A design and feasibility study of reactions comprising DNA molecular machine that walks autonomously by using a restriction enzyme

Abstract

In this paper, we propose an autonomous molecular walking machine using DNA. This molecular machine follows a track of DNA equipped with many single-strand DNA stators arranged in a certain pattern. The molecular machine achieves autonomous walk by using a restriction enzyme as source of power. With a proposed machine we can control its moving direction and we can easily extend walking patterns in two or three dimensions. Combination of multiple legs and ssDNA stators can control the walking pattern. We designed and performed a series of feasibility study with computer simulation and molecular biology experiments.

Appendix: Fok I as a nicking enzyme

We examined to achieve a function which is the same as a nicking enzyme by using Fok I and phosphorothioate-modified DNA. Fok I cleaves both of double-strand, this cleaving activity is not suitable for the molecular walking machine. We expected to block the cleaving activity at the cut point of one strand by phosphorothioate-modified DNA (Fig. 9). Remark that for our walking machine, only legs need phosphorothioate modification.

Fig. 9

Phosphorothioate-modified point. 1—Unmodified (F-13, F-20, F-33), a—before cleaving, b—after cleaving. 2—Phosphorothioate-modified (F-13, F-20, F-33s), a—before cleaving, b—after cleaving

We experimented to confirm the effect of phosphorothioate-modified DNA. We prepared five DNA strands for the experiment (Table 3). The five DNA strands compose the following four sets:

Table 3 DNA strands

• (1) unmodified and separated the recognition site and the cut point;

• (2) phosphorothioate-modified and separated the recognition site and the cut point;

• (3) unmodified and unseparated;

• (4) phosphorothioate-modified and unseparated.

Each four sets of DNA strands were mixed at 0.3 μM in hybridization buffer. NEBuffer4 from New England Biolabs was used as the hybridization buffer. 4 units of Fok I from New England Biolabs were added to each five sets 20 μl solution. The four sets were incubated at 37°C by 2 h.

We ran the resulting solutions in 16% PAGE (non-denaturing gel).

For this result, we found phosphorothioate-modified DNA could block the cleaving activity a little (Fig. 10). There were phosphorothioate-modified DNA with separate point in lane 4, and no-modified DNA with separate point in lane 2. To compare the two lanes, we confirmed that phosphorothioate-modified DNA was cleaved. The length 24mer band is the products of cleaving F-33 at the cut point, and there is the same band in lane 4. So this result shows phosphorothioate-modified DNA was cleaved.

Fig. 10

Result of experiment of phosphorothioate-modified. 1, 2—unmodified and separated (F-13, F-20, F-33); 3, 4—phosphorothioate-modified and separated (F-13, F-20, F-33s); 5, 6—unmodified and not separated (F-13 + 20, F-33); 7, 8—phosphorothioate-modified and not separated (F-13 + 20, F-33s). Odd number of lane: without Fok I, Even number of lane: with Fok I

In this regard, however there is the length 33mer band in lane 4. The band shows a few F-33s remained not to be cleaved. And there is the band which shows unreacted DNA strand. This band isn’t observed in lane 2. Unmodified DNA could not block the cleaving activity.

The effort to block the cleaving activity by phosphorothioate-modified DNA has failed. But it was observed phosphorothioate-modified DNA had the weak inhibition of the activity. We expect that using phosphorodithioate-modified DNA leads to stronger inhibition of the activity. The molecular machine may be able to get the cleaving function which it needs by phosphorodithioate-modified DNA.