A highly parallel method for synthesizing DNA repeats enables the discovery of ‘smart’ protein polymers

Abstract

Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of a glucagon-like peptide-1 analog. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover “smart” biopolymers that exhibit fully reversible thermally responsive behavior. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a “one-pot” parallel format.

Sequential growth of long DNA strands with user-defined patterns for nanostructures and scaffolds

“…However, in some cases, repeating patterns of sequence are desirable. Structural proteins like elastin, collagen and silk often contain repeating peptide motifs, and are therefore translated from repeating DNA sequences 7 . This has sparked an interest in engineered 'proteinpolymers,' developed with similar motifs from repeating DNA to optimize structural properties 8,9 .…”

“…Current methods to produce long, patterned DNA fall into three main categories: stepwise or parallel ligations, usually with intervening purification [12][13][14] or enzymatic selection 15 , overlap extension or related polymerase chain reaction (PCR) techniques, using many short overlapping fragments 1,16,17 , and enzymatic amplifications like rolling circle amplification (RCA) 7,18 . The first provides full control over the final product, but is time consuming and labour intensive.…”

Genetically engineered nanocarriers for drug delivery

“…When the identity of X is changed in the context of ELPs, many interesting properties can be imparted and precisely tuned, for example, reversible phase-separations in aqueous solutions. 2,15,29,30 One intriguing use of this guest residue-dependent modification of polymer properties has been the creation of ELP block copolymers, which have been constructed by genetically linking a hydrophobic block and a hydrophilic block together, for example, [VPGIG] n1 -[VPGSG] n2 . 4 These block copolymers have been verified to form stable nanoparticle structures ranging from 50-90 nm in diameter, which have various functions in drug delivery, and the formation of which is dependent on the difference between the transition properties of the hydrophilic and hydrophobic blocks.…”

“…4 These block copolymers have been verified to form stable nanoparticle structures ranging from 50-90 nm in diameter, which have various functions in drug delivery, and the formation of which is dependent on the difference between the transition properties of the hydrophilic and hydrophobic blocks. 2,6,16,18,23,[30][31][32] Figure 2 illustrates a series of ELP micelle nanoparticles formed by repetitive amino-acid sequences with different guest residues in hydrophobic and hydrophilic blocks. 4,23,33,34 …”

Design of Monodisperse and Well‐Defined Polypeptide‐Based Polyvalent Inhibitors of Anthrax Toxin

“…The ability to make monodisperse biomacromolecular scaffolds via protein engineering approaches has long been appreciated. [19][20][21][22][23] Kiick and co-workers have used a combination of biosynthetic and chemical approaches to design well-defined polyvalent molecules based on polypeptide scaffolds with different architectures. [24,25] Here we designed polypeptides in which multiple instances of the inhibitory peptide ligand (LIG -HTSTYWWLDGAP) were incorporated within the polypeptide sequence in a serial fashion, separated by flexible peptide linker sequences.…”