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Preparation of biomimetic gene hydrogel via polymerase chain reaction for cell-free protein expression

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  • ReceivedAug 8, 2019
  • AcceptedSep 19, 2019
  • PublishedNov 13, 2019

Abstract

Deoxyribonucleic acid (DNA) hydrogels, a three-dimensional (3D) network made from DNA chains, have attracted great attention because of its molecular programmability, excellent biocompatibility and wide biomedical applications. Construction of hydrogel incorporating genetic function is still a challenge because of the limitations in available preparation methods. Herein, we develop a polymerase chain reaction (PCR) based strategy to construct gene integrated hydrogel to mimic the biofunction of nucleus zone. DNA primers were chemically modified by methacrylamide, which were used as modular primers in PCR to hybridize with template plasmid DNA, yielding methacrylamide functionalized gene (Acry-gene). Afterwards, Acry-gene was chemically cross-linked and compressed via free radical polymerization of terminal group methacrylamide to form a three-dimensional gene network, namely gene hydrogel. The gene hydrogel retained the genetic function and expressed protein successfully in a cell free protein expression system. This work provides a general approach for the construction of biofunctional gene hydrogel which mimics bioprocesses, showing great potential in biomedicine and biomimetic fields.


Funded by

the National Natural Science Foundation of China(21621004,21575101,21622404)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (21621004, 21575101, 21622404).


Interest statement

The authors declare that they have no conflict of interest.


Contributions statement

These authors contributed equally to this work.


Supplement

The supporting information is available online at http://chem.scichina.com and http://link.springer.com/journal/11426. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Agarose gel electrophoresis analysis of PCR produced Acry-eGFP and the effect of concentration of APS on the free radical polymerization of Acry-eGFP. (a) Schematic diagram of the preparation of polymerized-gene. (b) Gel electrophoresis image of Acry-eGFP with 1,235 bp synthesized by PCR using pRset-eGFP plasmid as template. (c) Gel electrophoresis image of chemically cross-linked Acry-eGFP with 1,235 bp via free radical polymerization. Each electrophoresis band represented an initiator in different mass concentrations. (d) Acry-eGFP with 2,393 bp synthesized by PCR using pRset-eGFP plasmid as template. (e) Gel electrophoresis image of chemically cross-linked Acry-eGFP with 2,393 bp via free radical polymerization. Each electrophoresis band represented an initiator in different mass concentrations (color online).

  • Scheme 1

    Scheme of the preparation of gene hydrogel and protein expression in a CFPS system. Acry-primer sequences hybrided with plasmid DNA template and were extended by polymerase to synthesize Acry-gene via PCR process. Acry-gene was afterwards cross-linked via free radical polymerization of methacrylamide groups to form a 3D gene network, namely gene hydrogel. The gene hydrogel preserved the biological function of DNA and expressed protein in CFPS system (color online).

  • Figure 2

    Formation and rheological property of gene hydrogel. (a) The ultraviolet imaging of Hydrogel-1235 and Hydrogel-2393 formed with different concentrations of Acry-eGFP (1,235 bp) and Acry-eGFP (2,393 bp), respectively. The hydrogels were stained by SYBR Green I. (b) Time dependent development of the storage modulus G′ and the loss modulus G″ of Hydrogel-1235. (c) Time dependent development of the storage modulus G′ and the loss modulus G″ of Hydrogel-2393 (color online).

  • Figure 3

    SEM and fluorescence microscopy of gene hydrogel. (a, b) The morphology of Hydrogel-1235 (the arrow indicates nanofibers); (c) the fluorescence microscope image of the Hydrogel-1235 (stained with SYBR Green I); (d, e) the morphology of Hydrogel-2393; (f) the fluorescence microscope image of the Hydrogel-2393 (stained with SYBR Green I) (color online).

  • Figure 4

    Protein expression of gene hydrogel in a CFPS system. (a) Schematic illustration of protein expression in a gene hydrogel containing CFPS system; (b) time course of the eGFP expressed from Acry-eGFP Hydrogel-1235 in a CFPS system; (c) time course of the eGFP expressed from Acry-eGFP Hydrogel-2393 in a CFPS system (color online).

  • Table 1   Oligonucleotides used in PCR

    Name

    Sequence

    Use

    Primer 1

    TAACCGTATTACCGCCTTTGAGT

    Forward primerfor PCR

    Primer 2

    GCGGGCCTCTTCGCTATTA

    Reverse primer for PCR

    Primer 3

    GCGGGCCTCTTCGCTATTA

    Reverse primer for PCR

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