Table 1.
The aptamer sequences with the highest QGRS score, their GC-content percentage, and the RNAFold energy results.
Table 2.
Docking score and binding site of best five aptamers obtained in HDOCK and PatchDock servers.
Fig 1.
Secondary structure of five aptamers with the highest docking scores.
Structures are predicted by the RNAfold web server. Dash lines indicate G-G interactions responsible for forming of G-quadruplex structure.
Fig 2.
Interaction of Apt31 with the BVDV-E2 protein (chain A and B) with respect to interpolated charge surface representation.
H-bonding amino acids are shown in the magnified part. Interacting aptamer nucleotides are colored in red, and dashed black lines represent H-bonds.
Fig 3.
2D diagram of Apt31-E2 protein complex drawn by ligplot.
The molecule with purple bonds is the aptamer, and the E2 protein amino acids are shown in brown. Nine hydrogen bonds and two covalent bonds have formed between the Apt31 and protein E2. Also, several amino acids are involved in hydrophobic interactions.
Table 3.
Responsible amino acids and nucleotides for the interaction of Apt31 and E2 protein.
Fig 4.
Fluorescence intensity of the crystal violet (CV) in the presence of the Apt31 and BVDV.
The fluorescence changes in relative fluorescence units (RFU) for crystal violet dye against a) emission wavelengths (600–700 nm) in the presence of KCl, aptamer, and various dilutions of BVDV-infected plasmas: BVDV-negative plasma, 1:10, 1:100, 1:1000 and 1:10000. (b) various dilutions of BVDV-infected plasma at the emission wavelength of 631 nm. The experimental conditions: 0.25 μM CV, 0.125 μM aptamer, 1 mM KCl, 20 mM Tris–HCl buffer (pH 7.0), excitation wavelength: 580 nm, emission wavelength range:600–700 nm. Values with p < 0.05 (*) were considered statistically important.
Fig 5.
The spectroscopy of gold nanoparticles salt tolerance test.
The UV-Vis absorbance spectra for AuNPs in 400–700 nm wavelength in the presence of (a) final KCl concentrations of 9.5 mM to 47.6 mM, (b) different concentrations of aptamer and 23.8 mM KCl.
Fig 6.
Color change analysis of the aptasensor.
(a) visual color change of AuNPs from red to purple in the presence of aptamer, KCl, and different concentrations of the BVDV from 0.27 to 2.7×1010 copies/ml, (b) the absorbance spectra of the samples at 400–700 nm wavelength. (c) The corresponding plot of A520/A640 against log10 virus concentration (copies/ml) from I to VI. (I): 0.27, (II): 2.7×101, (III): 2.7×103, (IV): 2.7×105, (V): 2.7×108, (VI): 2.7×1010 copies/ml. Apt: Apt31, BLV: Bovine leukemia virus. Values with p < 0.05 (*) were considered statistically important.
Fig 7.
The mechanism of the action of the developed Apt31-biosensor.
In stage 1, adding 10 μl of 500mM KCl leads to aggregation of AuNPs, in stage 2, the optimum concentration of Apt31 electrostatically binds to AuNPs and inhibits aggregation of AuNPs after the addition of the KCl solution. In stage 3, if the added plasma sample is infected with BVDV, Apt31 binds to the virus and separates from the surface of AuNPs. Subsequently, the addition of the KCl can result in aggregation of AuNPs and purple-blue color.
Table 4.
Comparison of the present method with the recently reported methods for the detection of BVDV.