# US 8,569,252 B2 · Nucleolin specific aptamer and use thereof

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- Number: US 8,569,252 B2
- Document ID: US8569252B2
- Status: Granted
- Kind: B2
- Application number: 12/759,813
- Filed: 2010-04-14
- Published: 2010-12-16
- Granted: 2013-10-29
- Earliest claimed: 2009-04-15
- Assignees: Postech Academy-Industry Foundation; Posco
- Applicants: Jung-Hwan Lee; Soon-Hag Kim; Mi-Jin Kwon; Hyun-Gu Kang; Sung-Ho Ryu; Jong-In Kim; Youn-Dong Kim; Young-Chan Chae; Sung-Key Jang; Jong-Hun Im; Sun-Hak Lee; Hye-Jung Lee; Eun-Jung Jang; Ki-Seok Kim
- Inventors: Jung-Hwan Lee; Soon-Hag Kim; Mi-Jin Kwon; Hyun-Gu Kang; Sung-Ho Ryu; Jong-In Kim; Youn-Dong Kim; Young-Chan Chae; Sung-Key Jang; Jong-Hun Im; Sun-Hak Lee; Hye-Jung Lee; Eun-Jung Jang; Ki-Seok Kim
- Web page: https://patentgrep.com/patent/US8569252B2

## CPC

- G01N 33/57595: {involving intracellular compounds}
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12N 2310/16: Aptamers
- C12N 2310/335: Modified T or U
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Improved G-rich oligonucleotide (GRO) aptamers specific to nucleolin, a method of preparing the aptamers, and a use of the aptamers for diagnosing and/or treating a nucleolin-associated disease, are provided.

## Claims

### Claim 1

Nucleolin-specific aptamer, wherein the aptamer has the sequence of SEQ ID NO: 3, and two thymidines(T) present at at least 12th and 13th positions of the nucleotide sequence are independently substituted with a modified pyrimidine nucleoside, and wherein the modified pyrimidine nucleoside is deoxyuridine(dU), deoxycytidine(dC), uridine(U), or cytidine(C) having a hydrophobic group at 5 position:
NGGTGGTGGTGGTTGTGGTGGTGGTGGN
(SEQ ID NO: 3)
wherein each N is absent or 1 to 20 nucleosides, which is independently selected from the group consisting of adenosine(A), thymidine(T)/uridine(U), cytidine(C), and guanosine(G), and the positions are counted starting from ‘G’ immediately following ‘N’ at 5′-end,
wherein the hydrophobic group is selected from the group consisting of a benzyl group, a naphthyl group, or a pyrrolebenzyl group.

### Claim 2 (depends on 1)

The nucleolin-specific aptamer according to claim 1, wherein the aptamer has the sequence of SEQ ID NO: 1 or 2, and two thymidines(T) present at at least 15th and 16th positions of SEQ ID NO: 1 or at least 12th and 13th positions of SEQ ID NO: 2 are independently substituted with a modified pyrimidine nucleoside, and wherein the modified pyrimidine nucleoside is deoxyuridine(dU), deoxycytidine(dC), uridine(U), or cytidine(C) having a hydrophobic group at 5 position.

### Claim 3 (depends on 1)

The nucleolin-specific aptamer according to claim 1, wherein the modified pyrimidine nucleoside is selected from the group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine(called BzdU), 5-(N-naphthylcarboxyamide)-2′-deoxyuridine(called NapdU), 5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxyuridine(called 4-PBdU), 5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC), 5-(N- naphthylcarboxyamide)-2′-deoxycytidine (called NapdC), 5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PBdC), 5-(N-benzylcarboxyamide)-2′-uridine(called BzU), 5-(N-naphthylcarboxyamide)-2′-uridine(called NapU), 5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine(called 4-PBU), 5-(N-benzylcarboxyamide)-2′-cytidine (called BzC), 5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC), and 5-(N-4-pyrrolebenzylcarboxyamide)-2′-cytidine (called 4-PBC).

### Claim 4 (depends on 1)

The nucleolin-specific aptamer according to claim 1, wherein at least one thymidine in addition to the two thymidines(T) present at 12th and 13th positions of SEQ ID NO: 3 are additionally substituted with the modified pyrimidine nucleoside, and the positions are counted starting from ‘G’ immediately following ‘N’ at 5′-end.

### Claim 5 (depends on 2)

The nucleolin-specific aptamer according to claim 2, wherein at least one thymidine in addition to the two thymidines(T) present at 15th and 16th positions of SEQ ID NO: 1 or 12th and 13th positions of SEQ ID NO: 2 are additionally substituted with the modified pyrimidine nucleoside, myeloproliferative disorders, carcinomas of solid tissue, sarcomas, melanomas, adenomas, hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx, or lung, genitourinary cancers, hematopoietic cancers, head and neck cancers, and nervous system cancers, and benign lesions.

### Claim 6 (depends on 1)

A pharmaceutical composition containing the nucleolin-specific aptamer of claim 1.

### Claim 7 (depends on 1)

A method of inhibiting nucleolin, comprising the step of administering the nucleolin-specific aptamer of claim 1 to a subject or a sample comprising nucleolin-expressing cells.

### Claim 8 (depends on 1)

A method of inhibiting hyperproliferation of cell cased by nucleolin, comprising the step of administering the nucleolin-specific aptamer of claim 1 to a subject or a sample comprising nucleolin-expressing cells.