Selection of aptamers by non-SELEX method
20 μl of magnetic beads having Tosyl functional group (Dynabeads M280 tosyl activated beads, code no. 14203, purchased from Invitrogen, USA) were taken in five separate tubes and 10 μg of peptide sequence (KIPLRRVKTMRKTLSGKN) of PAG-18 was immobilized in on all tubes containing beads except first tube which was marked as control. The immobilization was done in presence of 150 μl borate buffer (0.1 M), pH 9.5. Further, 100 μl of 3.0 M ammonium sulphate (prepared in borate buffer, pH 9.5) was added to the mixture. The mixture was incubated at 37 °C for 22 h. The immobilized beads were blocked in 1.0 M tris for 3 h. Further the beads were blocked by adding 2% of BSA for 6 h at RT. Similarly, immobilized beads in control tube were also blocked in same fashion. After blocking, 50 ul of 1 × PBS was added to all tubes and were incubated overnight at 4 °C.
Next day 1.0 μM of 50 ul biotin labeled aptamer library (TAGGGAAGAGAAGGACATATGAT-N40-TTGACTAGTACATGACCACTTGA, Integrated DNA Technologies, Belgium) was taken and dissolved in 100 μl of 1 × PBS. 0.2 μl of magnesium chloride (1.0 M) was further added to the mixture. The mixture was snap cooled (heating at 96 °C for 15 min and quick cooling in ice for 5 min) and 30 μl of each of aptamer mixture was added to all tubes at room temperature for 1 h. After incubation, supernatant was discarded and a total of five washing with 1 × PBS was given to remove unbound aptamers. Streptavidin conjugated with horseradish peroxidase (ST-HRP, code-N100, purchased from Thermo Fisher, USA) were diluted to 1/30,000 times and was added to all the tubes for 20 min duration and was later washed with 1 × PBS before adding TMB (code-T0440, purchased from Sigma, Germany) solution for color development.
The tube that has given most intense blue color in shortest period of time was picked and washed with water. After washing, 2.0 μl of beads were taken in four PCR tubes and amplification was performed at following temperature: initial heating at 95 °C for 3 min 28 cycles of 95 °C for 30 s., 50 °C for 30 s, 72 °C for 30 s. and final extension of 72 °C for 5 min with cool down at 4 °C and checked at 2% agarose gel. The PCR product was purified and sent for amlicon sequencing. Similar protocol for aptamer selection was also followed for peptide sequence (KDSRGHCYTTFKEKRVRRS) of PAG-7 protein.
Sequence and structural analysis of selected aptamers
The sequences of PAG-7 and 18 aptamers generated by non-SELEX method were further studied for secondary structure analysis by online M-fold program. The predicted secondary structure based on lowest to highest free energy gives information regarding stem/loop structure of aptamers that might be involved in interaction with peptides. The G-quadruplex analysis of sequences was performed by using QGRS mapper.
Homology Modeling of PAG-7 and PAG-18
The amino acid sequences of Pregnancy Associated Glycoprotein 7 (PAG-7) and Pregnancy Associated Glycoprotein 18 (PAG-18) were retrieved from the protein sequence (UniPortKb) (https://www.uniprot.org) database. BLASTp tool of NCBI was used for identifying a suitable template for the computational modeling of PAG-7 and PAG-18. The secondary structures of PAG-7 and PAG-18 were predicted using PSIPRED server. The three dimensional (3-D) model of PAG-7 and PAG-18 was constructed using the homology modeling tool MODELLER v9.24. The best structure was selected according to the lowest discrete optimized protein energy (DOPE) score. After that, Proteins structure was subjected to energy minimization using GROMACS. Further, the model quality and validation was done through PROCHECK and ERRAT software’s. Further, Energy profile characterization was performed using ProSA.
Molecular docking
AutoDock v4.2 (http://autodock.scripps.edu/) tool was used for molecular docking analysis. The six aptamers were docked against the PAG-7 and four aptamers were docked against the PAG-18. In AutoDock tool, the polar hydrogen was added into the PAG-7 and PAG-18 proteins and Kollman charges were assigned for optimization of proteins. The Geister partial charges were applied on aptamers and non-polar hydrogens were merged to the aptamers. Then, the structures of the proteins and aptamers were saved in “.pdbqt” format that was used later for docking calculation. A grid maps was prepared for the docking calculation. In the PAG-7 protein, a 3-D grid box with dimensions X = 72, Y = 60, and Z = 72 was created with grid-point center X = − 0.167, Y = 0.194, Z = − 4.639 while the grid spacing was set to 1.000 ÅÅ. For PAG-18, a 3-D grid box with dimensions X = 68, Y = 60, and Z = 58 was created with grid-point center X = − 2.806, Y = − 1.528, Z = − 9.306 and the grid spacing was set to 1.000 Å. The docking conformation was analyzed by using the Lamarckian Genetic Algorithm (LGA). In the docking process, a maximum of 50 conformers was considered for each aptamers and the results were ranked based on their binding energy score and top aptamer with lowest binding energy score was carried out forward for further studies.
