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A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers - PubMed

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

Miriam Amiram et al. Nat Mater. 2011 Feb.

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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Conflict of interest statement

Competing financial interest. The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1

Schematic of snapshots depicting the evolution of an OERCA reaction. (a) A linear oligonucleotide is first circularized yielding a population of circular DNA, which is enriched by removal of the remaining linear DNA before the start of the OERCA reaction (b–h). (b) The circularized oligonucleotide is added to a conventional PCR mixture containing forward and reverse primers. (c) After annealing of the reverse primer, extension in cycle 1 (n=1) occurs primarily in the form of rolling circle amplification. (d) Upon DNA denaturation in cycle 2, linear repeats of the original circularized sequence become available as extension templates. (e–f) As the reactions proceeds, primer annealing and overlap extension preferentially take place to amplify the linear DNA. (g–h) Upon DNA denaturation the repetitive single-stranded DNA is capable of priming/overlapping, which further promotes the extension of the original repeat unit.

Figure 2
Figure 2

The effect of primer concentration and cycle number on the size range of the DNA product. The monomer gene for GLP-1 was used as a model. Lanes 1 and 2 are the products of 30 cycles with 10 and 40 pmol primer, respectively. All PCR reactions were column purified to remove excess primers and recover product. Products from lanes 1–2 were then subjected to 15 more cycles with no additional primer (lanes 3 and 5, respectively) or with 20 pmol primer (lanes 4 and 6). The size range of the DNA product can be further increased by 15 additional cycles without primer (lane 7 generated from lane 3).

Figure 3
Figure 3

Synthesis of polypeptide libraries by OERCA is simple and outperforms current synthesis methods. The product of an OERCA reaction (a) was ligated into a vector and positive colonies were screened by directional cPCR (b), wherein positive clones can be identified by the presence of a large DNA smear (*). Positive clones were subjected to restriction analysis and DNA sequencing to verify insert size and sequence fidelity (c). OERCA, unlike OE-PCR and concatemerization, enabled the synthesis of constructs with a wide size distribution (0.18 −1.5 Kbp). Insert sizes larger than 0.9 Kbp were estimated by restriction analysis.

Figure 4
Figure 4

Thermally responsive behavior of aELPs constructed by OERCA. (a) The turbidity profiles for all aELPs exhibit a sharp transition with temperature, characteristic of the inverse phase transition behavior displayed by canonical ELPs. We discovered 4 new hexapeptide motifs (b) that display reversible phase transition behavior. All polypeptides in (a–b) were prepared at a concentration of 50 μM in PBS (i.e., 0.14 M NaCl), except VAPGVG (0.64 M NaCl) and APGVG (2.14 M NaCl). Circular dichroism spectra at 25 °C revealed highly disordered conformations predominant in aELPs with both pentapeptide (c) and hexapeptide (d) motifs, similar to that of the canonical ELP. (e–h) The coacervation of aELPs with two distinct phase transition behaviors was studied by CD at various stages (arrows in e and g) of the coacervation process. The CD spectra and associated turbidity profiles were acquired in water at a polypeptide concentration of 5 μM, and θ indicates the mean residue ellipticity. The pentapeptide motif VPAVG (e–f) underwent quasi-irreversible phase separation (e) and lost its highly disordered conformers (i.e., negative peak at 197 nm) in the mature coacervate and upon cooling to 25 °C (f). In contrast, the hexapeptide motif VPGVAG (g–h) exhibited fully reversible phase transition behavior (g), highly disordered conformers were preserved in the mature coacervate, and the secondary structure of the polypeptide was completely recovered upon cooling (h). We also confirmed the reversible behavior of VPAVG upon cooling to 4 °C (Supplementary Fig. S7).

Figure 5
Figure 5

Gene synthesis, expression and characterization of a library of aELPs with the repeating sequence AVPGVG. Turbidity profiles for 5 constructs in this library in PBS (a) and PBS supplemented with NaCl to 1.14 M (b). The transition temperatures calculated from (b) varied linearly as the reciprocal of molecular weight of the aELP as expected for canonical ELP sequences (c). The wide distribution of molecular weights in this library synthesized by OERCA is illustrated at both the DNA and polypeptide level, which demonstrates the ability of OERCA to readily generate both low and large molecular weight protein-polymers (d).

Figure 6
Figure 6

Characterization of protease mediated cleavage of GLP-1 protein-polymers with variable thrombin recognition sequences. (a) Schematic illustration of GLP-1 polymers with variable protease cleavable sequences. Thrombin cleaves C-terminal to the Arginine (R), and in vivo the enzyme DPPIV can then cleave the “GA” dipeptide to leave a free N-terminal Histidine (H), the first amino acid in active GLP-1. (b) SDS-PAGE analysis of Alexa-488 labeled constructs incubated with 1 U thrombin for 2, 6 and 24 hours. (c) Differential activation of GLP-1 protein-polymers by incubation with mouse plasma demonstrated by cAMP production following GLP-1 binding to the GLP-1R in Baby Hamster Kidney cells.

Comment in

  • Protein polymer: Gene libraries open up.

    Ding S, Wang X, Barron AE. Ding S, et al. Nat Mater. 2011 Feb;10(2):83-4. doi: 10.1038/nmat2955. Nat Mater. 2011. PMID: 21258347 Free PMC article. No abstract available.

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