Lin28/let-7 crystal structures, purification protocols, and molecular probes suitable for screening assays and therapeutics

Patent metadata

Field	Value
Publication number	US-9593141-B2
Application number	US-201214357020-A
Country	US
Kind code	B2
Filing date	Nov 9, 2012
Priority date	Nov 9, 2011
Publication date	Mar 14, 2017
Grant date	Mar 14, 2017

Abstract

The invention provides compositions and methods for regulating microRNA (miRNA) biogenesis. The invention also relates to compositions and methods for treating or preventing cancer in a subject in need thereof.

First claim

What is claimed is:

1. An RNA oligonucleotide comprising:
a. a nucleotide sequence of formula 5′-N 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 -3′, wherein N 2 , N 4 , N 5 and N 7 are independently a purine; N 1 , N 3 , N 6 , N 8 , and N 9 are independently a pyrimidine; and
b. a nucleotide sequence of 5′-GGAG-3′, wherein the sequence 5′-GGAG-3′ is linked to the 3′ of the sequence of formula 5′-N 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 -3′, wherein the sequence 5′-GGAG-3′ is single-stranded,
wherein there are from 0 to 25 nucleotides between the 3′ end of 5′-N 1 N 2 N 3 N 4 N 5 N 6 N 7 N 8 N 9 -3′ and 5′ end of the sequence 5′-GGAG-3′, and

wherein the RNA oligonucleotide is 18-100 nucleotides in length.

2. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises a hairpin structure comprising a hairpin loop of at least 3 nucleotides and N 4 , N 5 , and N 6 are in the loop region of the hairpin.

3. The oligonucleotide of claim 2 , wherein the hairpin structure comprises a double-stranded stem of at least four nucleotide base pairs, wherein the stem is fully double-stranded.

4. The oligonucleotide of claim 3 , wherein the stem comprises at least one G-clamp:G or guanadinium-G-clamp:G base pair.

5. The oligonucleotide of claim 4 , the stem is terminated by a G:C, G:U, G-clamp:G or guanadinium-G-clamp:G base pair.

6. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises at least one modification selected from the group consisting of a sugar modification, a non-phosphodiester intersugar (or internucleoside) linkage, nucleobase modification, and ligand conjugation.

7. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises a fluorescent reporter.

8. The oligonucleotide of claim 1 , wherein the 5′ end of the oligonucleotide is covalently linked to the 3′ end of the oligonucleotide.

9. The oligonucleotide of claim 1 , wherein N 1 and N 3 are independently selected purines and N 7 , N 8 , and N 9 are independently selected pyrimidines.

10. The oligonucleotide of claim 1 , wherein N 2 and N 4 are guanosine and N 5 is adenosine.

11. The oligonucleotide of claim 1 , wherein N 1 and N 5 are adenosine; N 3 is adenosine or uridine; N 2 and N 4 are guanosine; and N 6 , N 7 , N 8 , and N 9 are uridine.

12. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises the

(SEQ ID NO: 16)

5′-GGGCAGAGAUUUUGCCCGGAG-3′

or

(SEQ ID NO: 17)

5′-GGGGUAGUGAUUUUACCCUGGAG-3′.

13. The oligonucleotide of claim 1 , wherein N 1 , N 3 , and N 6 are uridine; N 2 , N 5 , and N 7 are adenosine; N 4 is guanosine; and N 8 and N 9 are cytosine.

14. The oligonucleotide of claim 1 , wherein the oligonucleotide comprises the sequence

(SEQ ID NO: 18)

5′-GGGGUCUAUGAUACCACCCCGGAG-3′.

15. A method for promoting miRNA processing of pri-miRNA to mature miRNA in a cell, wherein the miRNA is a let-7 family member, the method comprising contacting a cell with an isolated oligonucleotide of claim 1 .

Assignees

• President And Fellows Of Harvard Univ
• Harvard College

Inventors

• Sliz Piotr
• Nam Yunsun

Classifications

• A61K31/713

  Double-stranded nucleic acids or oligonucleotides · CPC title

• A61K31/712

  Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose · CPC title

• C07H21/02 Primary

  with ribosyl as saccharide radical · CPC title

• A61K47/645

  Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT · CPC title

• A61K31/7105

  Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links · CPC title