Detection of PAG proteins by aptamers
Use of serum sample obtained from blood of bovine
5.0 μl of streptavidin coated gold nanoparticles (ST-GNP), (Cytodiagnostics, cat. no. AC-40-04-05, conc. 0.15 mg/ml) was taken and mixed with in 20 μl of 1 × PBS. Gold nanoparticle solution was further mixed with 25 μl of 1 μM of biotin labeled PAG7_2 aptamer (Sigma). The incubation was done for an hour at RT. Tube was centrifuged at 12,000×g for 5 min. The process was repeated thrice and each time unbound aptamers were discarded by removing the supernatant and finally 20 μl of 1 × PBS was added. Now 10 μl of serum solution extracted from blood of 0th day animal (cattle just before the artificial insemination was considered as 0th day animal) was taken and spotted at nitrocellulose membrane (NC), (pore size 0.45 μm) on spot no. 2 on membrane surface. Spot no. 1 on membrane was spotted with 10 μl solution of serum from an animal, 42 days post AI. The membrane was dried for an hour at RT and blocked in 5% BSA solution for 2 h. After the incubation, 5 μl of aptamer conjugated GNP was added on all the three spots on the membrane namely spot no. 1 and 2 while spot no.3 was the untreated (control) spot on membrane. The membrane was incubated for 45 min and then washed thrice by 1 × PBS for color development and observation.
In separate experiment, four spots were made on two strips of nitrocellulose membrane of 0.45 μm pore size. Spot no. 1 was left untreated while 4.0 μl solution of serum of an animal already passed 42 days after an AI was added at spot no. 2. 4.0 μl of serum solution extracted from blood of animal (0 day animal) was spotted at spot no. 3 while spot no. 4 had 4.0 μl of 10% BSA solution. The membrane was dried for an hour at RT and blocked in 5% BSA solution for 2 h. After the incubation, 5.0 μl of 1.0 μM biotin labeled aptamers (PAG7_2 and PAG7_29) was spotted at strip 1 and 2 respectively for 45 min at RT. Subsequently, strips were washed and immersed in streptavidin conjugated to HRP (5 μl of 1.25 mg/ml was dissolved in 10 ml 1 × PBS) for 30 min. A brief washing was again given to strips in 1 × PBS, followed by addition of 5.0 μl solution of 3,3′-Diamino benzidine (DAB purchased from GeNei, India) on each spot in dark. The brown color was observed after 15 min. Subsequently strips were also exposed to TMB for five minutes for enhanced blue color formation.
In another experiment carried with same gold nanoparticle, biotin labeled sequence PAG7_29 was coated on ST-GNP as mentioned earlier. 10 μl of serum solution extracted from animal blood (0th day animal) was taken and spotted on nitrocellulose membrane (spot no. 2) of pore size 0.45 μm which will also be considered as control here. Spot no. 1 was spotted with 10 ul serum solution of an animal, 42nd days post AI. The membrane was dried for an hour at RT and blocked in 5% BSA solution for 2 h. After the incubation 5.0 μl of aptamer conjugated GNP was added at spot no. 1 and 2. The membrane was incubated for 45 min and then washed thrice by 1 × PBS for color development and observation. In same manner PAG7_47, PAG7_62, PAG7_76 & PAG 18_91 were also tested for PAG recognition.
Use of serum sample obtained from blood of Water buffalo
PAG18_21 and PAG7_29 aptamers were also tested to detect PAG proteins in buffalo serum: from a buffalo 100 days post AI and from a buffalo that has just given birth to a calf (1 day post calving) by using ST-GNP approached as mentioned earlier. The intensity of interaction was further enhanced by using silver enhancer (Cytodiagnostics, cat no. SR-01–02).
In another experiment working with same ST-GNP approach, PAG18_8 aptamer was tested against serum of 0th day animal (n = 2) for detection of PAG protein. Further, aptamer detection test for PAG was done against serum samples from 28th, 35th and 42nd days of animal post AI. In similar manner aptamer PAG18_8 was tested for its interaction to PAG of an animal that had passed 28 days of AI in time dependent manner. The time given for interaction was set at 5, 10 and 20 min. respectively. Further on control spot no PAG was added, only membrane was incubated with aptamer. The condition of experiment was similar to what was followed in gold nanoparticle based detection method section. In another experiment on nylon membrane (Roche Diagnostics, cat. no. 11209299001) three spots were made by pencil. On spot no. 1 and 2, 5.0 μl of 1.0 μM aptamer PAG7_47 that contained amino group at 5’ end was spotted. The membrane was incubated for 5 min in UV light at 254 nm and subsequently blocked in 5% SDS for 12 h at RT. Next day, 2.0 μl of serum sample from 0 and 28th day animal post AI was spotted on spot no. 1 and 2 respectively for 20 min. The membrane was briefly washed in 1 × PBS. Afterwards 5’ biotin labelled sequence (which was complementary of random region of aptamer 47) was immobilized on ST-GNP (as mentioned above). 5.0 μl GNP conjugated aptamer 47 was added to each spots for 10 min. The membrane was washed and dried for colour development and observation.
