Design and synthesis of bispecific DNA-antibody conjugates
AssigneeThe Regents of the University of California
InventorsCharles R. Cantor, Roy S. Chuck, Doris B. Tse
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Abstract
The invention relates to bis-protein-DNA conjugates. A protein having an antigen specific binding activity is covalently linked to each end of a derivatized DNA molecule. The bis-protein-DNA conjugates can be used for immunoassays and measuring distances between proteins at up to 3.4 .ANG. 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 indirectly conjugated to an antibody or Fab' fragment, using a avidin/streptavidin-biotin linkage. The conjugates of the invention can be used in immunoassays and PCR assays.
Claims19
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, 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.
A method for preparing a double stranded DNA-protein conjugate comprising
- (a) preparing a first streptavidin derivatized single stranded DNA, wherein said streptavidin derivatized single stranded DNA is
- (1) about 10 to 100 bases in length, and
- (2) derivatized at either the 3' or 5' end;
- (b) preparing a second streptavidin derivatized single stranded DNA, said first and second streptavidin derivatized single DNA strands being complementary to each other, and wherein said second streptavidin derivatized single stranded DNA is
- (1) about 10 to 100 bases in length, and
- (2) derivatized at the same end as said first strand of DNA;
- (c) reacting said first streptavidin derivatized DNA strand with a biotinylated ligand binding first protein to form a first single stranded DNA-protein conjugate;
- (d) reacting said second streptavidin derivatized DNA strand with a biotinylated 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).
A method for preparing a double stranded DNA ligand binding bis-protein conjugate wherein a ligand binding protein is bound to either both 5' ends or both 3' ends of said double stranded DNA comprising
- (a) preparing a first single stranded DNA-protein conjugate wherein the DNA of said conjugate is 10 to about 100 nucleotides in length;
- (b) preparing a second single stranded DNA-protein conjugate wherein the DNA of said second single stranded DNA-protein conjugate is about 10 to about 100 nucleotides in length;
- said first and second DNAs being complementary to each other;
- (c) annealing said first and second single stranded DNA-protein conjugates to form a bis-protein double stranded DNA conjugate;
- (d) recovering the bis-protein double stranded DNA conjugate of step (c).
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, 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.
A method for preparing a double stranded DNA ligand binding protein conjugate wherein a ligand binding protein is bound to either both 5' ends or both 3' ends of said double stranded DNA comprising
- (a) preparing a first single stranded DNA-protein conjugate wherein the DNA of said conjugate is about 10 to about 100 nucleotides in length;
- (b) preparing a second single stranded DNA-protein conjugate;
- said first and second DNAs being complementary to each other;
- (c) annealing said first and second single stranded conjugates to form a bis-protein double stranded DNA conjugate;
- (d) recovering the bis-protein double stranded DNA conjugate of step (c).
Dependencies are inferred from the claim text.