Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale.
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This work was supported by the Ministry of Education, Science, Sports and Culture, with a Grant-in-Aid for Young Scientists (B), 19710111, 2007, and the Japan Securities Foundation, 2005–2007. The authors thank Prof. Hiroyuki Noji and Dr. Liza Lam of The Institute of Scientific and Industrial Research (ISIR), Osaka University, Japan for technical help with the DNA immobilization for the enzymatic reaction. The authors would also like to acknowledge Mr. Mauricio Cordero of the Institute of Industrial Science, The University of Tokyo for his critical reading of this manuscript.
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Yokokawa, R., Miwa, J., Tarhan, M.C. et al. DNA molecule manipulation by motor proteins for analysis at the single-molecule level. Anal Bioanal Chem 391, 2735 (2008). https://doi.org/10.1007/s00216-008-2125-6
- Molecular surgery
- Motor protein