Alkyl-linked nucleotide compositions
View Patent ↗Alkyl-linked nucleotide non-homogeneous solid supports and nucleotide affinity media comprising an alkyl-linked nucleotide are provided. The linker is generally a hydrophobic linker that can be a 3, 4, 5, 6, 7, 8, 9, 10, or a longer carbon chain. Also included in the invention are methods for synthesis of an alkyl-linked nucleotide, nucleotide affinity media and methods of use thereof for affinity chromatography and screening methods.
1. An alkyl-linked nucleotide non-homogeneous solid support consisting essentially of the general formula:
wherein Y is a solid support; x=1; R 1 is a covalent bond between Y and R 2 , or R 1 is a divalent acyl group —C(═Q)—, wherein Q is O or NH 2 + ; K is NH; R 7 is (P) n where P is a phosphate or a thiophosphate and n is at least one; and m is at least one; Z is a 5′-nucleosidyl group or a 5′-nucleosidyl group wherein the 5′-nucleosidyl group is not naturally occurring, or a derivative thereof; and —R 2 —K— is selected from the group consisting of:
wherein the solid support has a loading of an alkyl-linked nucleotide having a range of about 20% to about 50%.
2. The alkyl-linked nucleotide non-homogeneous solid support of claim 1 , wherein the solid support includes at least one member selected from the group consisting of an acrylamide, agarose, methacrylate, cellulose, nylon, silica, glass, ceramic, a magnetized particle, nitrocellulose, polystyrene, a thermoresponsive polymer, and derivatives thereof.
3. The alkyl-linked nucleotide non-homogeneous solid support of claim 1 , wherein the solid support is a beaded agarose.
4. The alkyl-linked nucleotide non-homogeneous solid support of claim 1 , wherein the 5′-nucleosidyl group is selected from the group consisting of a -5-deoxy-5′-adenosinyl radical, a -5-deoxy-5′-guanosinyl radical, a -5-deoxy-5′-cytidinyl radical, a -5-deoxy-5′-thymidinyl radical, and a -5-deoxy-5′-uridinyl radical, or an analog thereof.
5. The alkyl-linked nucleotide non-homogeneous solid support of claim 4 , wherein the 5′-nucleosidyl group is a -5-deoxy-5′-adenosinyl radical, said alkyl-linked nucleotide non-homogeneous solid support consisting essentially of the general structure:
or an ionized variant or a salt thereof.
6. The alkyl-linked nucleotide non-homogeneous solid support of claim 4 , wherein the 5′-nucleosidyl group is a -5-deoxy-5′-guanosinyl radical, said alkyl-linked nucleotide non-homogeneous solid support consisting essentially of the general structure:
or an ionized variant or a salt thereof.
7. The alkyl-linked nucleotide non-homogeneous solid support of claim 4 , wherein the 5′-nucleosidyl group is a -5-deoxy-5′-thymidinyl radical, said alkyl-linked nucleotide non-homogeneous solid support consisting essentially of the general structure:
or an ionized variant or a salt thereof.
8. The alkyl-linked nucleotide non-homogeneous solid support of claim 4 , wherein the 5′-nucleosidyl group is a -5-deoxy-5′-cytidinyl radical, said alkyl-linked nucleotide non-homogeneous solid support consisting essentially of the general structure:
or an ionized variant or a salt thereof.
9. The alkyl-linked nucleotide non-homogeneous solid support of claim 4 , wherein the 5′-nucleosidyl group is a -5-deoxy-5′-uridinyl radical, said alkyl-linked nucleotide non-homogeneous solid support consisting essentially of the general structure:
or an ionized variant or a salt thereof.
10. The alkyl-linked nucleotide non-homogeneous solid support of claim 1 , wherein:
R 1 is —C(═NH 2 + )—;
R 2 is:
P is phosphate; and
n is 3.
11. A method for synthesizing a nucleotide affinity medium consisting essentially of the general formula:
comprising the steps of:
a) coupling at least one linker to a solid support in a suitable coupling buffer, wherein said linker is R 2 or a combination of R 1 and R 2 ;
b) end-capping at least a portion of reactive sites remaining on said solid support after said coupling step; and
c) reacting a terminal phosphate or thiophosphate group of a nucleotide with said linker coupled to said solid support,
wherein Y is a solid support; x=1; R 1 is a covalent bond between Y and R 2 , or R 1 is a divalent acyl group —C(═Q)—, wherein Q is O or NH 2 +; K is NH; R 7 is (P) n where P is a phosphate or thiophosphate and n is at least one; Z is a 5′-nucleosidyl group or a 5′-nucleosidyl group wherein the 5′-nucleosidyl group is not naturally occurring, or a derivative thereof; and m is at least one;
and —R 2 —K— is selected from the group consisting of:
wherein the solid support has a loading of an alkyl-linked nucleotide having a range of about 20% to about 50%.
12. A method for screening a test compound comprising the steps of:
a) contacting a proteome with a nucleotide affinity medium consisting essentially of the general formula:
wherein Y is a solid support; x=1; R 1 is a covalent bond between Y and R 2 , or R 1 is a divalent acyl group —C(═Q)—, wherein Q is O or NH 2 +, K is NH; R 7 is (P) n where P is a phosphate or thiophosphate and n is at least one Z is a 5′-nucleosidyl group or a 5′-nucleosidyl group wherein the nucleoside is not naturally occurring, or a derivative thereof; and m is at least one;
and —R 2 —K— is selected from the group consisting of:
wherein the solid support has a loading of an alkyl-linked nucleotide having a range of about 20% to about 50%;
b) washing the nucleotide affinity medium with a buffer, whereby non-specifically bound components of the proteome are eluted from the nucleotide affinity medium and specific components of the proteome remain bound to the nucleotide affinity medium;
c) contacting the nucleotide affinity medium bound with specific components of the proteome with at least one test compound;
d) eluting from the nucleotide affinity medium components of the proteome that are specifically displaced by the test compound; and
e) identifying the components of the proteome that are specifically displaced by the test compound from the nucleotide affinity medium.