Parallel preparation of high fidelity probes in an array format
View Patent ↗The present invention provides massively parallel oligonucleotide synthesis and purification for applications that utilize large collections of defined high-fidelity oligonucleotides (e.g., from about 10 1 to about 10 5 different sequences, generally between 25-160 bases in length).
1. A method of fabricating a plurality of oligonucleotides having free 3′-hydroxyl groups from a high density oligonucleotide array, said method comprising the steps of
a) providing a solid substrate comprising a plurality of ribonucleotides attached thereto, one said ribonucleotide shown below
wherein PG 1 is protecting group 1, PG 2 is protecting group 2, B is a naturally or non-naturally occurring base, and said ribonucleotide is attached to said substrate through the 5′-hydroxyl group;
b) selectively removing PG 1 in pre-selected areas to provide a plurality of free 3′-hydroxyl groups on said ribonucloetide;
c) reacting said free 3′-hydroxyl groups with a 2′-deoxyribonucleotide having the structure
wherein PG 3 is protecting group 3 and RG is a reactive group to couple said 2′-deoxyribonucleotide to said ribonucleotide to provide the structure
d) selectively removing PG 3 from the 5′-hydroxyl of said 2′-deoxyribonucleotide in pre-selected areas to provide a plurality of free 5′-hydroxyl groups;
e) reacting said free 5′-hydroxyl groups with an additional 2′-deoxyribonucleotide having the structure
to yield a product of the structure
f) repeating steps d and e one or more times to provide said oligonucleotides attached to said solid substrate;
g) removing PG 2 from one or more of said ribonucleotides to provide a free 2′-hydroxyl group on each of said one or more ribonucleotides; and
h) transesterifying each of said one or more ribonucleotides to yield said solid substrate having a cyclic ester attached thereto and free oligonucleotides, each oligonucleotide having a 3′-hydroxyl group and having the structure
2. A method according to claim 1 wherein PG 2 is acetate.
3. A method according to claim 1 wherein PG 3 or PG 4 is a photolabile protecting group.
4. A method according to claim 3 wherein said photolabile protecting group is selected from the group consisting of NNPOC and MBPMOC wherein said photolabile protecting group is attached to the 5′-hydroxyl group of said 2′-deoxyribonucleotide as depicted by
5. A method according to claim 1 wherein the 2′-deoxyribonucleotide has a phosphoramidite reactive group as shown by
wherein R 1 is selected from cyanoethyl, methyl, t-butyl, trimethylsilyl or the like, and R 2 and R 3 are independently selected from isopropyl, cyclohexyl or the like.
6. A method according to any of claims 1 - 5 wherein B of the 2′-deoxyribonucleotide is selected from the group consisting of G, A, T, and C.
7. A method according to claim 1 wherein PG 3 or PG 4 is an acid labile protecting group.
8. A method according to claim 7 wherein PG 3 or PG 4 is dimethoxytrityl (DMT).
9. A method according to claim 8 wherein the DMT group is removed in selected areas by exposure to acid generated by a photoacid generator in the presence of electro magnetic radiation of an appropriate wavelength in the presence of an acid scavenger.
10. A method according to claim 9 wherein said acid scavenger is selected from the group consisting of organic bases and polymeric bases.
11. A method according to claim 10 wherein said acid scavenger is a polymeric base.
12. A method according to claim 1 wherein said plurality of oligonucleotides comprises between 10 1 to 10 5 sets of different sequences, each distinct set of sequences comprising a set.
13. A method according to claim 1 wherein said oligonucleotides are between about 80 to about 160 nucleotides.
14. A method according to claim 1 wherein said density is between 200-2000 pmol/cm 2 .
15. A method according to claim 1 wherein said transesterification is accomplished by raising the pH of the solution to between 9 and 12.
16. A method according to claim 1 wherein said tranesterification is initiated by the addition of metal ions.
17. A method according to claim 1 wherein PG 2 is selected from the group consisting of FPMP, CEE, TBDMS and TOM.
18. A method according to claim 1 wherein said oligonucleotides are probes.
19. A method according to claim 1 wherein said oligonucleotides are primers.
20. A method according to claim 1 wherein between steps d and e unreacted 5′-hydroxyls are capped.
21. A method according to claim 1 wherein said desired array has between 10 1 to 10 5 oligonucleotides of different sequences and between about 80-160 bases in length.
22. A method according to claim 1 wherein said release oligonucleotides have authentic 3′-hydroxy termini upon exposure to a distinct set of conditions or are further processed to have free 3′ hydroxyl termini.
23. A method according to claim 1 , further comprising the use of the photoprotective groups NPPOC or NNPOC to provide suitable primer purity and quantity.
24. A method according to claim 1 wherein DMT-based photoresist groups are used to provide fidelity primers.
25. A method according to claim 1 wherein ink-jet based in situ oligonucleotide synthesis is used to provide an oligonucleotide probe.
26. A method according to claim 1 wherein synthesis is initiated with a reverse-orientation RNA monomer that contains an orthogonal 2′-OH protecting group.
27. A method according to claim 7 wherein following conventional 3′→5′ probe synthesis, the 2′-OH protecting group is removed to allow base-induced intramolecular transesterification.
28. A method according to claim 8 wherein the rate of transesterification is enchanced by raising the pH of the aqueous solution to pH 9-12 or by the addition of particular metal ions.