Combinatorial selection of oligonucleotide aptamers
First Claim
Patent Images
1. A method of identifying an aptamer comprising the steps of:
- (a) synthesizing a random phosphodiester oligonucleotide combinatorial library wherein constituent oligonucleotides comprise at least a set of 5′ and
/or 3′
PCR primer nucleotide sequences flanking a randomized nucleotide sequence;
(b) amplifying the library enzymatically using a mix of four nucleotides, wherein at least a percentage of one of the nucleotides is modified, to form a modified oligonucleotide combinatorial library;
(c) contacting the modified oligonucleotide combinatorial library with a target molecule and isolating a subset of oligonucleotides binding to the target molecule;
(e) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotide substrates, wherein at least one nucleotide substrate is modified, to form a modified oligonucleotide sub-library; and
(f) repeating steps (c)-(e) iteratively until at least one aptamer comprising a modified oligonucleotide population of defined sequence is obtained.
0 Assignments
0 Petitions
Accused Products
Abstract
A random combinatorial selection method is disclosed for the construction of oligonucleotide aptamers in which nuclease resistance is conferred by the inclusion of modified nucleotides. The modified nucleotides are incorporated during PCR amplification to form achiral modified oligonucleotides. Thio-substituted aptamers are provided that bind tightly to the nuclear factor for human IL6 (NF-IL6).
-
Citations
35 Claims
-
1. A method of identifying an aptamer comprising the steps of:
-
(a) synthesizing a random phosphodiester oligonucleotide combinatorial library wherein constituent oligonucleotides comprise at least a set of 5′ and
/or 3′
PCR primer nucleotide sequences flanking a randomized nucleotide sequence;
(b) amplifying the library enzymatically using a mix of four nucleotides, wherein at least a percentage of one of the nucleotides is modified, to form a modified oligonucleotide combinatorial library;
(c) contacting the modified oligonucleotide combinatorial library with a target molecule and isolating a subset of oligonucleotides binding to the target molecule;
(e) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotide substrates, wherein at least one nucleotide substrate is modified, to form a modified oligonucleotide sub-library; and
(f) repeating steps (c)-(e) iteratively until at least one aptamer comprising a modified oligonucleotide population of defined sequence is obtained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 23, 25)
-
-
11. A method of thiophosphate aptamer selection comprising:
-
(a) generating a library of aptamers having a sequence of sufficient length to bind a desired target molecule, wherein at least a portion of the oligonucleotide phosphate groups are replaced with thio-modified nucleotides;
(b) contacting the aptamer library with the target under conditions sufficient to allow complexation between the aptamer library and the target;
(c) removing aptamer library members that bind weakly to the target molecule to yield an aptamer sub-library characterized by enhanced binding to the target;
(d) amplifying the aptamer sub-library enzymatically, wherein the amplifying includes incorporation thio-modified nucleotides into members of the amplified aptamer sub-library; and
(e) repeating steps (b) through (d), for a sufficient number of cycles to yield at least one aptamer of defined sequence and binding specificity for the target. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26)
-
-
27. An aptamer specific for NF-IL6 essentially homologous to the sequences of oligonucleotides selected from the group consisting of SEQ ID NOS.:
- 5 through 10.
- View Dependent Claims (28)
-
29. An NF-IL6 binding aptamer comprising a sequence of nucleotides essentially homologous to a nucleotide sequence of the formula:
-
5′
d GGGCCC GCTGN1 ACN2N3G C ACACGwherein substantially all dA nucleotides are phosphothioated and N1 is a C or T;
N2 is an A or G; and
N3 is a T or C.
-
-
30. An NF-IL6 binding aptamer comprising a sequence of nucleotides essentially homologous to a nucleotide sequence of the formula:
-
31. An NF-IL6 binding aptamer comprising the sequence of nucleotides:
-
G C T G T A C A T G (SEQ ID NO.;
12)wherein one or more nucleotides have at least one thio-modified phosphate group. - View Dependent Claims (32, 33)
-
-
34. A NF-IL6 binding oligonucleotide comprising at least the sequence of nucleotides:
-
G T T G T C C C A C (SEQ ID NO.;
13)wherein one or more nucleotides have at least one thio-modified phosphate group.
-
-
35. A NF-IL6 binding oligonucleotide comprising at least the sequence of nucleotides:
-
G T T G T T G T C C (SEQ ID NO.;
14)wherein one or more nucleotides have at least one thio-modified phosphate group.
-
Specification