A universal platform for building molecular logic circuits based on a reconfigurable three-dimensional DNA nanostructure

Integrating multiple components of a logic device into a 3D DNA nanoprism provides a universal platform for constructing diverse logic gates.

prism at a concentration of 33 nM. By making the final concentrations of Is and Es to be 33 nM and incubating the mixture at 37 °C for 1.0 h after every addition of Is or Es, reversible switch was achieved. After every addition of Is or Es and incubation, 100 L of sample was transferred to a cuvette for fluorescence measurement.
Assay of the serum stability of the DNA prism. For the study on the stability of DNA prism structure in serum, the DNA prism was mixed with fresh sample of undiluted human blood serum in 1×TAE-Mg 2+ buffer, and the final percentage of human blood serum was 10% (v/v). A control group without human blood serum was prepared with the same method except addition of serum. These samples were incubated at 37 °C. At time points of 0.0, 2.0, 4.0, 6.0, and 12.0 hr, 100 L of samples were taken out from the two groups respectively and measured immediately.
Before this, the influence of human blood serum on the fluorescence intensity of the fluorophore ROX was investigated, and no influence was observed.

Logic Gate Operation in Biological Matrix.
To prove that the DNA nanoprism is able to execute logic operation steadily in biological matrix, a binary OR logic gate was carried out in 10% (v/v) human blood serum. The logic gate was operated in the same manner described for that in pure buffered solution, except in this case a fresh sample of undiluted human blood serum was added with slight mixing to make the overall % of human blood serum 10% (v/v) before incubation.

