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Aptamer Chemistry Series III

Don Quixote spent four days coming up with a name for his horse, Rocinante, as the name had to befit a horse of such a great knight. The name Rocinante represents clever word play in Spanish, as “ante” means before, and “rocin” means a nag or low-level workhorse. The knight’s noble steed was previously a workhorse. In today’s blog I will discuss the use of biotin as a workhorse that has become a noble steed for aptamer analysis and applications.

Aptamers are synthesized from phosphoramidites. To label an aptamer with a biotin a biotin labeled phosphoramidite is used.

This means that there is no need for conjugation chemistry. The aptamer is synthesized with the biotin in place. Now, why is this a workhorse? The binding of the biotin to streptavidin is unusually strong. Streptavidin has four binding sites for biotin, so it is possible to associate four biotinylated aptamers to a single streptavidin molecule. This enables a wide array of applications:

  • Immobilization on a surface (diagnostics)

Conjugate aptamers to streptavidin and then load the streptavidin onto a surface such as a microtitre plate for ELISA analysis, or nitrocellulose for lateral flow analysis. We find that conjugating the aptamer to the streptavidin first and then passively immobilizing the complex works best.

  • Immobilization to resin (affinity purification)

Our preferred option for conjugating aptamers to resin is to use streptavidin agarose. We have found that the conjugation of a biotinylated aptamer to the streptavidin is improved by the addition of 20% DMSO. We have also shown that aptamers conjugated to streptavidin agarose retain 98% of their binding capacity for ochratoxin A after storage for a year at room temperature in 20% DMSO.

  • Signal detection (diagnostics)

Streptavidin/horseradish peroxidase (Strep/HRP) conjugate proteins are readily available commercially. These conjugates can be bound to biotinylated aptamers thus enabling HRP mediated colour development of  3,3’,5,5’-tetramethylbenzidine (TMB).

Rocinante enabled Don Quixote to accomplish many tasks some more imaginary than real. The use of biotin with aptamers has also enabled us to follow our imagination to develop real aptamer applications. Rocinante might have been a nag, but he has endured in history.