DNA Computing Circuits Using Libraries of. DNAzyme Subunits

Transcription

1 SUPPLEMENTARY INFORMATION Supplementary Information for the paper: DNA Computing Circuits Using Libraries of DNAzyme Subunits Johann Elbaz a, Oleg lioubashevski a, Fuan Wang a, Françoise Remacle b, Raphael D. Levine a and Itamar Willner a * nature nanotechnology 1

2 supplementary information The kinetics of the XOR gate operation is depicted in Figure S1. Figure S1: Time-dependent fluorescence changes of the XOR gate shown in Figure 2A in the paper: (a) no inputs, (b) input I1, (1,0), (c) input I2, (0,1), (d) the two inputs I1 and I2, (1,1). 2 nature nanotechnology

3 supplementary information Figure S2 depicts the AND gate and its experimental results. Figure S2: (A) Assembly of the DNAzyme construct for the AND gate. (B) Fluorescence intensities generated by the system in the presence of (a) no inputs, (b) input I9 (1,0), (c) input I10 (0, 1), (d) two inputs I9 and I10 (1, 1). Also, the same fluorescence intensities, in the presence of the different inputs, are presented in form of bars, and the AND gate truth table is presented. The system consists of the two DNAzyme subunits (8) and (9), the fluorophore/quencher-labeled substrate (12), that its cleavage provides the readout for the gate operation, and the inputs I9 and I10, (32) and (33), respectively. The inputs I9 and I10 include partial complementarity to the subunits, yet each of these inputs alone cannot assemble the subunits into an active DNAzyme structure, and thus the "0" output is generated. In the presence of both inputs, I9 and I10, cross hybridization between domain H, H' of the inputs allows the selection of the two DNAzyme subunits and the synergistically-stabilized supramolecular DNAzyme structure that leads to the cleavage of the substrate (12), and the generation of the fluorescence, F 1. nature nanotechnology 3

4 supplementary information Figure S3 depicts the InhibAND gate and its experimental results. Figure S3: (A) Schematic organization of the DNAzyme subunits and its fluorophore-tagged substrate upon interaction with the respective inputs that activate the InhibAND gate. (B) Fluorescence intensities generated by the system in the presence of (a) no input (b) input I11 (1,0) (c) input I12 (0, 1) (d) two inputs I11 and I12 (1, 1). Also, the fluorescence intensities in the prescence of different inputs are presented in form of bars, and the InhibAND gate truth table is presented. In the presence of I12 the DNAzyme structures are formed, leading to the cleavage of the substrate, leading to the generation of the fluorescence. Triggering the system with both inputs, I11 and I12, results in, however, the formation of the energetically favored duplex between (34) and (35) G ~ -60 kcal. mol -1, as compared to G ~ -14 kcal. mol -1 for the duplex structures between the inputs and the DNAzyme subunits. This prohibits the formation of DNAzyme structure and the generation of a fluorescence signals. Thus, a False output is observed upon triggering the system with the two inputs. Next, we use the DNAzyme assembly to build universal NAND and NOR gates. The operation of NAND and NOR gates is based on the co-addition to the library of DNAzyme 4 nature nanotechnology

5 supplementary information subunits of the nucleic acids (23) and (38), respectively, that pre-organize the active DNAzyme structures. Figure S4 depicts the configuration of NAND gate and the respective experimental results. Figure S4: (A) Schematic organization of the DNAzyme subunits and its fluorophore-tagged substrate upon interaction with the respective inputs that activate the NAND gate. (B) Fluorescence intensities generated by the system in the presence of (a) no input (b) input I13 (1,0) (c) input I14 (0, 1) (d) two inputs I13 and I14 (1, 1). Also, the fluorescence intensities in the presence of different inputs are presented in form of bars, and the NAND gate truth table is presented. In the absence of any input the co-added nucleic acid (23) bridges the DNAzyme subunits, leading to an active DNAzyme structure that cleaves the substrate and yield a TRUE output. In the presence of either input I13, (36), or I14, (37), that include, respectively, domains A 1 ' or B 1 ' complementary to the co-added strand (23), the DNAzyme structure between the co-added strand (23) and DNAzyme subunits is energetically favored (complementarity of 12 bases vs 10 bases, for A'-A VS A 1 '-A and 13 bases vs 11 bases B'-B vs B 1 '-B, respectively). This leads to the cleavage of the substrate and yields a TRUE output. In the presence of both inputs, I13 and I14, the hybridization of the inputs to the co-added nature nanotechnology 5

