Desthiobiotin, a Mild purification conditions for active reporter molecules in e.g. DNA/DNA or DNA/protein binding studies

Avidin, streptavidin and other biotin-binding proteins have the ability to form an intense association with biotin-containing molecules. This association has been used for many years to develop systems designed to capture biotinylated biomolecules. In the oligonucleotide field, probably the most common modification is biotinylation and reagents are available to modify oligos at the termini and within the sequence. A wide variety of tests and techniques are in routine use to exploit the extraordinary affinity of these biotin-binding proteins for biotinylated biomolecules. However, the intense affinity of biotin-binding proteins for biotin is also the biggest drawback in that the association is essentially irreversible. Indeed, extremely low pH and highly concentrated chaotropic reagents are required to break the association and these conditions are not entirely compatible with oligonucleotides. 2-Iminobiotin has been used as a reversibly binding biotin reagent since its association with biotin-binding proteins can be broken at pH4. However, 2-iminobiotin is not stable to the conditions of oligonucleotide deprotection. Another biotin derivative that exhibits lower binding to biotin-binding proteins like streptavidin is desthiobiotin (or dethiobiotin). This biotin analogue is lacking the sulfur group from the molecule and has a binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M)1 . Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Bio-Synthesis recommends substitution of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.



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