Automated system for polynucleotide synthesis and purification

• US 5,262,530 A
• Filed: 07/27/1990
• Issued: 11/16/1993
• Est. Priority Date: 12/21/1988
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Zon, Gerald, Andrus, William A., McCollum, Christie D.
• Primary Examiner(s): Brown, Johnnie R.
• Assistant Examiner(s): Kunz, Gary L.
• Application Number:
  US07/558,747
• Time in Patent Office: 1,208 Days
• Field of Search: 536/27, 536/25.3, 536/25.31, 935/88, 435/6
• US Class Current: 536/25.31
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/0068 Means for controlling the apparatus of the process
• CPC Class Codes: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

Claims

1. A method for synthesizing a polynucleotide of a predetermined sequence, the method comprising the steps of:

(a) providing a nonswellable porous polystyrene support which remains substantially mechanically rigid during synthesis and which has a crosslink ration in the range of 10 to 60 percent;

(b) providing a 5'"'"'-blocked protected nucleoside having a 3'"'"'-hydroxyl and a 5'"'"'-hydroxyl, the 5'"'"'-blocked protected nucleoside being attached to the nonswellable porous polystyrene support, such that the 5'"'"'-blocked protected nucleoside forms a protected correct-sequence chain;

(c) deblocking the 5'"'"'-hydroxyl of the correct-sequence chain;

(d) reacting with the 5'"'"'-hydroxyl of the correct-sequence chain a 5'"'"'-blocked protected nucleoside monomer selected from the group consisting of 5'"'"'-blocked protected nucleoside-3'"'"'-phosphoramidite and 5'"'"'-blocked protected nucleoside-3'"'"'-hydrogen phosphonate to form either a protected correct-sequence chain or a failure sequence, the failure sequence having a 5'"'"'-hydroxyl, wherein at least ninety-nine percent of the 5'"'"'-hydroxyls of the correct-sequence chains react with the 5'"'"'-blocked protected nucleoside monomer;

(e) repeating steps (c) and (d) until the polynucleotide of the predetermined sequence is obtained; and

(f) cleaving the polynucleotide from the nonswellable porous polystyrene support to form a cleavage mixture.

2. The method of claim 1 wherein said step of cleaving includes deprotecting said polynucleotide.

3. The method of claim 2 further including the step of capping said failure sequence by reacting a capping agent with said 5'"'"'-hydroxyl of said failure sequence.

4. The method of claim 3 further including the step of purifying said polynucleotide from said cleavage mixture.

5. The method of claim 4 wherein said step of purifying includes the steps of exposing said cleavage mixture to a nonswellable hydrophobic adsorbent, washing the nonswellable hydrophobic adsorbent to preferentially remove unblocked polynucleotides, deblocking the 5'"'"'-hydroxyl of said polynucleotide to form a deblocked polynucleotide, and eluting the deblocked polynucleotide from the nonswellable hydrophobic adsorbent.

6. The method of claim 3 wherein said 5'"'"'-blocked protected nucleoside monomer is 5'"'"'-blocked protected nucleoside-3'"'"'-hydrogen phosphonate, and wherein said step of capping occurs after said step of repeating.

7. The method of claim 3 wherein said 5'"'"'-blocked protected nucleoside monomer is 5'"'"'-blocked protected nucleoside-3'"'"'-phosphoramidite, and wherein said step of capping occurs after said step of reacting.

8. The method of claim 7 wherein said 5'"'"'-blocked protected nucleoside-3'"'"'-phosphoramidite is defined by the formula:

##STR5## wherein;

R₁ is hydrogen or lower alkyl;

R₂ is hydrogen, lower alkyl, lower alkoxy, lower aryloxy, or cyano-, nitro-, or sulfono-substituted lower aryloxy;

R'"'"' is methyl or beta-cyanoethyl;

DMT is dimethoxytrityl; and

B is adenine, guanine, thymidine, or cytidine.

9. The method of claim 8 wherein R₁ is hydrogen,

ethyl, or propyl, and wherein R₂ is hydrogen, methyl, ethyl, propyl, methoxy, ethoxy, isopropoxy, tert-butyloxy, phenoxy, napthoxy, or biphenoxy.

10. The method of claim 9 wherein R₁ is hydrogen or methyl, and wherein R₂ is hydrogen, methyl, ethoxy, or phenoxy.

11. The method of claim 1 wherein said step of reacting further includes exposing said 5'"'"'-hydroxyl of said correct-sequence chain to said 5'"'"'-blocked protected nucleoside monomer, said 5'"'"'-blocked protected nucleoside monomer being in less than or equal to ten molar excess of said 5'"'"'-hydroxyl of said correct-sequence chain.

12. The method of claim 11 wherein said 5'"'"'-blocked protected nucleoside monomer is a 5'"'"'-blocked protected nucleoside-3'"'"'-phosphoramidite.

13. The method of claim 11 wherein said step of reacting further includes delivering less than or equal to 4.0 mg of said 5'"'"'-blocked protected nucleoside monomer to less than or equal to 1 umole of 5'"'"'-hydroxyl of said correct-sequence chain in a reaction volume of between 50-200 uL.

14. The method of claim 13 wherein said 5'"'"'-blocked protected nucleoside monomer is a 5'"'"'-blocked protected nucleoside-3'"'"'-phosphoramidite.

15. In a method of synthesizing a polynucleotide of a predetermined sequence, the method of the type wherein nucleoside phosphoramidite monomers are coupled step-wise to a correct-sequence chain attached to a solid phase support in a solvent, an improvement comprising:

providing a nonswellable porous polystyrene support which remains substantially mechanically rigid during synthesis and which has a crosslink ratio in the range of 10 to 60 percent; and

coupling said nucleoside phosphoramidite monomer to said correct-sequence chain with a step-wise yield of at least ninety-nine percent.

16. The method of claim 15 wherein said nonswellable porous polystyrene support increases in volume in said solvent during synthesis by no more than ten percent over the dry volume of said nonswellable porous polystyrene support.

17. The method of claim 15 wherein said nonswellable porous polystyrene support increases in volume in said solvent during synthesis by no more than five percent over the dry volume of said nonswellable porous polystyrene support.