Development of Aptamer Specific for PfLDH and its Characterization
3.2 Experimental approaches .1 Materials
DNA oligonucleotides (primers, ssDNA library) were synthesized from IDT, USA.
The aptamers generated through the work were synthesized by Bioserve Biotechnologies, India Pvt. Ltd. PVDF membrane (Hybond P) was obtained from Amersham, UK. SYBR
74 Gold (10,000 X) nucleic acid stain was obtained from Invitrogen (USA), Restriction endonucleases, and streptavidin coated magnetic beads were procured from New England Biolabs (USA), pGEM-T easy TA cloning kit was obtained from Promega (USA). All other chemicals were of analytical grade.
3.2.2 Construction of the ssDNA library
The construction of the ssDNA library was such that a stretch of 40 randomized nucleotides was flanked by constant primer binding sites on both sides.
5ʹ-CACCTAATACGACTCACTATAGCGGATCCGA-N40-CTGGCTCGAACAAGCTTGC-3ʹ The primer sequences used for PCR amplification of the library are listed in table A5, appendix.
3.2.3 In vitro selection of ssDNA aptamers
PVDF membrane was cut to 0.5 cm x 0.5 cm and taken in a 2 ml Eppendorf tube.
The membrane was immersed in methanol for 10 min followed by equilibration in binding buffer for 20 min. A total of 1 ml (1 mg.ml-1) target protein (PfLDH) in binding buffer was added to the tube followed by gentle agitation on a rocking platform for 1 h. The membrane was then washed twice and stored in binding buffer at 4 °C. The amount of protein immobilized on the membrane was quantified using Bradford assay, by subtracting the amount of protein in the eluate from the original protein solution. The change in morphology of the PVDF membrane with subsequent protein immobilization was studied using atomic force microscopy (AFM) on an ambient air scanning probe microscope (Agilent Technologies 5500, USA) and a silicon nitride probe. Images were recorded with non- contact mode using Picoscan 5 software.
Before use, the library was denatured at 90 °C for 3 min, followed by incubation at 4 °C for 5 min. The ssDNA library (2.5 nmol) was suspended in 1 ml of the binding buffer, and then incubated with PfLDH immobilized onto PVDF membrane at RT for 1 h, on a rocking platform. The tube was spun down and the supernatant was stored as unbound library. Elution of bound candidates was performed in 200 µ l PCR grade water by heating the membrane at 90 °C for 5 min and crushing with a sterile microtip. Bound ssDNAs were amplified using BioMix Red DNA polymerase (Bioline) following the amplification protocol - initial denaturation at 95 °C for 10 min, followed by 18 cycles of 95 °C, for 15 s, 68 °C, for 15 s, 72 °C, for 3 s; and final extension at 72 °C for 3 s. To separate ssDNA from amplified dsDNA, the PCR product was incubated with 30 µl of streptavidin-coated magnetic beads in a coupling buffer at RT for 1 h. The beads were then washed with coupling buffer, and ssDNA strands were separated from biotinylated strands using 100 µl of 100 mM NaOH. The separated ssDNA strands were used for the next round of SELEX after neutralizing their pH with sodium dihydrogen phosphate (NaH2PO4).
The counter SELEX against hLDH A and hLDH B was carried out after cycle 4 and 8, respectively. The counter proteins were immobilized on PVDF in a manner similar to that for PfLDH and allowed to interact with the aptamer population. Counter SELEX against bare PVDF membrane was carried out twice, once at the very beginning and the second one after cycle 6. After every counter SELEX cycle, the unbound aptamer population was collected for the next cycle of operation. Details of buffers/solutions used have been listed in table A3, appendix.
76 3.2.4 Cloning of enriched aptamer population
At the end of the 10th positive SELEX cycle, the PCR product was cloned into pGEMT easy vector and transformed into competent E. coli DH5αcells. The positive clones carrying aptamer inserts were selected on the basis of Blue/White screening, and PCR. The recombinant plasmids from positive clones were sequences, and the aptamer sequences were aligned using Clustal X2, for comparison.
3.2.5 Electrophoretic mobility shift assay (EMSA)
10 % polyacrylamide gel with a cross linker ratio of 75:1 was prepared and pre-run in TBE buffer for 45 min at 120 V. PfLDH at various concentrations (0.2–12 µM) was mixed with 25 nM of selected aptamer candidate in 20 µl binding buffer and incubated for 1 h at RT. The mixture was loaded onto the pre-run gel, and separation was carried out for 2 h, at RT and 120 V. The gel was stained using SYBR gold dye. EMSA gel bands were quantified using ImageJ software, and the data were fit to the equation of rectangular hyperbola in SigmaPlot 13.0 to estimate the dissociation constant (Kd). The 2D structure of selected aptamer was determined using Mfold web server (Zuker, 2003).
3.2.6 Isothermal titration calorimetry
The ITC experiments were performed on a GE Healthcare UK, MICROCAL iTC 200 microcalorimeter. 20 µM of aptamer and 2 µ M of PfLDH protein solutions in 50 mM HEPES buffer, pH 7.4, were prepared to be used as ligand, and macromolecule, respectively.
The protein, and aptamer solution were degassed for 10 min before their use in the ITC experiment. Following the first injection of 0.4 µl aptamer solution, subsequent injections of 1.2 µl each were administered by the ITC syringe to the reaction cell in 20 titrations, at
150 s intervals, at 25 °C, and a stirring speed of 450 rpm. Control experiment without aptamer was performed to correct for heat of dilution. Analysis of ITC data was performed by Origin v 7.0 (OriginLab, USA).
3.2.7 Prediction of aptamer-protein interactions
The 3D structure of the aptamer was predicted using the RNA Composer web server (http://rnacomposer.cs.put.poznan.pl/) (Popenda et al., 2012), for which the Mfold dot- bracket notation was provided as an input. The sequence of the ssDNA aptamer was first converted to its corresponding RNA sequence. Docking studies involving PfLDH (PDB ID 4B7U) and aptamer were carried out on the PatchDock web server.
(http://bioinfo3d.cs.tau.ac.il/PatchDock/) (Duhovny et al., 2002; Schneidman-Duhovny et al., 2005) and analyzed using PyMOL. Chemical interactions at the protein-aptamer interface were studied using the PLIP (protein-ligand interaction profiler) web server (https://projects.biotec.tu-dresden.de/plip-web/plip/index) (Salentin et al., 2015).
3.3 Results and discussion