6 supplementary information strand (23) results in the formation of the T-shaped duplex, induced by the complementarity of P and P', that eliminates the active DNAzyme structure, leading to the FALSE output (the duplex P/P' synergistically stabilizes the A 1 /A 1 ' and B 1 /B 1 'duplexes). Figure S5 depicts the design of the NOR gate, and the respective experimental results. Figure S5: (A) Schematic organization of the DNAzyme subunits and its fluorophore-tagged substrate upon interaction with the respective inputs that activate the NOR gate. (B) Fluorescence intensities generated by the system in the presence of (a) no input (b) input I15 (1,0) (c) input I16 (0, 1) (d) two inputs I15 and I16 (1, 1). Also, the fluorescence intensities in the presence of different inputs are presented in form of bars, and the NOR gate truth table is presented. In the absence of any input the co-added strand (38) bridges the DNAzyme subunits, and an active DNAzyme structure cleaves the substrate, that yields a TRUE output. In the presence of either input I15, (39), or I16, (40), that include, respectively, domains A' or B', complementary to the co-added strand (38), or both inputs, the DNAzyme structure between the co-added strand (41) and DNAzyme subunits is eliminated by energetically favored 6 nature nanotechnology

7 supplementary information duplexes between co-added strand and inputs (complementarity of 27 bases vs 12 bases, for AC-A'C', A-A' and 30 bases vs 13 bases for BD-B'D', B-B', respectively). This leads to the FALSE output of the gate. nature nanotechnology 7

8 supplementary information Figure S6 shows the gel electrophoresis results following the AND gate operation. Figure S6: Acrylamide electrophoresis of the AND gate system: Control entries: (a) input I3, (b) input I4 (c) DNAzyme subunit (8) (d) nucleic acid substrate (15). Entries (e) to (f) correspond to different gate states: (e) no inputs, (f) input I3 only, (1,0) g) input I4 only, (0,1) (h) the two inputs I3 and I4, (1,1). One may realize that only the activation of the system with the two inputs I3 and I4 leads to disappearance of the bands corresponding to I3 and I4 and appearance of the bands corresponding to the I3/I4 hybrid, and the output M' of the AND gate. These results are consistent with the AND gate operation. 8 nature nanotechnology

9 supplementary information Figure S7 depicts the YES gates cascade and the respective experimental results. Figure S7: (A) The nucleic acid library consisting of the Mg 2+ -dependent DNAzyme subunits, their respective substrates and the input I5 that activate the serial gate cascade YES-YES-YES by the substrate metabolism mechanism.(b) The fluorescence spectra of the YES-YES-YES cascade gates upon the activation with input I5 : (a) no input (b) in the presence of I5. (C) Fluorescence intensity of the YES-YES-YES gate cascade in form of bars presentation: (a) control, in the absence of any component of the library of the Mg 2+ -dependent DNAzyme subunits or their respective caged substrates, in the presence of I5; (b) in the presence of all the component of the library of the Mg 2+ -dependent DNAzyme subunits and their respective caged substrates without I5; (c) in the presence of all the component of the library of the Mg 2+ -dependent DNAzyme subunits, their respective caged substrates and I5. nature nanotechnology 9

10 supplementary information Figure S8 shows the kinetics of YES gates cascade. Figure S7: Time-dependent fluorescence changes of the YES gates cascade: (a) single YES gate output activated by input; (a') single YES gate with no input; (b) two coupled YES gates activated by input; (b') two coupled YES gates with no input; (c) three wired YES gates activated with input; (c') three wired YES gates with no input. One may realize that longer set-on time intervals are observed as the number of wired gates increases. 10 nature nanotechnology

11 supplementary information Figure S9 shows the amplification feedback method: Figure S9 nature nanotechnology 11

