Amplified Analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires

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1 Supporting information for the paper: Amplified Analysis of DNA by the Autonomous Assembly of Polymers Consisting of DNAzyme Wires Fuan Wang, Johann Elbaz, Ron Orbach, Nimrod Magen and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Experimental Section: Materials. 4-(2-hydroxyethyl)piperazine-1 ethanesulfonic acid sodium salt (HEPES), sodium chloride and Magnesium chloride were purchased from Sigma-Aldrich. All oligonucleotide sequences were purchased from Integrated DNA Technologies Inc. (Coralville, IA). All DNA primers were HPLC-purified and freeze-dried by the company. They were used as provided and diluted in 10 mm phosphate Buffer (PB) to give stock solution of 100 μm. Table S1 depicts the sequences of the oligonucleotides used in the study. Ultrapure water from a NANOpure Diamond (Barnstead) source was used in all of the experiments. S1

2 Table S1 The DNA sequences used to construct the detection platform Number Sequence (1) 5 TC AAT TAG A AAG CAC CCA TGT TAC TCT3 (2) 5 GAT ATC AGC GAT CTT AG TCT TATG 3 (3) 5 Black Hole Quencher-1-AGA GTA TrAG GAT ATC-FAM 3 (4) 5 CATA AGA CTT CTA ATT GA 3 (5) (6) (7) (8) (9) 5 GAT ATC AGC GAT CTT CTA ATT GA AAG TTAT TAA TC AAT TAG AAG TCT TATG AAG CAC CCA TGT TAC TCT 3 5 GAT ATC AGC GAT CTT TTA ATAA CTT TC AAT TAG CATA AGA CTT CTA ATT GA AAG CAC CCA TGT TAC TCT 3 5 TC AAT TAG AAG CAA AAT TAT TTA TGA AGC TGT ATG GTT TCA GCA ACA GGG AGC AA CATA AGA CTT CTA ATT GA 3 5 TT GCT CCC TG T TGC TGA AAC CAT ACA GCT TCA TAA ATA ATT TTG CTT 3 5 CTT TTA ATAA CTT TC AAT TAG CATA AGA CTT CTA ATT GA AAG 3 Instrumentation: Light emission measurements were performed using a Cary Eclipse spectrometer (Varian inc). The FAM was excited at 495 nm. Fluorescence emission spectra were recorded from 500 to 600 nm. Atomic force microscopy (AFM) images were recorded using a Nanoscope 3A controller (Digital Instruments/Veeco Probes/USA) with NSC 15 AFM tips (Mikromasch, Germany) using the tapping mode at their resonant frequency. 10 μl of MgCl 2 (5mM) were deposited on freshly cleaved mica surface (Structure Probe Inc., USA) for 2 min, followed by their rinsing with double distilled water (DDW) and drying under a stream of nitrogen. The DNA sample was deposited on the mica surface, dried in air and gently washed with DDW. Images were analyzed using the WsXM SPIP software (Nanotec, Inc., Spain). S2

3 Experimental section: All the assays were prepared in 10 mm HEPES buffer containing 1 M NaCl and 20 mm MgCl 2. Each functional hairpin structure (1 μm) was heated to 95 C for 5 min and then allowed to cool to room temperature (25 C) for at least 2 hours before use. Then, different concentrations of the target DNA (4) were incubated with 0.5 μm of the prepared hairpin for 6 hours. Subsequently, the ribonucleobase (ra) containing substrate (3, 0.5 μm) was added to the sample and the time-dependent fluorescence changes upon analyzing different concentrations of target DNA (4) were monitored spectroscopically at 25 C. For the analysis of the target DNA (4) by using the Mg 2+ -dependent DNAzyme subunits, the concentration of the ribonucleobase (ra) containing substrate (3), and both DNAzyme subunits (1 and 2) were 0.5 μm. For AFM characterization of the DNA wires that were produced by cross-opening of the functional DNA hairpin structures (5 and 6), the concentration of both hairpins was 0.5 μm and the analyte DNA (4) was 10 nm. For the analysis of BRCA1 oncogene (8), the concentration of helper probe hairpin (7) was 0.3 μm, and the concentration of each of the Mg 2+ -dependent DNAzyme functionalized hairpins (5) and (6) was 0.5 μm. S3

4 Figure S1. Time-dependent fluorescence changes upon sensing different concentrations of the target analyte DNA, according to the Scheme shown in Figure 1(A): (a) 0 M, (b) 1 x 10-9 M, (c) 1 x 10-8 M and (d) 1 x 10-7 M. S4

5 The amplified analytical platform has an intermediate phase where one set of Mg 2+ -dependent DNAzyme subunit (I and I ) was removed from the functional hairpin (6). The analyte (4) opens hairpin (5) to yield structure a. The resulting structure opens hairpin (9) through a strand displacement process and yields structure b. The later structure opens hairpin (5) to yield structure c that includes the adjacent subunits of the Mg 2+ -dependent DNAzyme or active DNAzyme. By the repeated steps 2 and 3, the one-sided polymers consisting of the Mg 2+ -dependent DNAzyme subunits are formed, Figure S2(A). The time dependent fluorescence changes upon analyzing different concentrations of the target (4) are shown in Figure S2(B). The resulting calibration curve is shown in Figure S2(B), inset. The autonomous cross-opening of the hairpins (5) and (9) results in the amplified detection of the target (4). The sensitivity (1 x M) and the resulting fluorescence intensities generated by the one-sided DNAzyme wires is, however, lower than the results obtained with the two-sided DNAzyme polymer wires. S5

