DNA Aptamers

A workflow based on free bioinformatics tools validated by Oliviera et al. [13 (link)] in 2022 was utilized to predict the binding residues associated with the interaction of aptamers to target proteins of the Leptospira spp. (Figure 1). Using DNA aptamers as the starting point, the nucleotide sequence of aptamers was used to predict the secondary structure using the Mfold web server (http://www.unafold.org/mfold/applications/dna-folding-form.php, accessed on 4 July 2022) and default values and parameters [15 (link)]. Ionic conditions were set as [Na+] = 1.0 M and [Mg2+] = 0.0 M. For each aptamer, the most thermodynamically stable structure (lowest free Gibbs energy) was selected, and the corresponding Vienna file (dot bracket format [.b file]) was saved.
The tertiary structure of each aptamer was assembled using 3dRNA using the Vienna file input from the previous analysis [16 (link)]. However, the 3dRNA web server (http://biophy.hust.edu.cn/new/3dRNA, accessed on 4 July 2022) was developed for RNA structures; hence, the thymine (T) from the sequence was replaced by uracil (U). All analyses were performed using the Procedure Optimize, 5 predictions, 3dRNA-Lib1, and minimization parameters, and results were saved as PDB files.
Conversion of the RNA structures back to DNA was performed using Biovia Discovery Studio Visualizer software [17 ]. Conversion was performed by substituting the uracil (U) nucleotide to thymine (T) and by changing the pentose sugar from ribose to deoxyribose.
All structures were then imported to PyMOL to add hydrogen atoms, which play an important role in molecular docking and interaction. Prediction of the G-rich quadruplexes were carried out using the QGRS mapper (https://bioinformatics.ramapo.edu/QGRS/index.php, accessed on 3 August 2022). This software maps the location of potential G-quadruplexes, instrumental in the stability of the 3D structure of the aptamer, in each nucleotide sequence [18 (link)].
Finally, the 3D structures of the aptamers were saved as PDB files and used as the input for receptor–ligand docking. This process was also repeated for the protein targets prior to docking. The docking simulation was performed on the HDOCK web server (http://hdock.phys.hust.edu.cn/data/62bd3e9f971c2/, accessed on 6 July 2022), using the tertiary structure of the aptamer as the ligand input and the PDB file of the protein target as the receptor input [19 (link)].
The best docking model (lowest docking energy score) was selected and used for the identification of binding sites with the PLIP web server (https://projects.biotec.tu-dresden.de/plip-web/plip, accessed on 16 August 2022) [20 (link)]. Noncovalent interactions, such as hydrogen bonds and hydrophobic interactions, were recorded and interacting amino acids were identified.

Quartz crystals (Total Frequency Control, Storrington, UK), fundamental frequency 8 MHz, working area 0.2 cm² consisted of a thin gold layer, were cleaned before each measurement. Crystals were submerged in basic Piranha solution (29% NH₃, 30% H₂O and H₂O₂ with volumetric ratio 1:5:1, respectively) for 25 min. After this treatment, the crystal was washed 3 times with deionized water and stored in ethanol. The crystal was then submerged in 2 mM solution of 1-dodecanthiol (DDT) in ethanol and stored at room temperature for 16 h. After washing with ethanol and drying in a flow of nitrogen, the crystal was placed in an acryl flow cell (JKU Linz, Austria) connected to the syringe pump (Genie Plus, Kent Scientific, Torrington, CT, USA).
The lipid film was prepared using liposome fusion. For this purpose, the small liposomes were prepared using the sonication method. Briefly, 8 mg of the phospholipid mixture (DMPC: DMPG in a molar ratio of 1:1) was dissolved in a small quantity of chloroform and dried under nitrogen in order to deposit a layer on the walls of the flask. A 4 mL aliquot of PBS was then added, and after a 30 min incubation, the mixture was ultrasonicated for 20 min with a sonicator (Bandelin Sonorex RK31, Berlin, Germany) in a water bath at room temperature [36 (link)]. The liposomes in a concentration of 0.5 mg/mL were then added in a flow to a gold layer of quartz crystal modified by DDT (Figure 2).
The MUA self-assembled monolayers were prepared by chemisorption at the clean gold layers. Crystals were incubated in 2 mM solution of MUA dissolved in ethanol overnight. Crystals were then rinsed with MiliQ water and incubated for 35 min in a mixture of 20 mM EDC and 50 mM NHS. The crystals were then rinsed, dried under the nitrogen stream, and placed into the flow cell. The scheme of the MUA layer with covalently immobilized cyt c and the addition of AuNWs modified by DNA aptamers is presented in Figure 3.

For liposome and MUA monolayer preparation, the following chemicals were used: PBS (phosphate buffered saline composed of 10 mM Na₂HPO₄, 1.8 mM KH₂ PO₄, 137 mM NaCl, 2.7 mM KCl and 2 mM CaCl₂ diluted in MiliQ water, pH 7.4) and MiliQ water with a resistance of 18 MΩ.cm was prepared by Purelab Classic UV (Elga, High Wycombe, UK). The standard chemicals such as ethanol, NaCl, HNO₃, NH₃, H₂O₂, MgCl₂, and CaCl₂ were purchased from Slavus (Bratislava, Slovakia). Liposome solution was prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine—DMPC (Avanti Polar Lipids Inc., Birmingham, AL, USA) and 1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt—DMPG (Sigma Aldrich, Darmstadt, Germany). 1-dodecanethiol (DDT) (Sigma Aldrich, Darmstadt, Germany) was used to modify the gold layer on the quartz crystal surface. For covalent binding of cyt c, 11-mercapto-1-undecanoic acid (MUA) and bovine serum albumin (BSA) (Sigma Aldrich, Darmstadt, Germany) were applied.
Gold plating solution OROTEMP and silver plating solution TECHNI SILVER CY LESS II W (Italgalvano, Lodi, Italy), alumina powder (Schmitz-Metallographie GmbH, Herzogenrath, Germany), methylene chloride (Sigma-Aldrich, Darmstadt, Germany), nitric acid, isopropanol, ethanol (Slavus, Bratislava, Slovakia). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), and MES hydrate were purchased from Sigma Aldrich, Darmstadt, Germany.
DNA aptamer sensitive to cyt c was purchased from Biosearch Technologies (Risskov, Denmark). The aptamer (NH₂-Apt-cytc) of the following sequence: 5′-NH₂-TTTTTTTTTTATCGATAAGCTTCCAGAGCCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGCCGTAGAATTCCTGCAGCC-3′ was modified at the 5′ end by amino group [32 (link)]. A 10-mer thymidine spacer at the 5′ end was used for providing better aptamer flexibility. We also used a DNA aptamer that does not specifically bind to cyt c with the following sequence: 5′-NH₂-CTGAATTGGATCTCTCTTCTTGAGCGATCTCCACA-3′. This DNA aptamer was purchased from Generi Biotech Ltd. (Hradec Králové, Czech Republic).