Oligonucleotide fragments and methods of making rnai agents using the same
AssigneeEli Lilly and Company
InventorsCaleb Christopher CULY, Scott MAY, Eric MOHER, Sergey TSUKANOV
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Abstract
Intermediate compounds (i.e., oligonucleotide fragments) are disclosed for making RNAi agents, or pharmaceutically acceptable salts thereof. In addition, methods are disclosed for making single-stranded oligonucleotides by ligating two or more of the intermediate compounds herein via a hybrid chemical-enzymatic route.
Claims54
An oligonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS:5 to 37, or a pharmaceutically acceptable salt thereof.
A method of making a nucleic acid of SEQ ID NO:1, the method comprising the step of:
- ligating three oligonucleotide fragments selected from the group consisting of:
- (a) SEQ ID NOS:5, 6 and 7,
- (b) SEQ ID NOS:5, 10 and 11,
- (c) SEQ ID NOS:5, 12 and 13,
- (d) SEQ ID NOS:7, 14 and 15,
- (e) SEQ ID NOS:7, 18 and 19,
- (f) SEQ ID NOS:7, 22 and 23, and
- (g) SEQ ID NOS:5, 26 and 27.
A method of making a nucleic acid of SEQ ID NO:2, the method comprising the step of:
- ligating two oligonucleotide fragments selected from the group consisting of:
- (a′) SEQ ID NOS:8 and 9;
- (b′) SEQ ID NOS:16 and 17;
- (c′) SEQ ID NOS:20 and 21;
- and
- (d′) SEQ ID NOS:24 and 25.
A method of making a nucleic acid of SEQ ID NO:3, the method comprising the step of:
- ligating three oligonucleotide fragments of (a) SEQ ID NOS:7, 32 and 33, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:32 to SEQ ID NO:33 to SEQ ID NO:7.
A method of making a nucleic acid of SEQ ID NO:4, the method comprising the step of:
- ligating two oligonucleotide fragments of (a′) SEQ ID NOS:34 and 35, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:35 to SEQ ID NO:34.
A method of making a RNAi agent having a sense strand of SEQ ID NO:1 and an antisense strand of SEQ ID NO:2, the method comprising the steps of:
- forming the sense strand of SEQ ID NO:1 by ligating three oligonucleotide fragments selected from the group consisting of:
- (a) SEQ ID NOS:5, 6 and 7, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:5 to SEQ ID NO:6 to SEQ ID NO:7,
- (b) SEQ ID NOS:5, 10 and 11, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:5 to SEQ ID NO:10 to SEQ ID NO:11,
- (c) SEQ ID NOS:5, 12 and 13, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:5 to SEQ ID NO:12 to SEQ ID NO:13,
- (d) SEQ ID NOS:7, 14 and 15, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:14 to SEQ ID NO:15 to SEQ ID NO:7,
- (e) SEQ ID NOS:7, 18 and 19, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:18 to SEQ ID NO:19 to SEQ ID NO:7,
- (f) SEQ ID NOS:
- 7, 22 and 23, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:22 to SEQ ID NO:23 to SEQ ID NO:7, and
- (g) SEQ ID NOS:
- 5, 26 and 27, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:5 to SEQ ID NO:26 to SEQ ID NO:27;
- forming the antisense strand of SEQ ID NO:2 by ligating two oligonucleotide fragments selected from the group consisting of:
- (a′) SEQ ID NO:8 and 9, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:9 to SEQ ID NO:8
- (b′) SEQ ID NOS:16 and 17, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:17 to SEQ ID NO:16,
- (c′) SEQ ID NOS:20 and 21, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:21 to SEQ ID NO:20, and
- (d′) SEQ ID NOS:24 and 25, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:25 to SEQ ID NO:24;
- and
- forming the RNAi agent by annealing complementary nucleotides of the sense strand of SEQ ID NO:1 and the antisense strand of SEQ ID NO:2.
A method of making a RNAi agent having a sense strand of SEQ ID NO:3 and an antisense strand of SEQ ID NO:4, the method comprising the steps of:
- forming the sense strand of SEQ ID NO:3 by ligating three oligonucleotide fragments of (a) SEQ ID NOS:7, 32 and 33, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:32 to SEQ ID NO:33 to SEQ ID NO:7;
- forming the antisense strand of SEQ ID NO:4 by ligating two oligonucleotides of (a′) SEQ ID NOS:34 and 35, wherein the fragments are ligated from 5′ end to 3′ end as SEQ ID NO:35 to SEQ ID NO:34;
- and
- forming the RNAi agent by annealing complementary nucleotides of the sense strand of SEQ ID NO:3 and the antisense strand of SEQ ID NO:4.
Dependencies are inferred from the claim text.