In competitive color based experiment, 10 μl of serum solution of an animal (0th day animal) was spotted on two different strips of nitrocellulose membrane (pore size 0.45 μm) on spot no. 3 and 4 respectively. While on spot no. 1 and 2, 10 μl serum solution from a pregnant animal (42nd day post AI) was spotted. The membranes were dried for an hour at RT and blocked with 5% BSA solution for 2 h. 5.0 μl of streptavidin coated gold nanoparticles (ST-GNP) was taken in four tubes and mixed with 20 μl of 1 × PBS. The mixture was further mixed with 25 μl of 1 uM of biotin labeled PAG18_8R, PAG18_91R, PAG7_29R and PAG7_47R respectively. All the tubes were incubated for an hour at RT. Tubes were centrifuged at 12,000×g for 5 min. The process was repeated thrice and each time unbound aptamers were discarded by removing supernatant and in the final step 15 μl of 1 × PBS was added to all the tubes. 5 μl of aptamer (Seq. PAG18_91R) conjugated GNP was spotted at spot no. 1 and 3 while Seq. PAG18_8R was spotted on spot no. 2 and 4 of first strip of membrane. On second membrane strip, Seq. PAG7_29R was spotted on spot no. 1 and 3 while Seq. PAG7_47R on spot no. 2 and 4. Both the membrane strips were incubated 15 min. Subsequent washing was given with 1 × PBS so as to remove any unbound aptamers. In the final step, membrane strips were air dried and colour change was observed.
In the following experiment, nitrocellulose membrane (pore size 0.45 µm) was taken and three spots were made on it. 4.0 μl of serum sample from an animal (0th day post AI) was taken and spotted at spot no. 2. Spot no. 1 on membrane was spotted with 4.0 μl serum sample from an animal, 42nd day post AI while 4.0 μl of 10% BSA solution was taken as control and was spotted at spot no. 3. The membrane was dried for an hour at RT and blocked with 5% BSA solution for 2 h. After the incubation 5.0 μl of 1.0 μM aptamer (PAG18_91), conjugated to biotin at 5’ end was added on spot no. 1, 2 & 3 and was incubated for 45 min at RT. The membrane was washed with 1 × PBS. Streptavidin conjugated to HRP was prepared (5 μl of 1.25 mg/ml was dissolved in 10 ml 1 × PBS) and membrane strip was immersed in that solution for 30 min. A brief washing was given to membrane with 1 × PBS and subsequently 5.0 μl solution of 3,3′-Diamino benzidine (DAB) was added on each spot in complete darkness. Color change was observed after 15 min. The same set up of experiment was also performed by using ECL (enhanced chemiluminescence purchased from Bio-Rad Company) for PAG18_91. Test was also carried out to see aptamer (PAG18_91) interaction with PAG-18 peptide, recombinant PAG-7 and 18 proteins and BCM-7 (YPFPGPI) peptide.
Aptamer PAG7_47 interaction was checked with synthetic PAG-7 protein peptide as well as with natural PAG-7 protein present in pregnant sample by using DAB. In ECL experiment also (by maintaining same condition as mentioned above) PAG7_47 was used to see aptamer interaction with PAG protein of pregnant animal’s serum sample. Further by using same above mentioned protocol, 4.0 μl of recombinant PAG-7 and 18 proteins were spotted at nitrocellulose membrane (spot no. 1 and 3). While at spot no. 2, same volume of 10% BSA was spotted. In second strip of NC, synthetic PAG-7 protein peptide, BSA & BCM-7 peptides were spotted at spot no. 1, 2 and 3 respectively. The strips of membrane were dried for an hour at RT and blocked in 5% BSA solution for 2 h. After that 5.0 μl of biotin labeled 1.0 μM aptamer (PAG7_47) was added to all spots for 45 min. The strips were washed and streptavidin conjugated to HRP was added for 30 min. A brief washing was given to membrane with PBS and ECL was used for color development.
Ethical clearance
From the institute animal ethical committee of National Dairy Research Institute (IAEC- NDRI), approval was taken for blood sampling of animals vide letter no. 41-IAEC-18-50, Dated 27.1.2018. Further, all the experiments performed under this work were carried out in compliance with the ARRIVE guidelines & accordance to institutional guidelines of National Dairy Research Institute.
