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

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.

Folding Topology of a Short Coiled‐Coil Peptide Structure Templated by an Oligonucleotide Triplex

“…In the case of duplex systems, the attached peptide strands had no significant influence on the thermal stability, irrespective of whether one or two peptide strands were anchored (Table , entry 1–4). This is in stark contrast to the significant duplex stabilizing effect observed earlier by the conjugation of two 30‐mer peptide strands . We ascribe the loss of stabilizing cooperativity to the reduced intermolecular affinity between the truncated peptide sequences.…”

“…With POC1 , POC2 , POC3 and their corresponding ON controls ( ON1 – ON3 ) in hand, the thermal stability of duplex and triplex domains was evaluated to validate the fidelity of Watson–Crick and Hoogsteen recognition for 12 different assemblies with varying numbers of peptide strands attached (Table ). Whereas a cooperative stabilizing effect was noted when we employed the 30‐mer four‐heptad peptide strands because of the inherent stability of the corresponding three‐stranded coiled‐coil structures, the 23‐mer peptide used herein is too short to assemble into stable, monodisperse two‐ or three‐stranded helical coiled‐coil structures by itself . Therefore, one aim was to investigate whether the confinement of two or three of such shorter peptide strands to the terminals of hybridized ON domains would lead to a stabilizing effect on duplex and triple helical assemblies.…”

“…As expected, crude POC1 was water‐soluble. Interestingly, unlike the corresponding crude POCs containing the 30‐mer peptide (insoluble in Milli‐Q water), POC2 and the majority of POC3 (ca. 80 %) were easily dissolved in Milli‐Q water, although a minor portion of POC3 (ca.…”

“…Further details on ON and peptide synthesis are included in the Figures S1 and S2; Sections S2 and S3. For the POC synthesis, we have refined the method described in our previous work . Therefore, with the desired ONs and peptide ( ON1‐BCN , ON2‐BCN or ON3‐BCN , and azidopeptide, Figure b and Figure S2) at hand, the copper‐free azide–alkyne coupling reactions were carried out under microwave heating at micromolar concentrations by using one equivalent of BCN‐bearing ON and 1.25 equivalent azidopeptide in a solvent system of dimethyl sulfoxide (DMSO) and Milli‐Q water (1:1, v/v).…”

“Clicking” Gene Therapeutics: A Successful Union of Chemistry and Biomedicine for New Solutions

“…SPAAC has advantages of high reaction rate at ultramild conditions when the two molecules to be reacted have no steric hindrance. However, the rate of SPAAC can be lower compared to CuAAC when clicking longer peptides (> 20 amino acids) to oligonucleotides 31 .…”