6 Figure S2. (A) Amplified analysis of the target DNA (4) using two functional hairpins that upon the recognition of the target by (5) initiate an autonomous cross-opening of the hairpin structures (5 and 9) that lead to the formation of polymer wires consisting of one-sided Mg 2+ -dependent DNAzyme units, that lead to the cleavage of the substrate (3) and the generation of fluorescence. (B) Time-dependent fluorescence changes upon analyzing different concentrations of (4), according to the Scheme outlined in Figure S1(A): (a) 0 M, (b) 1 x M, (c) 1 x M, (d) 1 x M, (e) 1 x M, (f) 1 x 10-9 M, (g) 1 x 10-8 M, (h) 1 x 10-7 M. Inset: Resulting calibration curve. S6

7 Selectivity of analyzing the target DNA (4). Figure S3 demonstrates the selective analysis of the target (4) by the hairpins (5) and (6) by showing the performance of the system towards the discrimination of one base-, two baseand three-base mutations ((4a), (4b) and (4c)). For the detailed sequences of the mutants, see Table S2. Table S2 The DNA analyte with different base mutations Number Sequence (4a) 5 CATA AGA CTT GTA ATT GA 3 (4b) 5 CATA ACA CTT GTA ATT GA 3 (4c) 5 CATA ACA CTT GTA ATT CA 3 Figure S3: Time-dependent fluorescence changes upon analyzing the target DNA (4) and its mutants ((4a), (4b) and (4c)), 1 x 10-9 M, according to the platform shown in Figure 1(B): (a) the target (4), (b) one-base mutation (4a), (c) two-base mutations (4b) and (d) three-base mutations (4c). All systems consist of (5), 0.5 μm and (6) 0.5 μm in the presence of the respective analytes. S7

8 Optimization of the strands (5) and (6) for the sensing platform shown in Figure 1(B). For the effective development of the sensing platform shown in Figure 1(B) one should design the hairpins (5) and (6) to exhibit the following properties: (i) the hairpins (5) and (6) should be retained in fully closed structure prior to the interaction with the analyte (4) to eliminate cross-opening and a background signal. (ii) The hairpins (5) and (6) should exhibit a limited stabilizing energy to enable opening of (5) by the analyte and effective cross-opening process. Table S3 shows the respective sequences used to optimize the hairpin structures. Figure S4 shows the analysis of the respective targets, 100 nm, by the hairpins (5i)/(6i), (5)/(6), (5ii)/(6ii), (curves (a), (b) and (c)) and the performance of the systems in the absence of the analyte (curves (a ), (b ) and (c )). One may realize that only the (5)/(6) systems show high performance signals in the presence of the analyte and zero fluorescence in the absence of the analyte. Table S3 DNA sequences used to optimize the detection platform Number Sequence (4i) 5 C CATA AGA CTT CTA ATT G 3 (5i) 5 GAT ATC AGC GAT CTT CTA ATT G AAG TTTAT TAA C AAT TAG AAG TCT TATGG AAG CAC CCA TGT TAC TCT 3 (6i) 5 GAT ATC AGC GAT CTT TTA ATAAA CTT C AAT TAG CCATA AGA CTT CTA ATT G AAG CAC CCA TGT TAC TCT 3 (4) 5 CATA AGA CTT CTA ATT GA 3 (5) (6) 5 GAT ATC AGC GAT CTT CTA ATT GA AAG TTAT TAA TC AAT TAG AAG TCT TATG AAG CAC CCA TGT TAC TCT 3 5 GAT ATC AGC GAT CTT TTA ATAA CTT TC AAT TAG CATA AGA CTT CTA ATT GA AAG CAC CCA TGT TAC TCT 3 (4ii) 5 CAA AGA CTT CTA GTT GCG 3 (5ii) (6ii) 5 GAT ATC AGC GAT CTT CTA GTT GCG AAG GGT TAA CGC AAC TAG AAG TCT TTG AAG CAC CCA TGT TAC TCT 3 5 GAT ATC AGC GAT CTT TTA ACC CTT CGC AAC TAG CAA AGA CTT CTA GTT GCG AAG CAC CCA TGT TAC TCT 3 S8

9 Figure S4: (A) Time-dependent fluorescence changes of the amplified detection platform shown in Figure 1B using different hairpins that include different composition of the stem regions in the hairpins: (a ) and (a) hairpins (5i) and (6i) containing 10 base pairs in the stem regions, in the absence or presence of target (4i) (100 nm), respectively; (b ) and (b) hairpins (5) and (6) containing 11 base pairs in the stem regions, in the absence or presence of target (4) (100 nm), respectively; (c ) and (c) hairpins (5ii) and (6ii) containing 12 base pairs in the stem regions, in the absence or presence of target (4ii) (100 nm), respectively. S9

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