Determination of minimal sequence for binding of an aptamer. A comparison of truncation and hybridization inhibition methods

Abstract

Nucleic acid aptamers are attracting increasing interest as sensing and therapeutic molecules as a consequence of their high affinity and specificity for their target molecules. Aptamers are selected from large random libraries and where structural data are available it appears that only a small fraction of the sequence is actually involved in direct contact with the target. As there are many advantages to minimizing the size of the aptamer a rapid method that can determine those parts of the sequence critical for the target binding would be very useful. In this paper we describe mapping the effective binding region of an aptamer selected against electric eel acetylcholinesterase. As originally selected this aptamer is 77 nucleotides in length and was shown to bind the target with a high affinity (K_d = 174 ± 27 pM). Truncation to 39 nucleotides enhanced affinity for its target by an order of magnitude (K_d = 14 ± 1 pM). To further probe the relationship between sequence and affinity, we used two approaches: truncation and hybridization inhibition. Binding assays were performed with a number of truncated variants to determine a minimal binding sequence. A similar set of measurements for hybridization inhibition was also performed to allow a comparison of the two approaches. In general hybridization inhibition resulted in comparable conclusions to those found by truncation. The exception to this was where the former resulted in steric clashes between double stranded DNA regions and the target. In this case the effect on affinity was less pronounced than with truncation.