Operation of a DNA Robot Arm Inserted Into a 2D DNA Crystalline Substrate

Abstract

The success of nanorobotics requires the specific placement and subsequent operation of specific nanomechanical devices at particular locations. The structural programmability of DNA makes it a particularly attractive system for nanorobotics. We have developed a cassette that enables the placement of a robust sequence-dependent DNA robot arm within a 2D crystalline DNA array. The cassette contains the device, an attachment site and a reporter of state. We demonstrate by atomic force microscopy that the rotary device is fully functional following insertion. Thus, a nanomechanical device can operate within a fixed frame of reference.

Branched DNA has proved to be a very useful and exciting medium for nanotechnology (1). This is a consequence of the programmability of DNA topology and three-dimensional structure through sequence, combined with the well-defined local structure of intermolecular association that occurs via sticky ended cohesion (2). The development of stiff motifs (3) has enabled the self-assembly of DNA components to produce two dimensional arrays of high quality at atomic force microscopy (AFM) resolutions (4). In a separate but related thread, robust, sequence-dependent DNA nanomechanical devices have also been developed. The insertion of such nanomechanical devices into 2D arrays results in a nanorobotic system, wherein nanoscale moving parts can be controlled relative to a fixed frame of reference. We report the development of a cassette that contains both a rotary device and the features that enable its insertion into an array at a specific site. A change in the device control sequences or in the insertion sequences would result, respectively, in different controlling elements or in a different site of insertion, all within the context of the same cassette motif.

The PX-JX₂ device is a robust sequence-dependent DNA machine whose state is controlled by hybridization topology (5). It can assume two structural states (termed ‘PX’ and ‘JX₂’) that differ from each other by a half-turn rotation of one end of the molecule, relative to the other end. Two different pairs of set strands can bind to the framework of the device, thereby establishing which structural state it adopts. Different devices can be addressed independently, leading to 2^N structural states if N devices are present (6). We show by AFM that the PX-JX₂ device is functional after the cassette has been inserted into a 2D DNA array. The cassette used here also contains a component that reports its state, although that is not a general requirement.

The cassette that we have developed is shown schematically in both of its states in Figure 1. Panels (A) and (B) show the cassette plus a reporter hairpin in the PX state: (A) is perpendicular to its plane, and (B) is oblique; panels (C) and (D) show similar views of the JX₂ state. The cassette consists of three helical domains, one of which is much shorter than the other two. The short domain, shown on the bottom in Figures 1A-1D, is the insertion domain. It contains sticky ends that enable its cohesion roughly perpendicular (3 nucleotide pairs rotation, ~103°) to the array that supports it. This domain is connected to the middle domain by a DAO linkage (3). By contrast, the central domain is connected to the top domain by a PX linkage (7). Further right on the upper domains, the double helical continuity is interrupted by a pair of set strands (green in panels A and B, purple in panels C and D) that control the state of the PX-JX₂ device. Proceeding to the right, the two-helix motif continues for about four double helical turns. A long reporter hairpin has been attached so that it extends perpendicular to the plane of the cassette. Note that this hairpin points in opposite directions in the PX state and in the JX₂ state, enabling differentiation of the two states by means of AFM. The stability of the cassette in both states, with and without the reporter hairpin, is indicated by the presence of single bands on a non-denaturing gel in Fig. 1E.

Figure 1.

(A) A view perpendicular to the plane of the cassette in the PX state. The PX state is set by the green strands in the middle of the upper two domains. The reporter hairpin seen end on protruding from the plane. Note the sticky ends on the bottom domain that attach the cassette to the 2D array. (B) The same molecule is shown obliquely, so the reporter hairpin can be seen. (C) A view similar to (A), except that the cassette is in the JX₂ state, which is set by the purple strands. Note that the reporter hairpin is now behind the cassette, a point emphasized in (D). All drawings are in a virtual-bond representation produced by the program GIDEON (13). (E) This is a 5% polyacrylamide gel run in TAEMg buffer (3). The two different states are shown both for a cassette and for a cassette including a reporter hairpin. The single bands seen in each lane indicate that the motifs are stable and monodisperse.

