Aptamers for respiratory syncytial virus detection

Abstract

The identification of the infectious agents is pivotal for appropriate care of patients with viral diseases. Current viral diagnostics rely on selective detection of viral nucleic acid or protein components. In general, detection of proteins rather than nucleic acids is technically more suitable for rapid tests. However, protein-based virus identification methods depend on antibodies limiting the practical applicability of these approaches. Aptamers rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Although aptamers have been generated for virus identification in laboratory settings, their introduction into routine virus diagnostics has not been realized, yet. Here, we demonstrate that the rationally designed SELEX protocol can be applied on whole virus to select aptamers, which can potentially be applied for viral diagnostics. This approach does not require purified virus protein or complicated virus purification. The presented data also illustrate that corroborating the functionality of aptamers with various approaches is essential to pinpoint the most appropriate aptamer amongst the panel of candidates obtained by the selection. Our protocol yielded aptamers capable of detecting respiratory syncytial virus (RSV), an important pathogen causing severe disease especially in young infants, at clinically relevant concentrations in complex matrices.

Figure 1. Flow chart of virus selective aptamer generation.

See text for details. (FP, FBA and MEME stand for Fluorescence Polarization, Filter Binding Assay and Multiple Em for Motif Elicitation respectively).

Figure 2. Nucleotide sequence of the studied aptamers.

The sequence alignment of selected oligonucleotides resulted in identification of two aptamers in duplicated copies (B5, F6 and D10, D12); three pairs of aptamers holding an identical nucleotide sequence motif (H8, E6; E11, E10; B10, F10). The last studied aptamer was chosen randomly (H5) from the rest of the oligonucleotides that possessed unique sequences.

Figure 3

Fluorescence polarization change upon binding of various FAM-labeled aptamers (kept constant at 0.5 nM) to decreasing PFU RSV (A) and HRV (B) virus solution. Note that the HRV concentration in PFU equivalents is ca. two orders of magnitude higher than that of the RSV.

Figure 4. Functional analysis of aptamers by filter binding assay.

FAM labelled aptamers were incubated with varying concentrations of RSV or HRV formulated in either PBS or throat swab completed PBS and vacuum blotted onto nitrocellulose membrane. The bound aptamers were detected by fluorescence imaging. The selective binding of all studied aptamers were indicated by the distinctively high signal of RSV spiked samples.

Figure 5. Functional analysis of aptamers by ALPHA.

The biotin labelled aptamers were incubated with varying concentrations of RSV or HRV formulated in PBS. The reaction mixture was added to streptavidin coated donor beads and F protein selective antibody modified acceptor beads and the fluorescence intensities were measured.

Figure 6. Aptamer (H8) binding to RSV and competitive effect of the F-protein selective palivizumab monitored by fluorescence polarization.

The 0.5 nM Alexa Fluor 488 labelled H8 aptamer shows a polarization of ca. 50 mP that increases drastically in mixture with RSV indicating their binding. No change in the fluorescence polarization was obtained in the presence of a 100 × excess of F-protein selective antibody with respect of the aptamer.

Figure 7. Measurement of aptamer (H8)-G protein binding by fluorescence polarization.

The Alexa Fluor 488 labelled aptamer was mixed with various G-protein concentrations and the fluorescence polarization was determined. The experimental data were fitted with a 1:1 stoichiometry dose-response curve; the area highlighted with red is the 95% confidence band of the fit.

Figure 8. Detection of RSV at clinically relevant concentrations ALPHA.

Throat swab samples were spiked with different amount of RSV and the mixtures were supplemented with biotin labeled aptamer and F protein selective antibody. Fluorescence intensities were measured following addition of streptavidin coated donor beads and protein A acceptor beads. For purely aptamer-based detection RSV, the biotin labeled aptamer was coupled to streptavidin donor and acceptor beads. The modified beads were added to throat swab samples spiked with different amount of RSV and fluorescence intensities were measured. The ratios of sample fluorescence (I) and virus-free background fluorescence (I_o) are indicated. Both approaches provided distinctive fluorescence signal differences at clinically relevant RSV concentrations.

Figure 9. Aptamer stability analysis in throat swab samples.

Throat swab samples were infused with H8 aptamer and the aptamer concentration was determined at the indicated time points by real-time PCR.