CHEMICALLY MODIFIED POLYNUCLEOTIDES AND METHOD FOR PRODUCING CHEMICALLY MODIFIED POLYNUCLEOTIDES

• US 20200131512A1
• Filed: 09/12/2019
• Published: 04/30/2020
• Est. Priority Date: 08/26/2013
• Status: Abandoned Application

• Current Assignee: Universidade De Sã£O Paulo.
• Original Assignee: Universidade De Sã£O Paulo.
• Inventors: ULRICH, Alexander Henning, BASSANEZE, Vinicius, NERY, Arthur Andrade, KRIEGER, Jose Eduardo
• Application Number:
  US16/569,448
• Publication Number:
  US 20200131512A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: 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
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/344 Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/3533 Halogen
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention relates to chemically-modified polynucleotides of formula (I): 5′-CONS-SEQ.ID.n-CONS-3′ (I) containing one or more modified pyrimidines and at least one inverted nucleotide repeat, and to the method for producing the same.

Claims

1. Chemically-modified polynucleotides characterized by having the formula (I):

5′

-CONS-SEQ.ID.n-CONS-3′

 

 

(I),

wherein;

SEQ.ID.n is alternately or separately one or more of identifying sequences of SEQ. ID. 1 to SEQ. ID. 32 containing one or more modified pyrimidines made by replacing a 2-OH with a halogen atom, and at least one inverted nucleotide, wherein the polynucleotides of formula 5′

-CONS-SEQ.ID.n-CONS-3′

are organic molecules of molecular signature having a high binding affinity for mesenchymal stem cells from human adipose tissue and other cells of mesenchymal origin.

2. The polynucleotides according to claim 1, wherein modifications in one or more pyrimidines are made by the substitution of 2-OH with a halogen atom.

3. The polynucleotides according to claim 1, wherein the modified pyrimidines are 2′

F-dCTP and 2′

F-dCUTP.

4. The polynucleotides according to claim 1, wherein the polynucleotides are useful for the detection and identification of mesenchymal stem cells, preferably human mesenchymal cells.

5. A method of producing the chemically-modified polynucleotides of formula (I):

5′

-CONS-SEQ.ID.n-CONS-3′

 

 

(I)

which comprises the steps of;

(a) Providing a polynucleotide library;

(b) Incubating the polynucleotides of formula (I) in a selection buffer and possible binding targets;

(c) Subjecting the polynucleotides from step (b) to a series of washing cycles and collecting polynucleotides that remain bound after each washing cycle; and

(d) Amplifying the collected polynucleotides by polymerase chain reaction;

wherein SEQ.ID.n is independently and randomly a sequence among SEQ. ID, 1 to 32, and

wherein the amplified polynucleotides of formula 5′

-CONS-SEQ.ID.n-CONS-3′

are organic molecules of molecular signature having a high binding affinity for mesenchymal stem cells from human adipose and other cells of mesenchymal origin.

6. The method according to claim 5, wherein the targets of the polynucleotides of formula (I) 5′

-CONS-SEQ.ID.n-CONS-3′

are organic molecules recognizing the molecular signature of human lipoaspirate mesenchymal stem cell membranes.

7. The method according to claim 5, wherein step (a) occurs randomly, a polynucleotide of an ssDNA library is amplified;

the amplification occurs in cycles of between 4 to 6 minutes at a temperature between 90°

to 100°

C.;

followed by 4 to 6 minutes at a temperature between 40° and

45°

C. and 10 minutes at 72°

C.

8. The method, according to claim 5, wherein the binding event sequence to the target, selection and amplification is a SELEX cycle.