Selection, identification, and characterization of aptamers for pro-gastrin-releasing peptide (31-98), a tumor marker for small cell lung cancer

Abstract

Pro-gastrin-releasing peptide (31-98) (ProGRP31-98) is a highly reliable, sensitive, and specific tumor marker for small cell lung cancer (SCLC). DNA aptamers for ProGRP31-98 were selected in this study from an 89-mer DNA library with a random region of 48 nucleotides (nt) flanked with two primer hybridization sites, by using the systematic evolution of ligands by the exponential enrichment (SELEX) method. The DNA sequences binding to ProGRP31-98 immobilized magnetic beads were selected by running 12 SELEX cycles and 3 negative selection cycles. The selected DNA sequences were separated by cell based DNA cloning, and then sequence analyzed. The DNA sequences obtained were screened and investigated for binding specificity to ProGRP31-98 by using electrochemiluminescence (ECL) measurement with [Ru(bpy)2dppz]2+, a molecular light-switch, as the ECL probe. One DNA sequence (89 nt) and its random region (48 nt) were identified as specific aptamers for ProGRP31-98. Based on the secondary structures of the obtained DNA aptamers, two other truncated DNA aptamers, 40 nt and 15 nt long, respectively, were identified. The obtained aptamers have strong affinities to ProGRP31-98, with a Kd value of 16 nM, and can detect ProGRP31-98 with a detection limit of 17 nM using the label-free ECL measurement.