Interlocked DNA nanostructures controlled by a reversible logic circuit

Abstract

DNA nanostructures constitute attractive devices for logic computing and nanomechanics. An emerging interest is to integrate these two fields and devise intelligent DNA nanorobots. Here we report a reversible logic circuit built on the programmable assembly of a double-stranded (ds) DNA [3]pseudocatenane that serves as a rigid scaffold to position two separate branched-out head-motifs, a bimolecular i-motif and a G-quadruplex. The G-quadruplex only forms when preceded by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the head-motif containing dsDNA nanorings with respect to the central ring to which they are interlocked, triggered by release oligodeoxynucleotides. We employ these features to convert the structural changes into Boolean operations with fluorescence labelling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates. Such reversible logic circuits integrated into functional nanodevices may guide future intelligent DNA nanorobots to manipulate cascade reactions in biological systems.

Figure 1. Schematic for the synthesis and programmable assembly of dsDNA [3]pseudocatenane.

(a) The synthesized [3]pseudocatenane contains C-rich (cyan, C₆TC₆) and G-rich (purple, G₃ACG₃) branches. The rings L-M and M-R are held together by hybridization in short sections, to form the interlocked structure. (b) Addition of RO-L and RO-R (that is, ROs) triggers a structural conversion from [3]pseudocatenane to [3]catenane, where RO-L and RO-R (underlined: the active sequences) are hybridized with two gaps of ring M to displace L and R thereby allowing them to move freely. A bimolecular i-motif (cyan) can then form in the presence of H⁺, resulting in head-motif cyclization in the interlocked system.

Figure 2. Electrophoretograms of the synthesis and assembly processes of interlocked DNA nanostructures.

(a) Electrophoretogram of 1 pmol interlocked architecture and each components in 6% native polyacrylamide gel electrophoresis at pH 8. (1) M circle; (2) L and R circles; (3) raw product; (4) pure product. The new band in lane 3 with the slowest mobility corresponds to the formed [3]pseudocatenane. (b) Electrophoretogram of 1 pmol assembled [3]catenane in 2.5% agarose gel at pH 8. (1) [3]pseudocatenane; (2) [3]pseudocatenane plus 10 equivalent of ROs. The shift in the mobility of bands originates from the freely moving circles of the [3]catenane (lane 2 versus lane 1). (c–e) Electrophoretograms of 1 pmol interlocked DNA nanostructures (50 nM, 6% native polyacrylamide gels). (c) (1) [3]pseudocatenane at pH 8; (2) 1 plus 10 equivalent of ROs; (3) 2 plus 20 equivalent of cROs. An opposite shift in the mobility of bands is observed. (d) (1) [3]pseudocatenane plus H⁺; (2) [3]pseudocatenane plus ROs and H⁺; (3) 2 plus cROs. The two close bands in lane 2 correspond to [3]catenane before and after the cyclization of heads. (e) (1) Lane 2 in panel d plus OH⁻ (2) lane 2 in panel d plus cROs and OH⁻. The addition of OH^- results in a disappearance of the slowest band (lane 1), indicating the disruption of i-motif structure at pH 8.

Figure 3. AFM confirmation of the DNA nanoarchitectures.

(a) AFM image of [3]pseudocatenane at pH 8, with two clearly observed heads. (b) AFM image of [3]catenane with ROs at pH 8 in irregular shapes, implying the mobility of circles. (c) AFM images of [3]catenane with ROs at pH 5, demonstrating that two heads are linked together. It originates from the formation of bimolecular i-motif structure on the heads. The scale bars represent 50 nm. The whole images corresponding to (a–c) are provided as Supplementary Figs 2–4.

Figure 4. Fluorescence monitoring of the structural changes of DNA nanoarchitectures.

(a) Fluorescence signal of RG/BHQ1 FQ system in response to the structural changes. The toehold strand displacement is reflected by an increase in the RG fluorescence, while a decrease indicates the reverse process. (b) Kinetic data for the toehold-mediated release experiment in the working solution at different temperatures. The addition of ROs induces a sharp increase in the fluorescence in about 1 h at 37 °C, while in about 3 h at 25 °C (inset).

Figure 5. Logic behaviors of dye-labeled [3]pseudocatenane at four input modes.

Fluorescence intensity of (a) RG-BHQ1 FQ system and (b) Cy3-Cy5 FRET system was recorded at 534 nm (FI₅₃₄) and 663 nm (FI₆₆₃), respectively. The normalized FI₅₃₄ and FI₆₆₃ serve as outputs (1/0) with a threshold of 0.6, consistent with two-input ID and AND logic gates, respectively. Insets: corresponding truth tables (corresponding fluorescence spectra: Supplementary Fig. 5). (c) Logic circuit that consists of the two tandem gates. Here a one-input YES gate equivalent to the two-input ID gate is adopted. (d) Reversibility of the logic circuit in several working cycles measured by fluorescence.

Figure 6. Logically switching a G-quadruplex DNAzyme.

(a) Working principle of a DNAzyme nanoswitch built on the difference in robustness between the bimolecular i-motif (cyan) and the G-quadruplex (purple) structures on the head-motifs of the interlocked architecture. (b) Normalized absorbance of the DNAzyme switch on different inputs, consistent with the output of logic circuit. (c) Control experiment for the switch. The control structure is a [3]pseudocatenane with only G-quadruplex structure (that is, no i-motif) on the heads. (d) Working cycles of the nanoswitch between the inactive and active states in response to external stimuli.