Vaccinia virus (VACV, strain NYCDH, ATCC #VR-1536), VACV (strain Lister Elstree), cowpoxvirus (CPXV, strain 81/02), mousepoxvirus (ECTV strain Nü-1) and camelpoxvirus (CMLV, strain CP19) were propagated in Hep2 cells (ATCC CCL-23) at a multiplicity of infection (MOI) of 0.25 following standard procedures with Dulbecco's Modified Eagle Medium (DMEM) containing 1 g/L D-glucose, L-glutamine and 25 mM HEPES buffer. Infected cells were incubated for approximately 4 days until a pronounced cytopathic effect was observed. Virus-containing supernatant and cells were harvested and subsequently separated by centrifugation (10 min at 1000 × g) after freeze thawing. Virus-containing supernatant was titered according to Reed and Münch  and stored at -75°C until use. For inactivation prior to selection, the virus suspension was heat-treated at 60°C for 1 h and checked for infectious activity by cell culture as described above. As control virus for binding experiments, CMV was kindly provided by Stefanie Thulke, Charité Berlin, Germany.
Selection of high-affinity-binding aptamers by MonoLEX
A MonoLEX selection process comprised the following steps: Chemical coupling of the target (VACV) to the affinity column, incubation with the combinatory library, extensive washing to elute non-binding oligonucleotides, physical segmentation of the affinity column, PCR amplification of the aptamers bound to different column fragments and finally an aptamer dot blot assay to evaluate the binding specificity.
The selection experiments started from a combinatory library that was 64 bases long (5'-TACGACTCACTATAGGGATCC-N7-A-N7-A-N6-GAATTCCCTTTAGTGAGGGTT-3') including 20 random nucleotide positions and two terminal PCR primer sequences diluted in DMEM. For selection, an affinity capillary column was coated with heat-inactivated VACV (strain NYCDH) particles. To reduce unspecific binding, the complete template library was first applied to an affinity capillary column covalently coated with the cell culture supernatant of non-infected Hep2 cells. After application of the remaining combinatory library to the selection column, low-binding oligonucleotides were washed off with approximately the 5000-fold column volume of Tris buffer (pH 7.3, 0.1% TWEEN-20), DMEM (0.1% TWEEN-20) and phosphate buffer (pH 7.5, 0.1% TWEEN-20). The column was then cut into slices. Target-bound aptamers were desorbed from the column slices by denaturation of the target and amplified by quantitative real-time PCR on an iCycler (BioRad, Munich, Germany), using 2 pmol of the primers AP7 TACgACTCACTATAgggATCC and AP3: AACCCTCACTAAAgggAATT (Metabion, Martinsried, Germany) in a SybrGreen Mastermix (Cambrex, NJ, USA). The mixture of such amplified aptamers is called "polyclonal aptamers". Cycling conditions were as follows: Initial denaturation at 95°C for 5 min and 40 repeats of denaturation at 95°C for 15s, primer annealing at 50°C for 30s and elongation at 68°C for 30s. Subsequently to PCR, a fluorescence curve melting analysis (FCMA) was performed, cooling down the PCR reaction product to 50°C and increasing the temperature with a maximum ramping rate up to 90°C. Fluorescence was monitored continuously and melting peaks were calculating with the iCycler software.
Pyrosequencing was performed with a PyroMark ID System (Biotage, Sweden) using the Pyro Gold kit. Briefly, aptamer candidates were amplified by PCR as described above, applying one biotinylated primer AP3. Single-stranded DNA was prepared by denaturation in NaOH and binding to sepharose beads as recommended by Biotage. The single-stranded DNA was sequenced with primer AP7 in the sequencing mode of the PyroMark ID System. Usually, the complete aptamer sequence could be determined unambiguously.
Aptamer A38 was purchased as lyophilized oligonucleotide (Oligoservice, Berlin, Germany), either non-modified, 5' biotinylated or 5' tetramethylrhodamine isothiocyanate (TMR) labeled. All aptamers were reconstituted in Tris-HCL (10 mM, pH 8.4) as 100 μM stock solutions and stored at -20°C until use for further experiments.
Aptamer blotting assay
To validate the presence of target specific aptamers in the aptamers derived from the individual slices of the affinity column, a PCR-enhanced dot blot assay was performed. Briefly, the target VACV particles were chemically immobilized to a polypropylen tube. Polyclonal aptamers were purified by agarose gel electrophoresis from PCR mastermix components and incubated in the tubes overnight. After ample washing the immobilized aptamers were amplified by quantitative PCR as described above.