A three-domain tile (TX) array (8) has been selected for insertion. In this array, the TX tiles are connected so that the bottom domain of each tile is attached to the top domain of a tile in an adjacent column, as shown in Fig. 2. This arrangement produces slots that may be flanked by sticky ends on the termini of the middle domains of each TX tile. These sticky ends can be used to bind another tile with complementary sticky ends in that site (8). We form the TX array with eight unique tiles, so as to accommodate the cassette's long reporter hairpin (Fig. 1); the size of the hairpin needed to demonstrate motion has limited us to only two inserted elements. One of these elements is the cassette, containing the PX-JX₂ device, and the other is a TX marker tile, parallel to the cassette, that enables us to establish a reference frame on the array. The marker tile is in the same column as the cassette insertion domain (Fig. 2). The sequences of the cassette and the tiles are shown in Fig. S1; the presence of all strands in each state is shown in Figs. S2 and S3; conversion of state in solution is shown in Fig. S4 (9).

Figure 2.

The arrays are shown schematically to demonstrate the two states of the device in the cassette. The eight TX tiles that form the array are shown in differently colored outlined tiles. For clarity the cohesive ends are shown to be the same geometrical shape, although they all contain different sequences. The cassette and reporter helix are shown as red filled components; the marker tile is labeled ‘M’ and is shown with a black filled rectangle representing the domain of the tile that protrudes from the rest of the array. Both the cassette and the marker tile are rotated about 103° from the other components of the array (three nucleotides rotation). The PX arrangement is shown at the left and the JX₂ arrangement is on the right. Note that the reporter hairpin points towards the marker tile in the PX state, but points away from it in the JX₂ state.

The results of insertion and state conversion are shown by AFM in Fig. 3. Panel (A) shows an array of PX-state cassettes (left) that have been converted to JX₂-state cassettes (right); panel (B) shows the reverse conversion, where an array formed with JX₂-state cassettes (left) is converted to cassettes in the PX-state (right). It is important to recognize that these conversions occur after the cassettes have been inserted into the array (detailed methods are described in (9)). In addition to the arrays shown in Fig. 3, we have examined two other sets of inserted cassette arrays (Figs. S5 and S6) (9). As summarized in Table S1 (9), the AFM images are only good enough to ascertain the states of about half of the pre-transition cassettes, and slightly fewer of the post-transition cassettes. Amongst the three image sets (Figs. 3 and S5 and S6), we detect no errors in the pre-transition arrays. Following conversion from the PX state, 95 of 96 cassettes are seen to be correctly in the JX₂ state; following conversion from the JX₂ state, 85 of 86 cassettes are seen correctly in the PX state, suggesting a conversion error rate around 1%.

Figure 3.

Panel (A) shows the conversion of the array in the PX state to the array in the JX₂ state. Panel (B) illustrates the reverse motion, JX₂ to PX. The scales of the AFM images are indicated by a 100 nm scale bar in the upper right of each image. In both states, the cassette and the marker tile are visible as a doubly lobed blob-like region. In the PX state, the reporter hairpin is visible as a bright spot at the center of the blob. In the JX₂ state, the reporter arm is visible as a bright spot on one edge of the blob. We have emphasized these features on the four images: In each image, we have put a black box around the unit cell repeat in two cases, and a blue rounded figure within it around the blob-like region. In one of the two boxes we have emphasized the reporter arm by enclosing it in a red circle. Expanded, upright double scale copies of these boxes are shown adjoining the upper left edge of each image. In addition, in the lower left corner of each image, we have taken a 50 × 50 nm portion of the image and have circled the marker in a black ellipse and have enclosed the cassette with a red curve that has a protrusion corresponding to the reporter hairpin. The right side of each pair of images is from an aliquot taken from a solution of the material on the left and then converted to the other state.

It is key for nanorobotics to be able to insert controllable devices into a substrate, thereby leading to a diversity of structural states. Here we have demonstrated that a single device has been inserted and converted at a specific site. There is no reason to expect that the system is limited to a single device unit; as noted above, the specific addressability of the 2-state PX-JX₂ device has been demonstrated previously (6). It has been pointed out that two opposing PX-JX₂ devices could be used to produce complex patterns (10). The eight-tile TX array used here is technically difficult to obtain, but the recent advance in simplified 2D DNA patterning by Rothemund (11) should facilitate the construction of complex base planes for these systems. Similarly, DNA tubes (12) provide a means to incorporate nanomechanical devices into non-planar 2D arrangements.