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

Abstract

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

Self‐Assembly of DNA–Peptide Supermolecules: Coiled‐Coil Peptide Structures Templated by d‐DNA and l‐DNA Triplexes Exhibit Chirality‐Independent but Orientation‐Dependent Stabilizing Cooperativity

“…The programmed self‐assembly of oligonucleotides (ONs) through hydrogen bond and π‐stacking interactions has inspired the burgeoning development of elaborate two‐ and three‐dimensional DNA nanostructures during the last decades . With sizes ranging from nano‐ to micrometers, the user‐defined geometry and the facile chemical functionalization of DNA nanostructures offer ideal nanoscale molecular construction tools for peptide and protein engineering with unparalleled spatial accuracy down to the ångström‐scale level with various chemical and biological applications as long term goals . However, only a few examples have demonstrated bottom‐up construction of artificial protein mimics using the programmability and adaptability of nucleic acid secondary structures, including DNA triplexes, G‐quadruplexes and multiple DNA junctions .…”

“…We relied on our previous POC synthesis strategy, such that the bioconjugations between bicyclo[6.1.0]nonyne (BCN)‐functionalized ONs and azide‐modified peptides were accomplished via copper‐free ring‐strain promoted azide‐alkyne cycloaddition (Figure ). All involved ONs were synthesized from commercially available d ‐nucleoside or mirror‐image l ‐nucleoside monomers via phosphoramidite chemistry and standard automated DNA synthesis.…”

“…A 30‐mer peptide sequence was conjugated to each of three ONs capable of hybridization to form a triplex nucleic acid scaffold (Figure , previous work) . In a first step to dissect the self‐assembly process of such POC‐based complexes, we truncated the peptide from a 30‐mer to a 23‐mer sequence and observed that the POC was still able to self‐assemble into a monodisperse trimeric peptide bundle, with the formation of an ON triplex as driving force (Figure , previous work) …”

“…With sizes ranging from nano‐ to micrometers, the user‐defined geometry and the facile chemical functionalization of DNA nanostructures offer ideal nanoscale molecular construction tools for peptide and protein engineering with unparalleled spatial accuracy down to the ångström‐scale level with various chemical and biological applications as long term goals . However, only a few examples have demonstrated bottom‐up construction of artificial protein mimics using the programmability and adaptability of nucleic acid secondary structures, including DNA triplexes, G‐quadruplexes and multiple DNA junctions . The α‐helical coiled‐coil peptide domains are abundantly found in nature and play pivotal roles in proteins to mediate various biological processes .…”

Synthesis and purification of self‐assembling peptide‐oligonucleotide conjugates by solid‐phase peptide fragment condensation

“…DNAs are inherently charged and hydrophilic; however, peptides can have a wide variety of charge states and polarity characteristics depending on their primary sequences. Although quite a few studies on POCs have been reported, there is still limitations in the synthesis of POCs containing peptides with different properties for the following reasons: First, chemical incompatibility exists in the solid‐phase synthesis for DNAs and peptides. This is especially true during the final step of solid‐phase synthesis; DNAs are normally deprotected and cleaved from the solid support in basic conditions, while peptides are cleaved in acidic condition.…”