Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX.
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.
NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, D-10589 Berlin, Germany.
Axel
A
Vater
Florian
F
Jarosch
Klaus
K
Buchner
Sven
S
Klussmann
D000468:Alkalies; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D008024:Ligands; D008881:Migraine Disorders / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D051381:Rats; D015203:Reproducibility of Results; D013379:Substrate Specificity; D014158:Transcription, Genetic
D000468:Alkalies; D008024:Ligands; D009841:Oligonucleotides; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide