504. Neuronal Affinity of a C7C Loop Peptide Identified through Phage Display

Abstract

Background: Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. The C7C library is based on a randomized 7-mer sequence flanked by two cysteine residues. In contrast to linear peptides displayed in other libraries, these cysteine residues form a disulfide cross-link, resulting in a loop structure.Methods: We designed a two-tiered biopanning strategy initially selecting for ganglioside (GT1b) binding and subsequently selecting for binding to NGF-differentiated pheochromocytoma (PC12) cells. We next evaluated the binding characteristics of the predominant clones by the bound phage ratio and by immunofluorescence phage localization. Finally, the predominant clone was synthesized and fluorescein conjugated, in order to further assess its neuronal binding.Results: Sequencing random phage plaques revealed amplification of a dominant clone, TetC7C.1 (54.8%). Immunofluorescence binding studies using phage antibodies to detect the bound phage on the surface of the cells revealed selective binding of this clone to NGF-differentiated PC12 cells. Moreover, the synthetic peptide demonstrated specific binding to neuronal cell lines (SH-SY5Y, NSC-34 and NGF-differentiated PC12 cells) and neuronal tissue (DRG and spinal cord).Conclusions: The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Loop peptides are preferable for the modification of proteins through insertion at sites other than the terminal regions. Furthermore, small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. Our biopanning strategy favored the selection of a peptide with enhanced binding for the clostridial tetanus toxin receptor GT1b. The neuronal binding properties of the Tet.C7C.1 peptide may be applied in the development of neurotropic viral vectors and therapeutic fusion proteins with enhanced binding to axon terminals. Background: Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. The C7C library is based on a randomized 7-mer sequence flanked by two cysteine residues. In contrast to linear peptides displayed in other libraries, these cysteine residues form a disulfide cross-link, resulting in a loop structure. Methods: We designed a two-tiered biopanning strategy initially selecting for ganglioside (GT1b) binding and subsequently selecting for binding to NGF-differentiated pheochromocytoma (PC12) cells. We next evaluated the binding characteristics of the predominant clones by the bound phage ratio and by immunofluorescence phage localization. Finally, the predominant clone was synthesized and fluorescein conjugated, in order to further assess its neuronal binding. Results: Sequencing random phage plaques revealed amplification of a dominant clone, TetC7C.1 (54.8%). Immunofluorescence binding studies using phage antibodies to detect the bound phage on the surface of the cells revealed selective binding of this clone to NGF-differentiated PC12 cells. Moreover, the synthetic peptide demonstrated specific binding to neuronal cell lines (SH-SY5Y, NSC-34 and NGF-differentiated PC12 cells) and neuronal tissue (DRG and spinal cord). Conclusions: The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Loop peptides are preferable for the modification of proteins through insertion at sites other than the terminal regions. Furthermore, small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. Our biopanning strategy favored the selection of a peptide with enhanced binding for the clostridial tetanus toxin receptor GT1b. The neuronal binding properties of the Tet.C7C.1 peptide may be applied in the development of neurotropic viral vectors and therapeutic fusion proteins with enhanced binding to axon terminals.