Combinatorial selection of oligonucleotide aptamers
First Claim
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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
3′
PCR primer nucleotide sequences flanking a randomized nucleotide sequence;
(b) amplifying the library enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides comprises a mix of unmodified and thiophosphate-modified nucleotides, to form a partially thiophosphate-modified oligonucleotide combinatorial library;
(c) contacting the partially thiophosphate-modified oligonucleotide combinatorial library with a target molecule and isolating a subset of oligonucleotides binding to the target molecule;
(d) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotides, wherein at least a portion of at least one nucleotide is thiophosphate-modified, to form a thiophosphate-modified oligonucleotide sub-library; and
(e) repeating steps (c)-(d) iteratively with increased stringency of the contacting step between each iteration until at least one aptamer comprising a thiophosphate-modified oligonucleotide population of defined sequence is obtained.
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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).
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Citations
21 Claims
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1. A method of identifying an aptamer comprising the steps of:
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(a) synthesizing a random phosphodiester oligonucleotide combinatorial library wherein constituent oligonucleotides comprise at least a set of 5′ and
3′
PCR primer nucleotide sequences flanking a randomized nucleotide sequence;
(b) amplifying the library enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides comprises a mix of unmodified and thiophosphate-modified nucleotides, to form a partially thiophosphate-modified oligonucleotide combinatorial library;
(c) contacting the partially thiophosphate-modified oligonucleotide combinatorial library with a target molecule and isolating a subset of oligonucleotides binding to the target molecule;
(d) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotides, wherein at least a portion of at least one nucleotide is thiophosphate-modified, to form a thiophosphate-modified oligonucleotide sub-library; and
(e) repeating steps (c)-(d) iteratively with increased stringency of the contacting step between each iteration until at least one aptamer comprising a thiophosphate-modified oligonucleotide population of defined sequence is obtained. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of identifying a set of aptamers containing an optimal composition of thiophosphate-modified nucleotides such that the aptamers bind with high affinity to a target molecule, and have increased resistance to nuclease degradation, said method comprising the steps of:
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(a) synthesizing a random partially thiophosphate-modified oligonucleotide combinatorial library wherein at least a portion of the oligonucleotide phosphate groups are thiophosphate-modified nucleotides, and where no more than three of the four different nucleotides are substituted on the 5′
-phosphate positions by 5′
-thiophosphates in each synthesized oligonucleotide are thiophosphate-modified nucleotides;
(b) amplifying the library enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides comprises a mix of unmodified and thiophosphate-modified nucleotides, whereby no more than three adjacent phosphate sites in each amplified oligonucleotide are thiophosphate-modified nucleotides;
(c) contacting the amplified library with a target molecule under conditions favorable for binding of a binding oligonucleotide with said target molecule;
(d) isolating a subset of binding oligonucleotides from the library, that bind with higher affinity to the target molecule relative to the original amplified library; and
(e) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides comprises a mix of unmodified and thiophosphate-modified nucleotides. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18)
(f) repeating steps (c)-(e) iteratively, whereby an enriched subset of oligonucleotides binding with higher affinity to the target molecule relative to the original amplified subset, is isolated after each cycle.
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13. The method of claim 12, whereby each iteration is performed under conditions of increased stringency in the contacting step until a subset of high affinity binding oligonucleotides is identified.
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14. The method of claim 11, whereby synthesis of the combinatorial library is done using constituent oligonucleotides comprising at least a set of 5′
- and 3′
PCR primer nucleotide sequences flanking a randomized nucleotide sequence.
- and 3′
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15. The method of claim 11, whereby the subset of amplified oligonucleotides is cloned and where individual thiophosphate-modified oligonucleotides that bind to the target are isolated and sequenced.
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16. The method of claim 15, whereby the isolated aptamer is screened relative to its respective non-modified oligonucleotide, to possess:
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(a) increased affinity for the target, and (b) increased stability with respect to nuclease degradation.
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17. The method of claim 16, whereby the thiophosphate comprises a phosphorodithioate.
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18. The method of claim 16, whereby the thiophosphate comprises a phosphorothioate or phosphorodithioate.
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19. A method of identifying an aptamer that specifically recognizes NF-IL6, comprising the steps of:
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(a) synthesizing a random phosphodiester oligonucleotide combinatorial library wherein constituent oligonucleotides comprise at least a set of 5′ and
3′
PCR primer nucleotide sequences flanking a randomized nucleotide sequence;
(b) amplifying the library enzymatically using a mix of four nucleotides, wherein one or more of the nucleotides comprises a mix of unmodified and triphosphate (alpha-S) modified nucleotides to form a thiophosphate-modified oligonucleotide combinatorial library;
(c) contacting the thiophosphate-modified oligonucleotide combinatorial library with an NF-IL6 target molecule and isolating a subset of oligonucleotides binding to the NF-IL6 target molecule;
(d) amplifying the subset of binding oligonucleotides enzymatically using a mix of four nucleotide nucleotides, wherein a portion of at least one and no more than three of the nucleotide nucleotides in the mix is a nucleotide triphosphate, to form a thiophosphate-modified oligonucleotide sub-library; and
(e) repeating steps contacting and amplifying the oligonucleotides iteratively with increased stringency of the contacting step between each iteration until at least one aptamer comprising a thiophosphate-modified oligonucleotide population of defined sequence and site of thiophosphate substitution is obtained and only a portion of the oligonucleotide is substituted. - View Dependent Claims (20, 21)
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Specification