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Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

Nature Protocols volume 3, pages 655–662 (2008)Cite this article

Abstract

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase. The location of the scission site and the site-specificity are almost freely tunable, and there is no limitation to the size of DNA substrate. This protocol, which does not include the synthesis of pcPNA strands, takes approximately 10 d to complete. The synthesis and purification of the pcPNA, which are covered by a related protocol by the same authors, takes an additional 7 d, but pcPNA can also be ordered from custom synthesis companies if necessary.

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Figure 1: Strategy of ARCUT.
Figure 2: Construction of fusion vector using ARCUT.
Figure 3: Formation of recombinant DNA from WWOX fragment and EGFP fragment using a ligation joint.
Figure 4: Scission of a linear DNA substrate by ARCUT.
Figure 5: Emission of fluorescence from WWOX-EGFP fusion protein expressed in mammalian cell (Cos-7).

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Acknowledgements

This work was partially supported by a Grant-in-Aid for Specially Promoted Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (for Y.A.). Support by the Global COE Program for Chemistry Innovation is also acknowledged.

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Authors and Affiliations

  1. Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan

    Makoto Komiyama, Yuichiro Aiba, Yoji Yamamoto & Jun Sumaoka

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  1. Makoto Komiyama
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Correspondence to Makoto Komiyama.

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Komiyama, M., Aiba, Y., Yamamoto, Y. et al. Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity. Nat Protoc 3, 655–662 (2008). https://doi.org/10.1038/nprot.2008.7

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