DNA Hybridization-Based Differential Peptide Display Identified Potential Osteogenic Peptides

Abstract

A DNA hybridization-based differential peptide display (DPD) was developed for the screening of phage peptide library to find osteogenic peptides intended to bind to epigenetically induced osteogenic receptors on NIH/3T3 (3T3) cell surface. In the presence of DNA methylation inhibitor of 5-azacytidine (5AZC), an osteoblastic receptor of bone morphogenetic protein (BMP) receptor 1A (BMPR1A) was induced on the cell surface of NIH/3T3 fibroblasts. Cyclic heptamer-displaying phage library was screened against vehicle and 5AZC treated (Tx) 3T3 cells. Antisense oligo against library against library peptide coding DNA of control 3T3 cell bound phages were synthesized to subtract common binders from that of 5AZC-Tx 3T3 cell-bound phages that included 5AZC-induced receptor binders. The library peptide coding regions of conformational receptor binder-subtracted DPD were PCR-amplified and cloned into a plasmid vector specifically designed for short peptide expression. No unique binder was identified when 96 clones were randomly picked from the third round of panning against 5AZC-treated 3T3 cells, suggesting miscellaneous bindings to cell surface proteins. Unique binders showing homology to known function proteins were successfully identified when constitutive receptor binders were subtracted from 5AZC-induced protein binders. Some of identified peptides significantly increased alkaline phosphatase activity in 5AZC-Tx 3T3 cells. DPD can be a useful tool to screen functional peptide bindings to cell surface receptors.Graphic