DNA mediated coupling

This type of immobilization depends on the annealing of two complementary oligonucleotides modified with a tag. Clearly this method is more labor intensive to set up and the ligand has to be conjugated with biotin (1).

GraphDNA
DNA mediated capturing
1) Two complementary oligonucleotides are made. One 5'-thiol modified and one 5'-biotin oligonucleotide.
2) The 5'-thiol oligonucleotide is covalent bound to a recombinant streptavidin to form a DNA-Strp hybrid.
3) The ligand is biotinylated.
4) On a biacore SA chip the biotinylated oligonucleotide is captured by injecting 10 µM in 30 mM Sodium Phosphate, 150 mM NaCl, 3 mM NaCl, 0.025% Triton X-100, pH 7.4 for 4 minutes. Loosely attached material is removed with three injections of 10 µl 50 mM NaOH. Free biotin binding places are blocked with an injection of D-biotin.
5) The DNA-streptavidin hybrid is mixed with the biotinylated protein and incubated for 30 minutes at room temperature (20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.01% Triton X-100, 0.1 mg/ml BSA, pH 7.5). After dilution a small amount of D-biotin is added and incubated for 10 minutes at room temperature to block free binding places.
6) The Ligand-DNA mix is injected over the DNA-surface and allowed to hybridize. Different injection times and concentrations can control the amount of ligand immobilized on the surface.
7) Regeneration is done by injection of 10 µl 50 mM NaOH, which will denature the DNA bond and release the ligand-streptavidin-DNA hybrid.

References

(1) Niemeyer, C. M. et all ; DNA-Directed Immobilization: Efficient, Reversible, and Site-Selective Surface Binding of Proteins by Means of Covalent DNA-Streptavidin Conjugates; Analytical Biochemistry ;268: 54-63; (1-3-1999).