Methods and compositions for determining the purity of chemically synthesized nucleic acids
This application describes an antibody that specifically binds to a synthetic oligomer (e.g., an oligonucleotide or oligopeptide) having a organic protecting group covalently bound thereto, which antibody does not bind to that synthetic oligomer when the organic protecting group is not covalently bound thereto. Methods of making and using such antibodies are also disclosed, along with cells for making such antibodies and articles carrying immobilized oligomers that can be used in assay procedures with such antibodies.
1. A method for separating protected from fully deprotected synthetic oligonucleotides, comprising:
contacting a mixture of protected and fully deprotected synthetic oligonucleotides to an antibody, wherein said protected synthetic oligonucleotides have said organic protecting group covalently bound thereto, so that said protected synthetic oligonucleotides bind to said antibody; and then
separating said antibody from said fully deprotected synthetic oligonucleotides;
which antibody specifically binds to a synthetic oligonucleotide having an organic protecting group covalently bound thereto, which antibody does not bind to said synthetic oligonucleotide when said organic protecting group is not covalently bound thereto;
wherein said oligonucleotide contains a protected nucleotide according to Formula (I):
wherein:
(i) said protected nucleotide of Formula I is a 3′ nucleotide; R is a covalent bond to an adjacent nucleotide; R 1 is a protecting group; R 2 is H or —OH; R 4 is absent; and base is a purine or pyrimidine base; or
(ii) R is a covalent bond to an adjacent nucleotide; R 1 is a covalent bond to an adjacent nucleotide; R 2 is —OR 3 ; R 3 a protecting group; R 4 is absent; and base is a purine or pyrimidine base; or
(iii) R is a covalent bond to an adjacent nucleotide; R 1 is a covalent bond to an adjacent nucleotide; R 2 is H or —OH; Base is a purine or pyrimidine base; and R 4 is a protecting group bonded to an amino group of said base.
2. The method according to claim 1 , wherein said antibody is immobilized on a solid support.
3. The method according to claim 1 , wherein said protected synthetic oligonucleotide is a partially protected synthetic oligonucleotide.
4. The method according to claim 1 , wherein said contacting and separating steps are carried out by affinity chromatography.
5. The method according to claim 1 , wherein said oligonucleotide consists of from 3 to 20 nucleotides and has a 3′ nucleotide, and wherein said 3′ nucleotide is a protected nucleotide according to Formula (I):
wherein:
R is a covalent bond to an adjacent nucleotide;
R 1 is a protecting group;
R 2 is H or —OH; and
Base is a purine or pyrimidine base.
6. The method according to claim 1 , wherein said oligonucleotide consists of from 3 to 20 nucleotides, and wherein one of said nucleotides is a protected nucleotide according to Formula (I):
wherein:
R is a covalent bond to an adjacent nucleotide;
R 1 is a covalent bond to an adjacent nucleotide;
R 2 is
R 3 a protecting group; and
Base is a purine or pyrimidine base.
7. The method according to claim 1 , wherein said oligonucleotide consists of from 3 to 20 nucleotides, and wherein one of said nucleotides is a protected nucleotide according to Formula (I):
wherein:
R is a covalent bond to an adjacent nucleotide;
R 1 is a covalent bond to an adjacent nucleotide;
R 2 is H or —OH;
Base is a purine or pyrimidine base; and
R 4 is a protecting group bonded to an amino group of said base.
8. The method according to claim 1 , wherein said oligonucleotide consists of from 3 to 20 nucleotides, and wherein one of said nucleotides is protected with a photolabile protecting group.
9. The method according to claim 1 , which antibody is a polyclonal antibody.
10. The method according to claim 1 , which antibody is a monoclonal antibody.