12 supplementary information Amplification of the gate output signals in the layered circuits using feedback mechanism. The gate-generated outputs that act as inputs for subsequent layer are weakened with the depth of the circuit. This phenomenon is, however, natural for DNA computing schemes. Our computing paradigm that yields the layered circuits enables, however, to extend the design of the substrates to structures that lead to a feedback mechanism that amplifies and enriches the output signals for the subsequent gate, and enables the tuning of the appropriate stoichiometry of the inputs. One should note that in the layered circuit depicted in Figure 3 and Figure 4 the cleavage of the caged substrate led to a nucleic acid product acting as output for the next gate, and to a "waste" nucleic acid product that had no utility. One may, however, use the "waste" product as an activating unit of the feedback amplification as outlined in Figure 9S. The cleavage of the caged substrate shown in Figure 9S (A) leads to the output nucleic acid that acts as input for the next gate and to the nucleic acid strand ІІa that activates the feedback mechanism. The library of DNAzyme subunits includes now, as additional component, the hairpin structure (42) that is opened by the product strand N'D 1. The resulting open hairpin includes, however, the subunit (S' - I) that in the presence of subunit (41) assembles into the active Mg 2+ -dependent DNAzyme that cleaves the fluorophore-quencher-functionalized substrate, (5), (or re-cleaves the substrate for the subsequent layer), Figure 9S (B). The amplified generation of the output nucleic acid by feedback mechanism is schematically depicted in Figure 9S (C). The library consists of the subunits (10), (20) and (3), (4) that provide the elements for the assembly of the gate 1 and gate 2, respectively. The duplex (17)/( 18) represents the caged substrate for the layered circuit. The substrate (5) acts as the labeled substrate for the readout of the gate cascade output. The library of subunits includes, in addition, the hairpin structure (44) that upon interaction with the released strand ND leads to the assembly of the DNAzyme that re-cycles the formation of strand ND. Figure 9S (D) shows that the hairpin structure (42) is stable and does not lead to any fluorescence output in the presence of subunit (41), yet in the absence of strand ND, curve (a). 12 nature nanotechnology

13 supplementary information The addition of (43) triggers the cleavage of substrate (5), implying that the hairpin was opened, and this led to the self-assembly of the Mg 2+ -dependent DNAzyme, Figure 9S (D) curve (b). Figure 9S (E) shows the fluorescence intensity generated by the two layer circuit in the absence of the input I8 (a), in the presence of the input I8, but without the amplifying hairpin, (44), (b), and in the presence of the input I8 and the hairpin, (43), that leads to amplified cleavage of (5), (c). One may realize that the fluorescence intensity in the presence of (44), in a non-optimized configuration of the system, is ca. 35% higher than the intensity generated by the non-amplified system. The concentration of the components shown in Figure 9S (B) are: (5) 1μM; (41) 0.3μM; (42) 0.3μM and N'D 1 0.5μM. The concentration of the components shown in Figure 9S (C) are: All of the subunits: 1μM; the hairpin (44): 0.3μM; the caged substrate components (17) and (18) 1μM; the substrate (5) 1μM and the input I8 0.1μM. nature nanotechnology 13

14 supplementary information Figure S10 shows the gel electrophoresis results following the YES gate operation. Figure S10: (A) The caged substrates (15)/(16) and (17)/(18) for the YES gates cascade (the numbers in each colored region reflect the amount of nucleotides in each of the respective regions). (B) Acrylamide electrophoresis of the YES gate system: Control entries: (a) caged substrate (17)/(18) (b) caged substrate (15)/(16) Entries (c) to (f) correspond to the activities of the gate cascade: (c) single YES gate with no input I5 (the caged substrate (15)/(16) is visible). (d) single YES gate activated with the input I5 (one may realize the formation of a new band corresponding to the cleaved substrate).(e) two coupled YES gates with no input. (f) Two coupled YES gates activated with the input I5 (one may realize the formation of the two new bands corresponding to the products of the cleavage of substrates (15)/(16) and (17)/(18), and depletion of the bands corresponding to the substrates (15)/(16) and (17)/(18). 14 nature nanotechnology