Results and Discussion
Scheme S1 The detailed distribution of DNA sequence in motif A (left) and motif B (right), respectively. To confirm the self-assembly of the DNA triangular prism, all the well-formed DNA complexes were electrophoresed on a 6% PAGE. When the central strand L and the three short strands A1, A2 and A3 were mixed at 1:1:1:1 ratio to assemble the triangular face motif A (M A ), a major band was observed (lane 6); similarly, for the triangular face motif B (M B ), a single band appeared (lane 9) when L, B1, B2 and B3 were mixed together at 1:1:1:1 ratio for assembly. The identity of the two triangular base motifs can be confidently assigned from the purposefully prepared size marker L + A1 (1:1), L + A1+ A2 (1:1:1) and L + B1 (1:1), L + B1+ B2 (1:1:1).
Upon mixing the two triangular motifs together (M A + M B ), the bands of triangular motifs disappeared and a new dominant band appeared (lane 10), corresponding to a heterodimer of the two triangular motifs, namely the triangular prism.  The self-assembled triangular prism developed in this work has vertical sides with length about 3.4 nm (10 bp) and equilateral triangle bases with side length about 7 nm (21 bp). The size of the nanoprism perfectly matches the effective distance of FRET. S1,S2 Thus, in this work, we applied fluorescence resonance energy transfer (FRET) technique to indirectly prove the structure of the prism. The distance between the fluorophore and the quencher within the prism was measured by FRET study. The efficiency of energy transfer for a single donor-acceptor pair at a fixed distance is where is the separation distance between donor and acceptor, is the Förster distance of this 0 donor-acceptor pair. Given that the fluorophore ROX has a quantum yield Φ of 0.7, and according to their respective fluorescence and absorption spectra, an overlap integral J(λ) of 4.33 × 10 -13 M -1 cm 3 and Förster distance R 0 of 5.99 nm were calculated for the donor/acceptor pair of ROX/BHQ-2 ( Fig. S3 (a)), while the maximum FRET efficiency of this donor-acceptor pair was 62.5% (the self-assembled prism fluoresces at 37.5% the unquenched fluorescence, Fig. S2), thus the distance between ROX and BHQ-2 within the prism was calculated to be 5.5 nm. As shown in Fig. S3 (b), one helical turn of 10 bp is about 3.4 nm, and the DNA double helix diameter is about 2.0 nm, therefore the distance between nick A and nick B in this prism with 10 bp vertical edge was estimated to be 5.4 nm (2.0 nm÷2 + 3.4 nm + 2.0 nm÷2) according to the geometric models in Scheme 1 and Fig. S3. The distance between ROX and BHQ-2 is consistent well with the distance between the two nicks of the triangular motifs in this nanoprism, confirming the self-assembly of the DNA nanoprism. In our previous research to prepare the similar DNA nanoprisms with same size S3 , we have tried characterizing prisms directly by using atomic force microscope (AFM). Because the size of these triangular nanoprisms are too small, and they would probably partially collapse on the mica surface during AFM tip scanning due to all struts being single DNA duplexes, these nanoprisms appeared as dispersed spots or blobs under AFM (data not shown). The AFM image shows little structural information about the triangular nanoprisms, and it is not a solid evidence to demonstrate the prism structures in this case. Recent research work reported by Fan's group indicated that the bigger the 3D DNA nanostructure is the more detailed structural information can be obtained by AFM. S4 This fact enlightens us that it might be possible and easier to obtain the structural information of our nanoprisms by AFM if we prepare bigger nanoprisms with the side length of triangular bases about 3-4 turns. There may be two reasons for this phenomenon: firstly and dominantly, since three vertical sides are connected together by two triangular bases in the prism, a synergistic effect may exist between the three vertical sides, thus the vertical duplex as the component would be more thermally stable than it as solo duplex, which has been supported by that the temperature at which the prism starts to open its vertical side (43 °C) is remarkably higher than the Tm value of each vertical side of the nanoprism; Secondly, the increase of temperature leads to gradually open one, then two, and finally all three vertical sides, thus, at the "Tm" of the nanoprism, intact prism and different opened prisms with different fluorescent signals coexisted in the reaction system, including the mono-SD, di-SD, and tri-SD states (as shown in Fig. 1), which is somewhat different from the denaturation of a solo DNA duplex with only two states, the paired and the denatured. The melting curve clearly reflected the thermal stability of the prism, indicating that the prism was stable enough to be applicable at room temperature or physiological temperatures (37 °C). After one of the three input strands (C1, C2 and C3) was input to initiate DNA strand displacement, similar reconfigurated nanostructures were obtained from the prism ( Fig. 1 (a)).
These nanostructures (lane 4, 5 and 6) migrated at a slightly slower rate than the prism (lane 3).
When two of the three input strands were combined to reconfigurate the prism, nanostructures of two triangular motifs linked by a 10 bp DNA duplex were acquired ( Fig. 1 (b)). These nanostructures (lane 7, 8 and 9) had similar migration rates and migrated much slower than the prism (lane 3). The nanostructure (lane 9) derived from reconfiguration induced by C1 and C3 was unstable and part of this nanostructure disassembled to generate two separate motifs probably due to the heat generation during electrophoresis. The simultaneous input of C1, C2 and C3 leaded to a deconstruction of the prism structure, producing motif M A and motif M B with three paired tails. These experimental results of electrophoresis were in good agreement with that of fluorescence measurements (Fig. 1), confirming the successful reconfigurations of the triangular prism.

Fig. S10
The stability of the 3D DNA prism structure in serum. The results show that the DNA prism was able to keep its intact structure for about six hours in 10% (v/v) human blood serum.
For applications of the nanoprism in some specific cases, such as complex biological environments, the change of experimental conditions might make the structure of the prism unstable, for instance low concentration of cations, including Mg 2+ or Na + . In these cases, some strategies can be utilized to further enhance the stability of DNA duplex and facilitate the selfassembly of DNA nanostructures, such as small molecules that can intercalate into or complex with DNA duplex, artificial nucleic acid analogues, and modified DNA bases. S5 Additionally, it was reported that some molecules in cells such as polyamines also have a stabilizing effect on DNA duplexes. S6,S7 Together with the stability of 3D DNA nanostructure against nucleases degradation, these favorable factors would keep this nanoprism-based platform effective in varied user-specified situations.