Biotinylation of Oligonucleotides

Biotin undergoes specific strong interactions with proteins such as avidin or streptavidin in solution or tissue probes, which themselves can be linked to a reporter enzyme. Frequently, alkaline phosphatases or peroxidases are used for this purpose. To achieve optimum condi- 

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Figure 27.9 Biotin-ll-dUTP is perhaps the most popular nucleotide derivative used for enzymatic biotinylation of oligonucleotides. The 11 designation refers to the number of atoms in its spacer arm.

Figure 27.10 Biotinylation of oligonucleotides may be done at the 5 -phosphate end using a diamine derivative and reacting with NHS-LC-biotin.

Cubie, H.A., and Norval, M. (1989) Detection of human papilloma viruses in paraffin wax sections with biotinylated synthetic oligonucleotide probes and immunogold staining./. Clin. Pathol. 42, 988-991. 

Immobilisation of oligonucleotide probes onto the electrode surface 40 pL of 3 -biotinylated oligonucleotide probes (0.5ng/pL) is left on the electrode surface for 15 min. Finally, the electrodes are rinsed with 2 x SSC buffer pH 7.2 containing 1% of BSA. 

Tris(hydroxymethyl)methylamine (TRIS), sodium chloride, sodium citrate, ethylenediamine tetraacetic acid disodium salt (EDTA), lithium chloride, Tween 20, streptavidin 10 nm colloidal gold labelled, hydrochloric acid (37%), nitric acid, streptavidin-coated paramagnetic beads (MB) with a diameter of 2.8 pm, Dynabeads M-280 Streptavidin (Dynal Biotech, Oslo, Norway) biotinylated probe oligonucleotides which sequences are shown in Table 53.1. 

W, M. PMdrige and R. I. BOftdO- Enhanced oeRular uptake of biotinylated antisense oligonucleotide or peptide mediated by avidin, a cationic protein. PEBS Lett. 288 30-32(1991). 

Several groups have created multifunctional streptavidins with reversible linkers to solid surface. Scouten and Konecny [87] used an immobilized oligonucleotide to capture a biotinylated complementary oligonucleotide bound to streptavidin, which was free to react with biotinylated protein. A captured biotinylated antibody was released by denaturation of the duplex DNA by incubation at low ionic strength. Morris and Rany [88] reported the release of streptavidin bound to the 3 (but not the 5 ) end in an ATP-dependent helicase reaction. 

An alternative reagent (216) has been prepared for the synthesis of biotinylated oligonucleotides. In this case the protection used for the biotin moiety is base labile rather than the conventional dimethoxytrityl. The photo-crosslinking behaviour of oligonucleotide constructs, incorporating photoactive residues 217a-d at a defined position, has been examined in the presence of their DNA and RNA complementary targets. The X-ray crystal-structure of the photoproduct formed between 4-thiothymidine and adenosine upon near UV irradiation has been reported. 

Wu, D., Boado, R., and Pardridge, W. (1996) Complete inactivation of target mRNA by biotinylated antisense oligonucleotide-avidin conjugates.. /. Pharm. Exp. Therap. 276, 206-211. 

Consists of Streptavidin mixed with biotinylated DNA oligonucleotide capture probe that is complementary to section of target molecule... 

Fig. 5. Sandwich-hybridization assay (A) A biotinylated DNA oligonucleotide, which is complementary to one portion of the target sequence, is mixed with streptavidin and applied to form the capture zone 1.5 cm from the base of the membrane. An unmodified DNA oligonucleotide, which is complementary to the reporter probe sequence on the liposomes, is applied to form the control zone 2.5cm from the base of the membrane. (B) In the presence of target, a sandwich hybridization complex forms with dye-encapsulating liposomes resulting in a magenta-colored band at the capture zone. (C) In the absence of target, only the control band is visible as no sandwich complex has formed.

The most common alternative for immobilization of oligonucleotides involves the use of biotinylated derivatives. These are conveniently made with standard reagents for oligonucleotide synthesis and biotin can be added at both the 3 and 5 ends. Immobilization of the biotinylated oligonucleotide to avidin-modified sensor surfaces is performed in neutral buffer conditions and is normally efficient. 

Guitteny, A. F, Fouque, B., Mongin, C., Teoule, R., and Boch, B. (1988) Histological detection of mRNA with biotinylated synthetic oligonucleotide probes. J. Histochem Cytochem. 36, 563-571. 

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