Fig 1.
Obtaining and purification of His-tagged GHRH (1–44) ligand for the aptamer selection.
A GHRH (1–44) expression of bacterial colonies expressing His- and His-GHRH vectors demonstrated by immunoblotting. 1 E. coli HB101 His vector clone, 2 E. coli HB101 His-GHRH vector clone. B Purified His tagged bacterial GHRH 1–44 ligand was demonstrated by immunoblotting via anti-GHRH and anti-His tag antibodies, respectively. 1 His-GHRH expressing total bacterial lysate, 2 His-GHRH expressing solubilized pellets. Eukaryotic GHRH NH2 (1–44) expression in His-GHRH vector-transfected HEK293 cells was identified by C qRT-PCR, D immunoblotting, and E immunofluorescence. 1 HEK293 wt 2 HEK293 His-transfected 3 HEK293 His-GHRH transfected. F GHRH 1–44 concentration in the media of HEK293 cells was measured by Human GHRH ELISA. GAPDH was used as a loading control. 18S was used as an internal control. DAPI was used to indicate the nuclei of the cells.
Fig 2.
Synthesis, selection of putative x-aptamers against Growth Hormone Releasing Hormone.
A First bead based aptamer selection against GHRH NH2 (1–44) target and B second bead based aptamer selection against GHRH NH2 (1–29) target was performed by using the X-aptamer kit. Tube #1: Cleaved oligunucleotide pool, Tube #2: Cleaved oligonucleotide pool+Magnetic beads+Prokaryotic GHRH (1–44) target, Tube #3: Cleaved oligonucleotide pool+Magnetic beads+Eukaryotic GHRH (1–44) target, Tube #7: Cleaved oligonucleotide pool+Magnetic beads. Cycle course PCR (up) and last PCR (down) amplification of the x-aptamer selection process were given. C Sequences of putative x-aptamers were determined by next-generation sequencing. D. Analysis for all 24 putative aptamers were performed by using MEME program (https://meme-suite.org/meme/).
Fig 3.
Determination of binding affinity, dissociation constant (Kd) of putative x-aptamers.
The binding affinity of putative X-aptamers was determined by dot blot assay against A GHRH NH2 (1–44) target and B GHRH NH2 (1–29) target. C Dose-dependent dot blot assay was performed in increasing doses (0–1000 nM) of X-aptamers. The scramble aptamer was used a negative control for dot-blot analysis. Dot intensities were measured by Image J (imagej.nih.gov/ij/) and analyzed by GraphPad Prism 8.0. Nonlinear regression analysis was performed by Sigma Plot v14.0 and Kd was calculated. D. The specific binding affinity of selected aptamers against GHRH peptide was determined by both GHRH sandwich ELISA assay (left panel) and dot-blot analysis (right panel) in a dose-dependent manner.
Fig 4.
Determination of dissociation constant (Kd), and serum stability of putative x-aptamers.
A. Stability of each putative aptamer detected by polyacrylamide gel electrophoresis of serum and aptamer mixture at 37°C in time-dependent (0–120 h) manner. Each experiment was performed and repeated at least three times, given figures are the representative figure of one of the three assay repeat results. ns nonspecific, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 B. Binding affinity of selected x-aptamers to their specific target was demonstrated by Surface Plasmon Resonance analysis. After loading the CM-5 chip with the dose-dependent target molecule and incubation period, each aptamer was run in BiaCore T200 in triplicate for 120–12000 seconds.
Fig 5.
Demonstration of GHRH expression in different cancer cell lines.
A GHRH expression was figured out in MIA PaCa-2 pancreatic cancer, HT-29 colorectal cancer, PC3 prostate cancer, and PNT1a normal prostate epithelium cell lines by immunofluorescence. DAPI was used to observe the nuclei of the cells. B Translational expression of GHRH in MIA PaCa-2, HT-29, PC3, and PNT1a cells was determined by immunoblotting. ß-actin was used as a loading control. C. GHRH concentrations in media of MIA PaCa-2, HT-29, PC3, and PNT1a cells determined by Human GHRH ELISA.
Fig 6.
Investigation of the binding location of X-aptamers in MIA PaCa-2 cells.
The binding position of A TKY2.T1.08, C TKY2.T1.13, E TKY.T2.08, G TKY.T2.09 x-aptamers were investigated by immunofluorescence. Streptavidin-Alexa Fluor 488 conjugate was used to specify biotin-labelled x-aptamers. Cells were treated with X-aptamers in a dose-dependent manner (0–500 nM). Following incubation, cells were examined under a fluorescence microscope (Olympus) and images were taken and analyzed. The binding position of B TKY2.T1.08, D TKY2.T1.13, F TKY.T2.08, H TKY.T2.09 x-aptamers investigated by co-immunofluorescence. Streptavidin-Alexa Fluor 488 conjugate was used to specify biotin-labelled X-aptamers. Streptavidin-Alexa Fluor 588 conjugated anti-GHRH was used to specify GHRH. Cells were treated with x-aptamers in a dose-dependent manner (0–500 nM). Following incubation, cells were examined under a fluorescence microscope (Olympus) and images were taken and analyzed. GHRH siRNA was used as a negative control.
Fig 7.
Blockage of GHRH signaling by selected aptamers.
A. The effect of TKY2.T1.08, TKY2.T1.13, TKY.T2.08, TKY.T2.09 x-aptamers on the intracellular cAMP levels were determined by cAMP assay kit in both HT29 and MIA Paca-2 cells. B. The impact of TKY2.T1.08, TKY2.T1.13, TKY.T2.08, TKY.T2.09 x-aptamer on GH/GHRHR expression profile was investigated by co-immunofluorescence assay under a fluorescence microscope (Olympus). GHRH peptide (500 nM) was used as a positive control. Each experiment was performed and repeated at least two times, given figures are the representative figure of one of the three replicated assays. ns: non-specific, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.