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

Nature Materials volume 10, pages 141–148 (2011)Cite this article

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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, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. 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 behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a ‘one-pot’ parallel format.

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Figure 1: Schematic of snapshots depicting the evolution of an OERCA reaction.
Figure 2: The effect of primer concentration and cycle number on the size range of the DNA product.
Figure 3: Synthesis of polypeptide libraries by OERCA is simple and outperforms current synthesis methods.
Figure 4: Thermally responsive behaviour of aELPs constructed by OERCA.
Figure 5: Gene synthesis, expression and characterization of a library of aELPs with the repeating sequence AVPGVG.
Figure 6: Characterization of protease mediated cleavage of GLP-1 protein-polymers with variable thrombin recognition sequences.

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Acknowledgements

A.C. acknowledges the financial support of NIH grants R21 EB009904 and R01 GM61232, M.A. acknowledges the support of a graduate fellowship from the Center for Biologically Inspired Materials and Material Systems, and F.G.Q. acknowledges the support of a fellowship from the Medtronic Foundation.

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Author notes

  1. Miriam Amiram and Felipe Garcia Quiroz: These authors contributed equally to this work

  2. Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708-0281, USA

Authors and Affiliations

  1. Ashutosh Chilkoti

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  1. Miriam Amiram
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  2. Felipe Garcia Quiroz
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  3. Daniel J. Callahan
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Contributions

A.C. designed experiments, analysed data and prepared the manuscript. M.A. and F.G.Q. designed and performed experiments, analysed data and prepared the manuscript. D.J.C. designed experiments.

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Correspondence to Ashutosh Chilkoti.

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Amiram, M., Quiroz, F., Callahan, D. et al. A highly parallel method for synthesizing DNA repeats enables the discovery of ‘smart’ protein polymers. Nature Mater 10, 141–148 (2011). https://doi.org/10.1038/nmat2942

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