A38 was tested at various concentrations against VACV, non-infective cell culture supernatant and CMV. 30 μL of sucrose-cushion-purified VACV (~107 particles) were spotted onto a 0.2 μm PVDF membrane (Schleicher & Schuell, Dassel, Germany). After incubation with blocking buffer (PBS, 0.1% Tween 20 und 10% low-fat milk powder), the membrane was incubated with varying amounts of biotin-conjugated A38 (0.01–10 nM) for 1 h at RT. Following repeated washing with PBS and incubation with a streptavidin peroxidase conjugate (Dianova, Hamburg, Germany) for 1 h, the detection was performed by chemiluminescence using SuperSignal® West Pico (Pierce, USA) according to the manufacturer's instructions.
Fluorescence Correlation Spectroscopy (FCS)
The binding of the aptamer to VACV was assayed by Fluorescence Correlation Spectroscopy (FCS). We used a confocal microscope ConfoCor (Carl Zeiss, Jena; Evotec Biosystems, Hamburg, Germany) and analyzed the diffusion of dye-labeled monomers and complexes by the normalized autocorrelation function (ACF), as described previously . The fluorescence fluctuations of the marker dye, tetramethylrhodamine isothiocyanate (TMR), were recorded for 5 min at RT and submitted to autocorrelation analysis using the hardware and software by Evotec Biosystems (Hamburg, Germany). The autocorrelation function was analyzed in terms of particle diffusion through the confocal volume and the intrinsic triplet state dynamics of the fluorophor TMR . The analysis yielded the number of particles and the mean dwell time (diffusion time) of each particle species in the confocal volume. Total particle numbers between 0.5 and 1.5 were observed. Data were rescaled to a total particle number of one for better comparison. The calculated diffusion times were characteristic for TMR, A38-TMR and VACV-TMR. Saturation of the binding of TMR-labeled A38 to non-labeled VACV was indicated by a complete shift of the autocorrelation function (ACF) from a shorter to a longer correlation time characteristic for VACV-TMR. The following concentrations were applied: 3 nM TMR, 5 nM A38-TMR, 108 PFU/ml VACV-TMR, 108 PFU/ml non-labeled VACV and 108 PFU/ml non-labeled CMV in PBS buffer.
Surface Plasmon Resonance measurements (Biacore)
Binding experiments were done with the SPR-based instrument Biacore™2000 (Biacore, Freiburg, Germany) and sensor chips CM4, using the control software version 2.1 and evaluation software version 3.0 (Biacore AB, Uppsala, Sweden). The running buffer was PBS containing 0.005 % (v/v) Tween 20. This buffer was also used for sample dilution. The temperature of the flowcells was 25°C.
For the immobilization of Neutravidin, the carboxymethyl dextrane matrix of the chip was activated for 7 min with 35 μl of a mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.05 M N-hydroxysuccinimide (NHS). Neutravidin (Pierce, Rockford, USA) was diluted to 20 μg/ml in 10 mM phosphate pH 5.5 and injected into the activated flowcell 2 for 5 minutes. Non-reacted sites were blocked with 1 M ethanolamine pH 8 for 1 min. The amount of immobilized Neutravidin was 2100 resonance units (RU) for flowcell 2. The control flowcell 1 was only activated and blocked.
Binding experiments were performed with aptamer A38 that was injected into flowcell 2 at a concentration of 1 μM in PBS for 5 min, yielding 480 RU for bound aptamer. After 5 min of washing with buffer, the dissociation of aptamer was constantly slow, and the virus suspension (109 particles/ml) was injected into flowcells 1 and 2 for 5 min. Bound particles were then eluted with 50 mM sodium carbonate for 1 min. Finally, a tenfold diluted virus suspension was injected (Fig. 3b, left panel). Specific binding sensorgrams were obtained by normalization to the curve of the control flowcell 1. The background was set to zero, and the injection start was synchronized (Fig 3b, right panel). Kinetic evaluation of the binding was not possible, as the binding of virus particles is a multisite attachment to the aptamer layer.
Immunofluorescence Assay (IFA)
5 × 103 Hep2 cells were propagated in 96-well plates (Nunc, Wiesbaden, Germany) for 24 h at 37°C. Cells were either infected with VACV at a MOI of 0.1 alone or together with 2.5 μM A38 or 2.5 μM of the combinatory library. Cells were incubated for another 24 h at 37°C. For visualization OPV-infected cells were fixed in 4% formalin and stained with a polyclonal human anti-pox IgG (1:500, Omrigam, USA), followed by a FITC-conjugated goat anti-human IgG (1:50, Caltag Laboratories, USA) according to standard procedures and contrasted by Evans Blue staining. The number of infected cells was determined by fluorescence microscopy. Briefly, four representative pictures ((??)) were taken. The total cell number, stained with Evans Blue, and the number of infected cells, stained with the anti-poxvirus serum, were enumerated by application of the software package ImageJ http://rsb.info.nih.gov/ij/.