15 supplementary information Table S1 depicts the instructions of inputs design and their rules for individual and parallel gates operations. A B C D E Gates Number of complementary Domain between input-subunits (I1/I2) Number of complementary Domain Between inputs (I1/I2) Number of DNAzyme subunits Number of Substrates Schematic design of the inputs 1 (I1/I2) OR 2/2 0/0 4 1 AND 1/1 1/1 2 1 XOR 2/2 4/4 4 1 InhibAND 2/0 3/3 2 1 NOR 2 2/2 0/0 2 1 NAND 2 1/1 1/1 2 1 OR+AND 3/3 1/1 6 2 OR+XOR 4/4 4/4 8 2 OR+InhibAND 4/2 3/3 6 2 OR+NOR 4/4 0/0 8 2 OR+NAND 3/3 1/1 6 2 AND+XOR 3/3 4/4 6 2 AND+InhibAND 3/1 3/3 4 2 AND+NOR 3/3 1/1 4 2 AND+NAND 2/2 1/1 4 2 XOR+InhibAND 4/2 5/5 6 2 XOR+NOR 4/4 4/4 6 2 XOR+NAND 3/3 4/4 6 2 NOR+NAND 3/3 1/1 4 2 OR+AND+XOR 5/5 4/ InhibAND+AND+XOR (HA+HS) 5/3 5/5 8 3 NAND+AND+XOR 5/5 4/4 8 3 NAND+InhibAND+AND+XOR 5/4 5/ An and An' represents the domains and their complementary domain, respectively. 2. For the gates the input domains hybridize with a helper nucleic acid that bridges the DNAzyme subunits (see S4 and S5). nature nanotechnology 15

16 supplementary information Table S1 summarizes for all individual and set of two, three and four parallel gates: - The number of complementary domains between each of the inputs and the DNAzyme subunits (entry A). - The number of inter-inputs complementary domains between inputs (entry B). - The number of DNAzyme subunits (entry C). -.The number of substrates (entry D). - The schematic design of the inputs (entry E). The design of the inputs rests on two basic types of complementary nucleic acid sequences: One type includes domains complementary to the respective subunits of the DNAzyme, where the second type includes inter-inputs complementary domain. For the construction of individual and parallel gates, the number of complementary domains is designed using the following instructions: 1. The number of complementary domains (entry A and B) for each individual gates is a mandatory requirement, and these represent the minimums number of domains to activate all of the gates individually. 2. The number of inter-inputs complementary domains between inputs (entry B) is divided into three groups: the AND and NAND, the XOR and InhibAND and the OR and NOR gates. For the first group, the number of complementary domains is only one (as the complementary domains have to stabilize the formation of the duplex between inputs at room temperature, that bridge two DNAzyme subunits into an active DNAzyme). For the second group, the complementary domains need to provide strong hybridization energy, as they destabilize two DNAzyme structures and one DNAzyme structure, for the XOR and for the InhibAND gate, respectively. The third group doesn t require any of these domains in the inputs sequence. 16 nature nanotechnology

17 supplementary information Following this rules, in order to design parallel gates, the number of inter-inputs complementary domains is a combination of the inter-inputs domains of the individual gates. In fact, upon the activation of a parallel combination of gates from group one and two, the number of inter-inputs complementary domains required to activate these gates, correspond to the number of inter-inputs complementary domains required to activate the individual gate of the second group. For example, in order to activate the AND-XOR gate (HA), the number of inter-inputs complementary domains required is 4 in each input, yet this originate from the XOR gate design requirement. Thus, the inter-inputs complementary domains requirement for the AND gate is provide by the design of the XOR gate. On the other hand, upon activating two parallel gates from group two or two parallel gates of group one, one of the inter-inputs complementary domains can be eliminated. For example, by activating the AND-NAND gates, only one inter-inputs complementary domain is required in each of the inputs (the inter-inputs complementary domains requirement to activate both individual gates, can be design in common and have the same function requirement). 3. In order to form and activate a DNAzyme, the bridging of two DNAzyme subunits is mandatory. However, it is possible to use a common DNAzyme subunit to construct and activate two different DNAzyme that will cleave two different substrates. For example, by bridging DNAzyme subunits 1+2 and 1+3, two different DNAzyme will be activated with a common DNAzyme subunit 1. This property is used in the combination and activation of parallel gates. For example by activating the Half- adder and Half-substractor systems (InhibAND-AND-XOR gates), one of the DNAzyme subunits used for the InhibAND and AND gates are identical (the input domain requires for the hybridization with this DNAzyme subunit is also identical for both gates). Nonetheless, the instructions given in this paragraph can be applied only after implementation of the instructions nature nanotechnology 17

