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Abstract
We developed an integrated method to identify aptamers with only 10 fixed nucleotides
through ligation and removal of primer binding sites within the systematic evolution
of ligands by exponential enrichment (SELEX) process. This Tailored-SELEX approach
was validated by identifying a Spiegelmer ('mirror-image aptamer') that inhibits the
action of the migraine-associated target calcitonin gene-related peptide 1 (alpha-CGRP)
with an IC50 of 3 nM at 37 degrees C in cell culture. Aptamers are oligonucleotide
ligands that can be generated to bind to targets with high affinity and specificity.
Stabilized aptamers and Spiegelmers have shown activity in vivo and may be used as
therapeutics. Aptamers are isolated by in vitro selection from combinatorial nucleic
acid libraries that are composed of a central randomized region and additional fixed
primer binding sites with approximately 30-40 nt. The identified sequences are usually
not short enough for efficient chemical Spiegelmer synthesis, post-SELEX stabilization
of aptamers and economical production. If the terminal primer binding sites are part
of the target recognizing domain, truncation of aptamers has proven difficult and
laborious. Tailored-SELEX results in short sequences that can be tested more rapidly
in biological systems. Currently, our identified CGRP binding Spiegelmer serves as
a lead compound for in vivo studies.