Design and synthesis of bispecific reagents: use of double stranded DNAs as chemically and spatially defined cross-linkers
First Claim
1. A method for preparing a double stranded DNA-protein conjugate comprising(a) preparing a first thiolated single stranded DNA, wherein said first thiolated single stranded DNA is(1) about 10 to 100 bases in length, and(2) thiolated at either the 3'"'"' or 5'"'"' end;
- (b) preparing a second thiolated single stranded DNA, said first and second thiolated single DNA strand being complementary to each other, and wherein said second thiolated single stranded DNA is(1) about 10 to 100 bases in length, and(2) thiolated at the same end as said first thiolated DNA strand;
(c) reacting said first thiolated DNA strand with a thionitrobenzoate derivatized ligand binding first protein to form a first single stranded DNA-protein conjugate;
(d) reacting said second thiolated DNA strand with a thionitrobenzoate derivatized ligand binding second protein to form a second single stranded DNA-protein conjugate;
(e) annealing said first and second single stranded conjugates; and
(f) recovering the double stranded DNA-protein conjugate product of step (e).
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Abstract
The invention relates to bis-protein-DNA conjugates. A protein having a specific ligand binding activity is covalently linked to each end of a derivatized DNA molecule. These bis-protein-DNA conjugates can be used for immunoassays, PCR assays and measuring distances between proteins at up to 3.4 A resolution. The invention also relates to methods of synthesizing these bis-protein-DNA conjugates. Synthesis of the conjugates entails derivatizing the 5'"'"' or 3'"'"' end of a DNA oligonucleotide and covalently linking that DNA to a protein. The DNA can be conjugated to the proteins, including antibodies or Fab'"'"' fragments, using disulfide bond linkage.
119 Citations
15 Claims
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1. A method for preparing a double stranded DNA-protein conjugate comprising
(a) preparing a first thiolated single stranded DNA, wherein said first thiolated single stranded DNA is (1) about 10 to 100 bases in length, and (2) thiolated at either the 3'"'"' or 5'"'"' end; -
(b) preparing a second thiolated single stranded DNA, said first and second thiolated single DNA strand being complementary to each other, and wherein said second thiolated single stranded DNA is (1) about 10 to 100 bases in length, and (2) thiolated at the same end as said first thiolated DNA strand; (c) reacting said first thiolated DNA strand with a thionitrobenzoate derivatized ligand binding first protein to form a first single stranded DNA-protein conjugate; (d) reacting said second thiolated DNA strand with a thionitrobenzoate derivatized ligand binding second protein to form a second single stranded DNA-protein conjugate; (e) annealing said first and second single stranded conjugates; and (f) recovering the double stranded DNA-protein conjugate product of step (e). - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method for preparing a bis(Fab'"'"')-DNA conjugate comprising
(a) preparing a first thiolated single stranded DNA, wherein said first thiolated single stranded DNA is (1) about 10 to 100 bases in length, and (2) thiolated at either the 3'"'"' or 5'"'"' end; -
(b) preparing a second thiolated single stranded DNA, said first and second thiolated single DNA strand being complementary to each other, and wherein said second thiolated single stranded DNA is (1) about 10 to 100 bases in length, and (2) thiolated at the same end as said first thiolated DNA strand; (c) reacting said first thiolated DNA strand with a thionitrobenzoate derivatized first antibody Fab'"'"' fragment to form a first single stranded DNA-Fab'"'"' conjugate; (d) reacting said second thiolated DNA strand with a thionitrobenzoate derivatized second antibody Fab'"'"' fragment to form a second single stranded DNA-Fab'"'"' fragment conjugate; (e) annealing said first and second single stranded conjugates, and (f) recovering the bis(Fab'"'"')-double stranded DNA conjugate product of step (e). - View Dependent Claims (8, 9)
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- 10. A bis-protein DNA conjugate comprising a ligand binding first protein cross-linked by a double stranded DNA to a ligand binding second protein, wherein said ligand binding first protein is linked to a 5'"'"' end and said ligand binding second protein is linked to the other 5'"'"' end or said ligand binding first protein is linked to a 3'"'"' end and said ligand binding second protein is linked to the other 3'"'"' end of said double stranded DNA via disulfide bonds, wherein said double stranded DNA is about 10 to about 100 base pairs in length, and wherein said ligand binding first protein and said ligand binding second protein retain the ability to specifically recognize and bind to their respective ligands when cross-linked to said double stranded DNA and the strands of said double stranded DNA are complementary to each other.
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15. A bis-protein DNA conjugate comprising a ligand binding first protein cross-linked by a double stranded DNA to a ligand binding second protein, wherein, via disulfide bonds, said ligand binding first protein is linked to a 5'"'"' end and said ligand binding second protein is linked to the other 5'"'"' end or said ligand binding first protein is linked to a 3'"'"' end and said ligand binding second protein is linked to the other 3'"'"' end of said double stranded DNA, and wherein said double stranded DNA is about 10 to about 100 base pairs in length and the DNA strands of said double stranded DNA are complementary to each other.
Specification