Bio-Synthesis offers several different oligonucleotide biotinylation synthesis options.
Avidin, streptavidin, and other biotin-binding proteins can form an intense association with biotin-containing molecules. Biotinylation allows affinity capture of biotinylated molecules via the avidin, streptavidin system. Many applications are in routine use to exploit the extraordinary affinity of these biotin-binding proteins for biotinylated molecules. However, the biggest bottleneck of biotin-binding proteins for biotin is that the association is essentially irreversible. Extremely low pH or highly concentrated chaotropic reagents are required to break the association, and these conditions are not entirely compatible with oligonucleotides. The biotin analog desthiobiotin has a lower affinity to biotin-binding proteins. This biotin analog lacks the sulfur group from the molecule and has a dissociation constant (Kd) several orders of magnitude less than biotin/streptavidin. Biomolecules containing desthiobiotin dissociate from streptavidin in the presence of a buffered biotin solution.
- Biotinylated Oligonucleotides are versatile molecules useful for many applications. For example, the avidin/streptavidin system allows the capture of biotinylated oligonucleotides that are hybridized to a target sequence.
- Biotin allows the attachment of avidin-conjugated enzymes used in chemiluminescent and colorimetric detection protocols.
- Solid-phase differential display protocols and solid-phase genomic and plasmid sequencing utilize biotinylated oligonucleotides as well.
- Solid-phase capture methods using streptavidin-coated magnetic beads enhance restriction mapping, PCR-based genomic walking, differential display detection of unique mRNA species, and DNA sequencing.
- Oligonucleotides can be biotinylated on their 5’- and 3’-end as well as internally.
Oligonucleotide biotin modification includes:
Standard C6 Spacer Biotin: This biotin modification is attached to the 5’- or 3’-ends of an oligo using a C6 (standard) spacer. This Biotin version is recommended for most applications.
Biotin dT. Biotin-dT: This modification can be inserted at any position within an oligonucleotide.
Biotin TEG: Biotin-TEG increases the oligo–biotin distance to 15 atoms using a triethyleneglycol (TEG) spacer. Biotin-TEG is commonly used to avoid steric hindrance and can be beneficial for attaching oligonucleotides to nanospheres, magnetic beads or long oligonucleotide sequences with strong secondary structure.
Dual Biotin. Dual Biotin is a modification resulting in two functional biotin groups which act to increase biotin–streptavidin binding affinity, and are used for applications requiring high sensitivity, e.g., the Serial Analysis of Gene Expression (SAGE) assays.
Photocleavable (PC) Biotin and DesthioBiotin-TEG: One of the most challenging, and often frustrating, aspects of applications employing biotin is the nearly irreversible biotin–streptavidin interaction. The bond is stable over a broad pH and temperature range. Conditions necessary to release biotin can be potentially harmful or negatively affect downstream procedures. There are two biotin modifications that provide binding and controlled release: PC Biotin employs a photocleavable spacer arm which can be cleaved when exposed to UV light of specific wavelength (300–350 nm). One benefit of the PC modification is that upon cleavage, the resulting DNA oligo will have a free phosphate group available for subsequent ligase reactions.
DesthioBiotin-TEG, is a biotin analog missing the sulfur atom, offering another option for post-binding release. This analog binds tightly to streptavidin, but more weakly than standard biotin. Because of this, rinsing streptavidin bound oligos with buffered solutions containing free biotin will result in the displacement of desthiobiotin with the free biotin, allowing the oligonucleotide to be removed and collected.
Contact us for Biotinlyated Oligonucleotide Synthesis Services.
References:
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Misra, R. R.; Chiang, S. Y.; Swenberg, J. A. Carcinogenesis, 1994, 15, 1647.
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Conrad, F.; Krupp, G., Nucleic Acids Res., 1992, 20, 6423.
-
Rosenthal, A.; Jones, D. S. C., Nucleic Acids Res., 1990, 18, 3095.
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Rostok, O.; Odeberg, J.; Rode, M.; Stokke, T.; Fundruck, S.; Smeland, E.; Lunderberg, J., Biotechniques, 1996, 21, 114.
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Huitman, T.; Stahl, S.; Hornes, E.; Uhlen, M., Nucleic Acids Res., 1989, 17, 4937.