1 His-GHRH expressing total bacterial lysate, 2 His-GHRH expressing solubilized pellets. the production and secretion of Growth Hormone (GH) . Although a full-size GHRH peptide comprises 44 amino acids, its biological activity resides in the first 29 amino acids in the N-terminal . Instead of the neuroendocrine function, the peripheral expression of GHRH and its receptor is evident in various surgical samples of the prostate, Rabbit polyclonal to ATF2 breast, ovarian, and endometrial cancers . The expression and secretion of GHRH in non-pituitary cell BCDA types imply the effect of GHRH on the regulation of cell proliferation, differentiation, and carcinogenesis . GHRH peptide antagonists have been shown to trigger apoptotic cell death inhibiting the GHRH signaling in the prostate, endometrial, colon, lung cancer and [6, 10]. In order to generate GHRH antagonists, we select aptamers due to their excessive specificity, high binding affinity, low toxicity, and non-immunogenic properties . Aptamers are single-strand nucleic acid molecules (DNA or RNA) that can bind to target molecules such as proteins, peptides, carbohydrates, bacteria, viruses, and cancer cells for detection and diagnosis . However, they are generally used as biosensors for detecting and diagnosing target molecules. VEGF aptamer (Macugen) is used for the treatment of macular degeneration . The synthesis of aptamers is commonly performed by the SELEX (systemic evolution of ligands by exponential enrichment) method, a repeating amplification of nucleic acid with high binding affinity against the target molecule . Nucleic acid modifications, truncations, labeling of aptamers increased the binding affinity of aptamers . Recently, new generation aptamer synthesis has been performed using a magnetic bead-dependent modified ssDNA library for target binding and amplifying candidate sequences termed X-aptamer technology . The most improved advantage of X-aptamer technology is synthesizing up to 5 different targets simultaneously with one SELEX method. Besides, modified nucleotides are included in the X-aptamer library to increase the stability and specificity of BCDA aptamers. However, the limitation of X-aptamer technology is that the molecular size of the target molecule or targets longer than 10 amino acid is assumed to be more preferred . As there is no GHRH antagonist aptamer, we preferred to BCDA synthesize aptamers against GHRH 1C44 and 1C29. To synthesize X-aptamers that can capture BCDA every epitopic region, we selected full GHRH protein NH2 (1C44). Besides, as GHRH peptide antagonist select 1C29 region, we used GHRH NH2 (1C29) peptide as a target to synthesize X-aptamers. To illustrate, by comparing the binding affinity of GHRH antagonist X-aptamers, we aimed to synthesize, select X-aptamers against both GHRH NH2 (1C44) and NH2 (1C29) peptides. Materials and methods Cell lines and antibodies HEK293 (CRL-1573), MIA PaCa-2 (CRL-1429), HT-29 (HTB-38), PC3 (CRL-1435), LNCaP (CRL-1740), and PNT1a (CRL-11609) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Each cell line was grown in MEM, DMEM, McCoys 5A, and RPMI medium (PAN Biotech, Aidenbach, Germany) completed by 10% fetal bovine serum (Gibco, Paisley, PA4, United Kingdom), 10000 U/mL penicillin, and 10 mg/mL streptomycin (PAN Biotech, Aidenbach, Germany) at 37C in 5% CO2 incubator (HERAcell 150, Thermo Scientific (Paisley, PA4, United Kingdom), respectively. Anti-His tag (1:1000), anti-GAPDH (1:1000) primary antibodies, HRP-conjugated anti-rabbit secondary antibody (1:3000), and HRP-conjugated anti-goat secondary antibody (1:3000) were obtained from Cell Signaling Technologies (Danvers, MA, USA). Anti-GHRH primary antibody (1:500) and anti-GHRHR antibody (1:1000) were purchased from Origene (Rockville, USA) and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively. His-tagged (pCMV3-SP-N-His-NCV; CV023) and BCDA His-tagged GHRH (pCMV3-SP-His-ORF His-GHRH) plasmids were purchased from Sino Biological (Wayne, PA, USA). Bacterial His-GHRH (1C44) protein isolation and.