Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

Abstract

Background Growth Hormone Releasing Hormone (GHRH), 44 amino acids containing hypothalamic hormone, retains the biological activity by its first 29 amino acids. GHRH (NH2 1–29) peptide antagonists inhibit the growth of prostate, breast, ovarian, renal, gastric, pancreatic cancer in vitro and in vivo. Aptamers, single-strand RNA, or DNA oligonucleotides are capable of binding to target molecules with high affinity. Our aim in this study is to synthesize and select X-aptamers against both GHRH NH2 (1–29) and GHRH NH2 (1–44) and demonstrate synthesized aptamers’ target binding activity as well as serum stability. Methods and results Aptamers against GHRH NH2 (1–44) and NH2 (1–29) peptides were synthesized, and binding affinity (Kd) of 24 putative X-aptamers was determined by the dot-blot method, co-immunofluorescence staining and, SPR analysis. The serum stability of TKY.T1.08, TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers was 90–120 h, respectively. The dose-dependent binding of TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers on GHRHR in MIA PaCa-2 was approved by co-IF assay results. Moreover, SPR analysis indicated the Kd (4.75, 1.21, and 4.0 nM) levels of TKY2.T1.13, TKY.T2.08, TKY.T2.09 putative X-aptamers, respectively. Conclusion Our results illustrate the synthesis of 24 putative X-aptamers against both GHRH NH2 (1–44) and NH2 (1–29) peptides and TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers have high serum stability, high target binding potential with low Kd levels.

Application of Aptamer-based Biosensors in Early Diagnosis of Gastric Cancer: A Promising Method for Early Detection

“…21 The x-aptamers against GHRH 1-44 and its first 29 amino-acids (GHRH 1-29) have been synthesized using magnetic bead selection. 22 Epithelial cell adhesion molecule (EpCAM) is another non-specific biomarker for GC that is related to worse prognosis, larger tumor size, and metastasis to the lymph nodes. EpCAM is a transmembrane protein that regulates cell adhesion and plays a role in epithelialto-mesenchymal transition of carcinoma cells in various cancers.…”