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

Abstract

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 K(d) of 40 nM and to inhibit (IC50 of 45 nM) l-substance P-mediated Ca2+ release in a cell culture assay.

Figure 1

The selection robot. (A) Picture of the RoboAmp robot with accessories under a temperature-controlled hood. (B) Schematic work surface used for the selection; numbers refer to the participation of workstations during the selection as depicted in Figure 2B.

Figure 2

The automated selection process. (A) Course of the selected RNA pool and its intermediates through two consecutive selection rounds. Blackened wells indicate the positions where the processed nucleic acids are located during the respective steps. Yellow plates with simple wells: Thermosprint reaction plates. Black plates: for fluorescent assays. White concentric circles on red plate: plastic columns for partitioning in vacuum manifold L. White circles on blue plate: ultrafiltration devices on vacuum manifold R. Step I: adjustment of buffer conditions, RNA denaturing and re-folding, and pre-selection. Step II: incubation of RNA and target, fishing of RNA•target complexes. Step III: partitioning of bound from unbound RNA. Step IV: semi-quantitative RT–PCR. Step V: in vitro transcription. Step VI: purification of RNA. (B) Robot devices involved in each selection step. Numbers refer to workstations as shown in Figure 1B.

Figure 3

Progress of the in vitro selection against substance P. Rounds 1–3 were carried out manually; in rounds 4 and 5, the target concentration was 10 μM for all three selection strands while varying the wash volume (CV, column volumes); rounds 6–15, varied target concentrations while washing with a fixed wash volume. Starting with round 4, the ratio of fluorescence intensity with/without polymerase is given.

Figure 4

Aptamer/Spiegelmer sequences. (A) DNA sequences that were obtained after cloning and sequencing the 14th round of selection. (B) Truncated versions of the most frequent clone A11. (C) Secondary structure (minimum free energy conformations) as predicted by mfold (19).

Figure 5

Binding and inhibition of substance P by Spiegelmer l-SUP-A-004. (A) Calorimetric profile of Spiegelmer l-SUP-A-004 binding to substance P (representative data from one ITC experiment). The dissociation constant was determined to be ∼40 nM. (B) Inhibition of substance P-induced AR42J cell stimulation by l-SUP-A-004 (filled squares) and non-functional control Spiegelmer (filled circles). The IC₅₀ was determined to be ∼45 nM at a stimulatory substance P concentration of 3 nM.