymidine, 2′-O-methyl inosine, 2′-O-methyl cytidine, 2′-O-methyl diamino purine, 2′-fluoro guanine, 2′-fluoro uracil, 2′-fluoro adenosine, 2′-fluoro thymidine, 2′-fluoro inosine, 2′-fluoro cytidine, or 2′-fluoro diamino purine.

### Claim 4 (depends on 1)

The method of claim 1, wherein step (ii) comprises binding said headpiece directly to said first component.

### Claim 5 (depends on 1)

The method of claim 1, wherein step (ii) comprises binding said headpiece indirectly to said first component via a bifunctional linker.

### Claim 6 (depends on 1)

The method of claim 1, further comprising
(iv) binding a single-stranded oligonucleotide second building block tag to the 5′-terminus or 3′-terminus of said complex; and
(v) binding a second component of said chemical library to said first component, wherein said steps (iv) and (v) can be performed in any order.

### Claim 7 (depends on 6)

The method of claim 6, wherein said second building block tag comprises a 2′-substituted nucleotide at one or more of the 5′-terminus, the 3′-terminus, and/or the internal position of said second building block tag.

### Claim 8 (depends on 6)

The method of claim 6, wherein said step (iv) is carried out using enzymatic ligation.

### Claim 9 (depends on 1, 6)

The method of claim 1 or 6, wherein said method further comprises separating said complex from any unreacted tag or unreacted headpiece before any one of binding steps (ii)-(v) and/or purifying said complex before any one of binding steps (ii)-(v).

### Claim 10 (depends on 1)

The method of claim 1, wherein said method further comprises binding one or more additional building block tags to said complex and binding one or more additional components to said complex.

### Claim 11 (depends on 1)

The method of claim 1, wherein said headpiece comprises a hairpin structure.

### Claim 12 (depends on 1, 10)

The method of claim 1 or 10, wherein said headpiece, said first building block tag, said second building block tag, and/or said one or more additional building block tags, if present, further comprises a first library-identifying sequence, a use sequence, and/or an origin sequence.

### Claim 13 (depends on 1)

The method of claim 1, wherein said method further comprises binding a first library-identifying tag, a use sequence, an origin sequence, and/or a tailpiece to said complex.

### Claim 14 (depends on 1)

The method of claim 1, wherein said method comprises a plurality of headpieces.

### Claim 15 (depends on 1, 8)

The method of claim 1 or 8, wherein said enzymatic ligation is carried out using RNA ligase.

### Claim 16 (depends on 15)

The method of claim 15, wherein said RNA ligase is T4 RNA ligase.

### Claim 17 (depends on 1, 8)

The method of claim 1 or 8, wherein said enzymatic ligation is carried out using DNA ligase.

### Claim 18 (depends on 17)

The method of claim 17, wherein said DNA ligase is ssDNA ligase.