Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic.

Chenguang Lou,Jesper Wengel,Søren Roi Midtgaard,Knud J Jensen,Peter W Thulstrup,Jørgen Kjems,Manuel C Martos-Maldonado,Rasmus P Thomsen,Charlotte S Madsen,Niels Johan Christensen

doi:10.1038/ncomms12294

Abstract

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide–oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

Highlights

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions
Efficient and high-yielding preparation of peptide-ON conjugates (POCs) containing long peptide fragments (Z30-mers) or proteins is challenging and only very few successful examples have been published via chemical synthesis[35,36,45], but this method furnished the desired conjugates POC1, POC2 and POC3 in satisfactory yields for milligram-scale preparation
These POCs formed nano-assemblies which were characterized by ultraviolet thermal denaturation and annealing studies, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS) and TEM

Summary

Introduction

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. Formation of nanoscale objects and artificial proteins from peptide-ON conjugates (POCs) as building blocks with combined involvement of two separate self-assembly principles have, to our knowledge, not yet been realized. POCs have not been explored in de novo protein design using two orthogonal self-assembly principles

Methods

Results

Conclusion