18 supplementary information given in paragraph (2). This common complementary domain between input-dnazyme subunit should not be use for the inter-inputs complementary domains design requirement. S11 Material and method Material- 4-(2-hydroxyethyl)piperazine-1 ethanesulfonic acid sodium salt (HEPES), sodium chloride, Magnesium cloride were purchased from Sigma-Aldrich Inc. DNA oligonucleotides (5, 11, 12, 15, 17, 26 and 29) were purchased from Integrated DNA Technologies Inc. (Coralville, IA). All other oligonucleotide sequences were purchased from Sigma-Genosys. Ultrapure water from NANOpure Diamond (Barnstead) source was used in all of the experiments. DNA sequences: (1) 5' T AGG TAT TTG TAG GTTA CAC CCA TGT TAC TCT 3' (2) 5' GAT ATC AGC GAT TAAC ACT CAG GAT TCG 3' (3) 5' G TGA TGT GTC ATA GTTA CAC CCA TGT TAC TCT 3' (4) 5' GAT ATC AGC GAT TAAC AG TAG TAG TCT GC 3' (5) 5 FAM-AGA GTA TrAG GAT ATC-Black Hole Quencher-1 3' (6) 5' CGA ATC CTG AGT CTA CAA ATA CCT AG TGA TGT GTC ATA AG TAG TAG TCT GC 3' (7) 5' GC AGA CTA CTA CT TAT GAC ACA TCA CT AGG TAT TTG TAG ACT CAG GAT TCG 3' (8) 5' CTG CTC AGC GAT TAAC AAC TGG TGC TA 3' (9) 5' GA CTC GTA TGC GTTA CAC CCA TGT TAG AGA 3' (10) 5' ATC TGT CGA GTG GTTA CAC CCA TGT TCG TCA 3' (11) 5' Cy5.5-TGA CGA TrAG GAG CAG-Iowa Black RQ 3' (12) 5' ROX-TCT CTA TrAG GAG CAG-Black Hole Quencher-2 3' 18 nature nanotechnology

19 supplementary information (13) 5' CGA ATC CTG AGT CTA CAA ATA CCT AG TGA TGT GTC ATA AG TAG TAG TCT GC ATC TGT CGA GTG GCA TAC GAG TC 3' (14) 5' TAG CAC CAG TT CAC TCG ACA GAT GC AGA CTA CTA CT TAT GAC ACA TCA CT AGG TAT TTG TAG ACT CAG GAT TCG 3' (15) 5' A TAC GCT TAT CGG CAC ATG AGA TCT CTA TrAG GAG CAG GAG TG AA CTG 3' (16) 5' TA GCA TCA GTT CAC TCG ACA GAT TCT CAT GTG CCG ATA AGC GTA T 3' (17) 5' C TGG TCT GGT GCA GCA CTG GTA TGA CGA TrAG GCA AGA TCA TA AG TAG 3' (18) 5' GC AGA CTA CTA CT TAT GAC ACA TCA C TAC CAG TGC TGC ACC AGA CCA G 3' (19) 5' GA CTC GTA TGC GTTA CAC CCA TGT TCG TCA 3' (20) 5' TCT TGC AGC GAT TAAC AAC TGA TGC TA 3' (21) 5' CTG CTC AGC GAT TAAC ACT CAG GAT TCG 3' (22) 5' T AGG TAT TTG TAG GTTA CAC CCA TGT TAG AGA 3' (23) 5' CGA ATC CTG AGT CTA CAA ATA CCT A 3' (24) 5' ATC TGT CGA GTG GCA TAC GAG TC 3' (25) 5' G TGA TGT GTC ATA AG TAG TAG TCT GC 3' (26) 5' A TAC GCT TAT CGG CAC ATG AGA TGA CGA TrAG GCA AGA TGT AG AC TCA 3' (27) 5' CGA ATC CTG AGT CTA CAA ATA CCT A TCT CAT GTG CCG ATA AGC GTA T 3' (28) 5' TA GCA TCA GTT CAC TCG ACA GAT 3' nature nanotechnology 19

