Development of a sphingosylphosphorylcholine detection system using RNA aptamers

Abstract

Sphingosylphosphorylcholine (SPC) is a lysosphingolipid that exerts multiple functions, including acting as a spasmogen, as a mitogenic factor for various types of cells, and sometimes as an inflammatory mediator. Currently, liquid chromatography/tandem mass spectrometry (LC/MS/MS) is used for the quantitation of SPC. However, because of the complicated procedures required it may not be cost effective, hampering its regular usage in a routine practical SPC monitoring. In this report, we have generated RNA aptamers that bind to SPC with high affinity using an in vitro selection procedure and developed an enzyme-linked aptamer assay system using the minimized SPC aptamer that can successfully distinguish SPC from the structurally related sphingosine 1-phosphate (S1P). This is the first case of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process being performed with a lysosphingolipid. The SPC aptamers would be valuable tools for the development of aptamer-based medical diagnosis and for elucidating the biological role of SPC.

Figure 1

Chemical structures of SPC and S1P.

Figure 2

Binding analysis of the selection pools using SPR. The interactions between the selection pools (round 0, 12, 13) and the immobilized SPC on a SA sensor chip were investigated. The concentration of each pool was adjusted to 2 µM in the selection buffer.

Figure 3

Binding of the enriched clones (C1–C9) to the immobilized SPC. Forty microliters of each 500-nM aptamer in the selection buffer with 0.005% Tween 20 was injected individually at a flow rate of 20 µL/min. All clones bound to SPC were removed completely except for C8 (dotted line) by adding 20 µL of 10 mM EDTA at a flow rate of 60 µL/min.

Figure 4

Predicted secondary structures of the SPC aptamers. The estimated stabilities, ΔG (kcal/mol), are shown in parentheses. The conserved hairpin structure is highlighted with gray backgrounds. All RNA structures were visualized using the PseudoViewer3 program [20].

Figure 5

The overlaid SPR sensorgrams of the truncated variants (m009–m012) derived from the initially determined clones (C1 and C2). The measurements were performed under the same conditions as for Figure 3.

Figure 6

ELAA. (a) Sensitivity of the m012 aptamer for biotinylated SPC. The amount of biotinylated SPC captured by immobilized m012 was shown as the absorbance at 405 nm. The experiment was repeated three times with each data point measured in duplicates, and a representative result is shown. (b) Competitive ELAA using m012 and biotinylated and unmodified SPC. The biotinylated and serially diluted unmodified SPC were added to the m012-immobilized plate. The amount of biotinylated SPC captured by the immobilized m012 was shown as the absorbance at 405 nm. The experiment was repeated three times with each data point measured in duplicates, and a representative result is shown. (c) Specificity of the m012 aptamer for SPC and S1P. The amount of the biotinylated m012 reacted with the immobilized biotinylated SPC and S1P was shown as the absorbance at 405 nm. The experiment was repeated three times in triplicates, and a representative result is shown. Error bars show standard deviations.

Figure 7

Bindinganalysis of the m012 aptamer to SPC and S1P using SPR. The biotinylated SPC and S1P were immobilized on each flow cell at similar amounts (~250 RU). 400 nM aptamer was injected over the ligands for 2 min.