Methods, kits, and reactive supports for 3' labeling of oligonucleotides
First Claim
1. A reactive support having a free or protected hydroxyl group useful for synthesis of an oligonucleotide, said support comprising a label moiety covalently bonded via a stable bond to a trifunctional spacer containing the hydroxyl group to form a labeled linker complex, the labeled linker complex being covalently bonded to a solid support via a cleavable bond.
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Abstract
In a first aspect, the invention involves a reactive support useful for automated synthesis of oligonucleotides. The reactive support comprises a label moiety (e.g. hapten) covalently bonded via a stable bond to a trifunctional spacer. The labeled trifunctional spacer complex is covalently bonded to a solid support via a cleavable bond. One arm of the trifunctional spacer attaches the solid phase; another arm attaches the label; while the third arm provides a hydroxyl group useful for synthesizing a labeled oligonucleotide. Upon synthesis, the cleavable bond is broken, yielding the labeled oligonucleotide. Methods for labeling oligonucleotides and useful kits are also described.
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Citations
27 Claims
- 1. A reactive support having a free or protected hydroxyl group useful for synthesis of an oligonucleotide, said support comprising a label moiety covalently bonded via a stable bond to a trifunctional spacer containing the hydroxyl group to form a labeled linker complex, the labeled linker complex being covalently bonded to a solid support via a cleavable bond.
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10. A reactive support having a free or protected hydroxyl group useful for synthesis of an oligonucleotide, said support comprising a label moiety covalently bonded via a stable bond to a trifunctional spacer containing the hydroxyl group to form a labeled linker complex, the labeled linker complex being covalently bonded to a solid support via a cleavable bond;
- wherein said trifunctional spacer is a cyclic or heterocyclic molecule.
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11. A process of preparing a reactive support having a label and a hydroxyl group useful for synthesizing a labeled oligonucleotide, said process comprising:
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a. reacting the second functionality of a trifunctional linker having three functionalities, a first one of whose functionalities is a hydroxyl group or a protected hydroxyl group and each of whose functionalities has or can be made to have a differential reactivity, with a label moiety having a reactive group capable of reacting with the second functionality to form a stable bond connecting the label moiety to the trifunctional linker; and b. reacting the third functionality of the trifunctional linker with one or more reagents capable of imparting a cleavable bond to the trifunctional linker, and with a functionalized solid support under conditions such that the trifunctional linker is covalently attached to the solid support via the cleavable bond. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19)
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20. A method for labeling the 3'"'"' end of an oligonucleotide synthesized on a solid support, said method comprising:
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a. reacting the second functionality of a trifunctional linker having three functionalities, a first one of whose functionalities is a protected hydroxyl group and each of whose functionalities has or can be made to have a differential reactivity, with a label moiety having a reactive group capable of reacting with the second functionality to form a stable bond connecting the label moiety to the trifunctional linker; b. reacting the third functionality of the trifunctional linker with one or more reagents capable of imparting a cleavable bond to the trifunctional linker, and with a functionalized solid support under conditions such that the trifunctional linker is covalently attached to the solid support via the cleavable bond. c. deprotecting the hydroxyl group; and d. synthesizing an oligonucleotide from the deprotected hydroxyl group. - View Dependent Claims (21, 22, 23, 24)
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Specification