20 supplementary information (29) 5' GTG GTC CAG TGC TGC AGT GCA ATC TCA TGT ACC GAT AAG CGT AT AGA GTA TrAG GAT ATC GG TTG GTG TGG TTG G 3' (30) 5' CAC CAA CC AT ACG CTT ATC GGT ACA TGA GAT TGC ACT GCA GCA CTG GAC CAC 3' (32) 5' ATC TGT CGA GTG GCA TAC GAG TC 3' (33) 5' TAG CAC CAG TT CAC TCG ACA GAT 3' (34) 5' GTA GTA GTC TGC ATC TGT CGA GTG AA CTG GTG CTA 3' (35) 5' TAG CAC CAG TT CAC TCG ACA GAT GCA GAC TAC TAC 3' (36) 5' ATG ACA CAT CAC ACT CAG GAT T 3' (37) 5' GG TAT TTG TAG GTG ATG TGT CAT 3' (38) 5' ATG CAG ACT ACT ACT CGA ATC CTG AGT CTA CAA ATA CCT ATA TGA CAC ATC ACG AT 3' (39) 5' ACT CAG GAT TCG AGT AGT AGT CTG CAT 3' (40) 5' AT CGT GAT GTG TCA TAT AGG TAT TTG TAG 3' (41) 5' GAT ATC AGC GAT ATT GGT GAG 3' (42) 5' AT TGG TGA TAT GA TC TTG CTC ACC AAT CAC CCA TGT TAC TCT 3' (43) 5' TCT TGC AGC GAT TAA CCA CTC TAT GA TC TTG AGT GGT TA 3' Instrumentation: Light emission measurements were performed using a photon counting spectrometer (Edinburgh Instruments, FLS 920) equipped with a cooled photomultiplier detection system, connected to a computer (F900 v 6.3 software). The excitation of Cy5.5 (F 1 ), FAM (F 2 ) and ROX (F 3 ) was done at 700 nm, 495 nm and 648 nm, respectively. 20 nature nanotechnology

21 supplementary information Experimental section: Logic gate systems: All the assays were prepared into 10 mm HEPES buffer containing 1M NaCl and 20 mm MgCl 2. They include 1µM of appropriate DNAzyme subunits, substrates and 0.9 µm of the appropriate inputs describe in each figures for each state and respective gate. The solution was then heated to 90 C for 5 minutes and cooled instantly to 25 C and holded at this temperature for 1 hour and 30 minutes for simple gate (AND, XOR, InhibAND, NAND and NOR gates) and for the Half adder/ Half substractor systems and 3 hours for the cascade and fan-out systems. Inhibition of thrombin by a DNA-based machine: The assay was done into a 10 mm HEPES buffer containing 1M NaCl, 20 mm MgCl 2 and 7mM KCl. We add 1µM of the appropriate DNAzyme subunits, substrate and 0.9 µm of the input. The solution was then heated to 90 C for 5 minutes and cooled instantly to 25 C and holded at this temperature for for 1 hour and 30 minutes. After that we add 20 nm of Thrombin for half an hour in order to form the aptamers/substrate complex. For the fluorescence measurements chromogenic substrate Rhodamine110 bis(p-tosyl-gly-pro-arg), 5.8x10M -6 was used. Nondenaturing polyacrylamide gel electrophoresis: Gels contained 30% polyacrylamide (acrylamide/bis-acrylamide). Tris-borate-EDTA (TBE) consisting of Tris base (89 Mm, ph= 7.9), boric acid (89 mm) and EDTA (2 mm) was used as the separation buffer and Gels were run on a Hoefer SE 600 electrophoresis unit at 25 C (300 V, constant voltage) for 3 hours. After electrophoresis, the gels were stained with SYBR Gold nucleic acid gel stain (Invitrogen) and scanned. nature nanotechnology 21