Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist

• Published in: Nucleic acids research Vol. 33; no. 4; p. e45
• Main Authors: Eulberg, Dirk, Buchner, Klaus, Maasch, Christian, Klussmann, Sven
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2005; Oxford Publishing Limited (England)
• Subjects: Aptamers, Nucleotide; Base Sequence; Binding Sites; Calorimetry; Cell Line; Directed Molecular Evolution - instrumentation; Directed Molecular Evolution - methods; Humans; Methods Online; Molecular Sequence Data; Oligoribonucleotides - chemistry; Oligoribonucleotides - metabolism; Oligoribonucleotides - pharmacology; Polymerase Chain Reaction; RNA - chemistry; RNA - isolation & purification; RNA - metabolism; RNA - pharmacology; Robotics; Substance P - antagonists & inhibitors; Substance P - chemistry; Substance P - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gni044

We have developed an automated SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process that allows the execution of in vitro selection cycles without any direct manual intervention steps. The automated selection protocol is designed to provide for high flexibility and versatility in terms of choice of buffers and reagents as well as stringency of selection conditions. Employing the automated SELEX process, we have identified RNA aptamers to the mirror-image configuration (d-peptide) of substance P. The peptide substance P belongs to the tachykinin family and exerts various biologically important functions, such as peripheral vasodilation, smooth muscle contraction and pain transmission. The aptamer that was identified most frequently was truncated to the 44mer SUP-A-004. The mirror-image configuration of SUP-A-004, the so-called Spiegelmer, has been shown to bind to naturally occurring l-substance P displaying a Kd of 40 nM and to inhibit (IC50 of 45 nM) l-substance P-mediated Ca2+ release in a cell culture assay.