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US 2003/0113769 A1Published application
Aminooxy functionalized oligomers, oligomer arrays and methods of using them
AssigneeIsis Pharmaceuticals, Inc.
InventorsMuthiah Manoharan, Harri Lonnberg, Harri Salo, Pasi Virta
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Abstract
The present invention provides oligomers which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least one of the nucleoside moieties of the oligomer is modified to include an aminooxy linkage. These oligomers are useful for diagnostic, therapeutic and investigative purposes.
Claims44
An oligomer comprising a plurality of nucleotide units of the structure:
- 5
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further comprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 6
- wherein:
- each Z is, independently, a single bond, O or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6.
15 more elements
A method of identifying a target oligonucleotide in a sample comprising the steps of:
- (a) selecting an oligonucleotide comprising a plurality of nucleotide units of the structure:
- 7
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further ccmprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 8
- wherein:
- each Z is, independently, a single bond, 0 or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6;
- (b) attaching said oligonucleotide to a solid support material;
- (c) treating said oligonucleotide with a target oligonucleotide to form a hybridization mixture, said target oligonucleotide being labeled with a marker;
- (d) detecting the binding of said oligonucleotide with said target oligonucleotide in said hybridization mixture;
- and
- (e) determining the amount of said oligonucleotide bound to said target oligonucleotide.
21 more elements
A method of deprotecting an aminooxy functionality comprising the steps of:
- (a) providing an oligomer bearing an aminooxy functionality protected with a phthaloyl protecting group;
- (b) treating said oligomer with a hydrazine acetate solution to form a deprotected oligomer;
- and
- (c) washing said deprotected oligomer.
1 more element
A method of identifying a gene in a sample comprising the steps of:
- (a) selecting a first oligonucleotide which is complementary to a first region of said gene;
- (b) attaching said first oligonucleotide to a solid support;
- (c) adding said first oligonucleotide to said sample to form a hybridization complex between said first oligonucleotide and said gene;
- (d) selecting a second oligonucleotide which is complementary to a second region of said gene;
- wherein said first and second oligonucleotides comprise a plurality of nucleotide units of the structure:
- 9
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further comprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 10
- wherein:
- each Z is, independently, a single bond, 0 or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6;
- said second oligonucleotide further comprising a marker thereon;
- (e) adding said second oligonucleotide to said complex to form a sandwich hybrid;
- and
- (f) detecting said marker of said second oligonucleotide in said sandwich hybrid.
24 more elements
A method of identifying a target gene amenable to antisense modulation comprising the steps of:
- (a) selecting a plurality of oligonucleotides, each of said oligonucleotides comprising a plurality of nucleotide units of the structure:
- 11
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further comprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 12
- wherein:
- each Z is, independently, a single bond, 0 or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6;
- (b) attaching each of said oligonucleotides to individual solid supports to form an array;
- (c) adding said target gene to said array to allow the binding of said target gene to at least one oligonucleotide of said array;
- and
- (d) detecting the binding of said target gene to said oligonucleotide of said array.
20 more elements
A method of identifying a full-length oligonucleotide in a mixture of oligonucleotides comprising the steps of:
- (a) selecting a target oligonucleotide complementary to said full-length oligonucleotide, said target oligonucleotide comprising a plurality of nucleotide units of the structure:
- 13
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further comprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 14
- wherein:
- each Z is, independently, a single bond, 0 or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6;
- said target oligonucleotide further having a donor fluorophore at one end and an acceptor fluorophore at the other end;
- (b) attaching said target oligonucleotide to a solid support;
- (c) adding said mixture of oligonucleotides to said target oligonucleotide to form a hybridization complex between said target oligonucleotide and said full-length oligonucleotide;
- (d) washing said complex to remove unbound oligonucleotides;
- and
- (e) detecting said full-length oligonucleotide bound to said target oligonucleotide.
22 more elements
A method of detecting binding of an oligonucleotide to a target oligonucleotide comprising the steps of:
- (a) selecting a target oligonucleotide having a donor fluorophore and an acceptor fluorophore such that both of said fluorophores are in close proximity, said target oligonucleotide comprising a plurality of nucleotide units of the structure:
- 15
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further comprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 16
- wherein:
- each Z is, independently, a single bond, 0 or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6;
- (b) adding an oligonucleotide to said target oligonucleotide to form a hybridization complex;
- and
- (c) detecting the fluorescence of said hybridization complex.
19 more elements
A method of determining accessibility of a target oligonucleotide comprising the steps of:
- (a) selecting a plurality of oligonucleotides, each of said oligonucleotides comprising a plurality of nucleotide units of the structure:
- 17
- wherein:
- Bx is a purine or pyrimidine heterocyclic base;
- each T1 and T2 is, individually, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
- T3 is H, OH, a protected hydroxyl or a sugar substituent group;
- said oligomer further comprising at least one group, R, therein;
- said R group occurring at the 5′-end, the 3′-end, in lieu of at least one T3 or as a substituent on at least one Bx;
- said R group having one of the formulas:
- 18
- wherein:
- each Z is, independently, a single bond, 0 or a phosphate;
- each Q is, independently, H, C1-C10 alkyl or a nitrogen protecting group;
- each T0 is, independently, a bond or a linking moiety;
- each L is, independently, a chemical functional group, a conjugate group or a solid support material;
- or Q, T0 and L, together, are a chemical functional group;
- each m is, independently, an integer from 1 to about 10;
- and
- each n is, independently, an integer from 1 to about 6;
- selecting a target oligonucleotide;
- (b) attaching each of said oligonucleotides to individual solid supports to form an array;
- (c) selecting a target oligonucleotide;
- (d) attaching a label to said target oligonucleotide to form a labeled target;
- (e) contacting said labeled target to said array;
- and
- (f) detecting the label of said labeled target.
23 more elements
Dependencies are inferred from the claim text.