C-3' protected monomeric nucleotides and synthesis of oligonucleotides on solid support
AssigneeLinden Technologies, Inc.
InventorsYih Huang, Tai-Nang Huang, Ming Shen
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Abstract
Immobilized nucleotide primers of this invention include a modified nucleotide tethered to a support substrate through a linking group. In particular, the modified nucleotide is constructed such that the C-5′ end of the nucleotide is tetherable to the linking group and the protected C-3′ end is available for further controlled modification, e.g., addition of other nucleotides in specific sequences to the immobilized nucleotide primer. Additionally, the linking group is of sufficient length to allow the immobilized nucleotide primer to be used to synthesize and screen arrays of oligonucleotides, e.g., enzymatic C-3′ primer extension.
Claims52
A method of producing an immobilized oligonucleotide on a substrate comprising:
- covalently attaching a first nucleotide or an oligonucleotide via a C-5′ oxygen of the nucleotide to the substrate.
A method of synthesizing an oligonucleotide on a substrate, comprising:
- (a) contacting a modified nucleotide via a C-5′ oxygen to an activated immobilized hydroxyl group to produce a covalently attached nucleotide, wherein the modified nucleotide includes a C-3′ photolabile protecting group and a C-5′ hydroxyl group, and wherein the activated immobilized hydroxyl group is activated with a phosphorous activating group;
- (b) irradiating the covalently attached nucleotide to remove the C-3′ photolabile protecting group and form a C-3′ hydroxyl group;
- (c) contacting the C-3′ hydroxyl group of the covalently attached nucleotide with a phosphorous activating group to produce an activated immobilized hydroxyl group at the C-3′ position of the covalently attached nucleotide;
- and
- (d) repeating steps (a) to (c).
A method of synthesizing an oligonucleotide, comprising:
- (a) providing a nucleotide or an oligonucleotide having a free terminal C-3′ hydroxyl and a terminal C-5′ that is blocked by a group, wherein the free terminal C-3′ hydroxyl is activated with a phosphorous activating group;
- (b) covalently coupling a modified nucleotide via a C-5′ oxygen to the activated hydroxyl group, wherein the modified nucleotide includes a C-3′ photolabile protected group and a C-5′ hydroxyl group;
- (c) irradiating the covalently attached nucleotide to remove the C-3′ photolabile protecting group and form a C-3′ hydroxyl group;
- (d) contacting the C-3′ hydroxyl group of the covalently attached nucleotide with a phosphorous activating group to produce an activated hydroxyl group at the C-3′ position;
- (e) covalently coupling a modified nucleotide via a C-5′ oxygen to the activated hydroxyl group of a previously covalently attached nucleotide;
- and
- (f) repeating steps (c) to (e).
An oligonucleotide array, comprising:
- A substrate having a plurality of addressable sites;
- each of the sites of the plurality having an oligonucleotide covalently attached to the substrate via its C-5′ oxygen atom, wherein each site of the plurality is directly adjacent to at least one other site, and wherein the sequence of each oligonucleotide is unique among the plurality.
A compound of the formula
- 15
- wherein X is selected from the group consisting of a photolabile protecting group and a chemically labile protecting group;
- R is selected from the group consisting of hydrogen, hydroxyl, and alkoxyl;
- P is selected from the group consisting of hydrogen, a phosphorous activating group, a nucleotide monomer or oligomer, and a phosphate or derivative thereof;
- and Base is selected from the group consisting of pyrimidine, purine, and derivatives thereof.
A compound of the formula
- 16
- wherein R1, R2, R3 each independently is selected from the group consisting of hydrogen, C1-C10 alkyl, C2-C10 alkenyl, aryl, benzyl, and C1-C10 alkoxyl;
- R4 is selected from the group consisting of C1-C10 alkyl, C2-C10 alkenyl, aryl, and benzyl;
- and P is selected from the group consisting of hydrogen and [(i-Pr)2N]POCH2CH2CN.
A compound of the formula
- 17
- where Y is a C-3′ oxygen of a nucleic acid and R1 is methyl.
Dependencies are inferred from the claim text.