Supporting Information

Transcription

1 Supporting Information An Enzyme-Powered Three Dimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing Xiaolong Yang, Yanan Tang, Sean D. Mason, Junbo Chen, Feng Li * Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, Canada, L2S 3A1 Analytical & Testing Center, Sichuan University, Chengdu, Sichuan, China, *Corresponding authors. fli@brocku.ca 1

2 Table S1. DNA sequences and modifications DNA name Sequences DNA probes for 3-D DNA nanomachine DNA probes for MTB DNA nanosensor DNA targets and controls for differentiating single point mutation (T = target C = control; numbers represent corresponding reverse toehold length) DW-57 DW-42 SR Poly dt DW 5 -SH-T-50-T-CCTCAGC-3 5 -SH-T-35-T-CCTCAGC-3 5 -SH- T-10-T- GC*TGA GGAT-FAM-3 (*cleavage site) 5 -SH-T-19-T-3 5 -SH-T-40-T-ATTC ATG GGC CAG AACA CCTCAGC-3 Target 5 - AGC TGA GCCA ATTC ATG GGC CAG AACA-3 DNA Scramble 5 - CGC AGT CCC CAA CCT CC A ATC ACT CAC-3 DNA P TG AGG TGTT CTG GCC CAT GAAT TGG CT-3 P TG AGG TGTT CTG GCC CAT GAAT TGG CTC-3 P TG AGG TGTT CTG GCC CAT GAAT TGG CTC A-3 P TG AGG TGTT CTG GCC CAT GAAT TG GCT CAG-3 P TG AGG TGTT CTG GCC CAT GAAT TGG CTC AGC- 3 P TG AGG TGTT CTG GCC CAT GAAT TGGC TCA GCT- 3 T GCTG AGC CA ATTC ATG GGC CAG AAC-3 C GCTG AGC CA ATTC ATG GAC CAG AAC-3 T GCTG AGC CA ATTC ATG GGC CAG AA-3 C GCTG AGC CA ATTC ATG GAC CAG AA-3 T GCTG AGC CA ATTC ATG GGC CAG A-3 C GCTG AGC CA ATTC ATG GAC CAG A-3 T GCTG AGC CA ATTC ATG GGC CAG-3 C GCTG AGC CA ATTC ATG GAC CAG -3 T GCTG AGC CA ATTC ATG GGC CA-3 C GCTG AGC CA ATTC ATG GAC CA-3 2

3 Supporting Figures Figure S1. (A) Schematic illustration the principle of nicking cleavage on the 3-D DNA nanomachine. (B, C) Native PAGE characterization of the 3-D DNA nanomachine. Lane 1 contains low molecular weight DNA standards. Lane 2 and 3 contain DNA sequences that were released by DTT from 12.5 nm DW-SR-AuNP (Lane 2) or from a reaction mixture containing 12.5 nm DW-SR-AuNP and 20 U/100 µl nicking endonuclease at 37 C for an hour (Lane 3). The gel was first imaged directly to visualize FAM labeled SR (B) and then stained with SYBR Green I to visualize all DNA oligonucleotides (C). To ensure a sufficient signal to noise ratio for SYBR Green staining, the gel was irradiated by UV light to photo bleach the FAM labels before staining. 3

4 Figure S2. Fluorescence increase as a function of time from DW-SR-AuNP with DW densities n varying from 19 to 60 per AuNP. 4

5 Figure S3. Concentration effect of nicking endonuclease on the kinetics of 3-D DNA nanomachine, DW-SR-AuNP. Each reaction mixture contained 100 pm DW-SR-AuNP (l = 57 nt, n = 19, and m = 350) and varying concentrations of nicking endonuclease. (a) Fluorescence increase as a function of time from DW-SR-AuNP with enzyme concentrations [E] varying from 0.3 U to 20 U per 100 µl reaction mixture; (b) Quantitative relationship between [E] and corresponding initial rates V of DW-SR- AuNP, where [E] is between 0.3 U and 20 U; (c) Quantitative relationship between [E] and V, where [E] is between 0.3 U and 5 U; (d) Quantitative relationship between [E] and V, where [E] is between 5 U and 20 U. Error bars represent one standard deviation from triplcated analyses. 5

6 Figure S4. Concentration effect of DNA nanomachine on the kinetics of DW-SR-AuNP. Each reaction mixture contained 20 U nicking endonuclease and varying concentrations of DW-SR-AuNP (l = 57 nt, n = 19, and m = 350). (a) Fluorescence increases as a function of time from DW-SR-AuNP with nanomachine concentrations [AuNP] varying from 6.25 pm to 200 pm; (b) Quantitative relationship between [AuNP] and corresponding initial rates V of DW-SR-AuNP. Error bars represent one standard deviation from triplicated analyses. Figure S5. Size effect of AuNP on the performance of the 3-D DNA nanomachine. Each reaction mixture contained 100 pm DW-SR-AuNP and 20 U nicking endonuclease. For the 20 nm DW-SR-AuNP, the length of DW l was 57 nt, coverage of DW n was ~20 per AuNP, and the coverage of SR m was ~350 per AuNP. For the 40 nm DW-SR-AuNP, the length of DW l was also 57 nt, coverage of DW n was ~25 per AuNP, and the coverage of SR m was ~500 per AuNP 6

7 Figure S6. Two different protecting DNA designs to illustrate the mechanism of protecting DNA on deactivation of the 3-D DNA nanomachine. (a) Protecting DNA is designed to hybridize to DW but does not block any nicking recognition DNA sequence. This protecting DNA is able to slow down the DNA nanomachine by ~ 8 times (V DW/P(+0)- SR-AuNP = Ms -1 V.S. V DW-SR-AuNP = Ms -1 ); (b) Protecting DNA is designed to hybridize to DW and partially block nicking recognition DNA sequence by 5 nt. This design is able to fully deactivate the DNA nanomachine to a background level (V DW/P(+5)-SR-AuNP = Ms -1 ). 7

8 Figure S7. Effect of incubation time that is used to carry out toehold exchange reactions between 20 nm target DNA and 100 pm DNA nanosensor on the regeneration of the activity of the 3-D DNA nanomachine. Error bars represent one standard deviation from triplicated analyses. Figure S8. Specificity of DNA nanosensor analysis of a scrambled DNA control and a mixture of salmon sperm genome DNA fragments. 8

9 Figure S9. Tuning the length of the reverse toehold to achieve the specificity of single point mutation differentiation. (A) Effect of reverse toehold length on the initial rates of DNA nanosensors triggered by 20 nm target DNA or 20 nm control sequence that is identical to the target except for bearing a single point mutation. The length of the forward toehold is fixed at 9 nt. (B) Real-time monitoring the fluorescence increases from 100 pm DNA nanosensors containing 10-nt reverse teohold in the presence of 20 nm target DNA (blue curve), 20 nm mutation control (red curve), or blank (green curve). Error bars represent one standard deviation from triplicated analyses. Figure S10. Quantification of MTB target DNA using 50 pm DNA nanosensor and endpoint fluorescence measured at 2 hr. Error bars represent one standard deviation from triplicated analyses. 9

10 Figure S11. (a) Real-time monitoring of fluorescence increases of DNA nanosensors in the presence of varying concentrations of target DNA spiked in 10-fold diluted human serum samples; (b) Quantification of targets using end-point fluorescence measured at 1 hr. Figure S12. Detection of MTB target DNA from undiluted human serum samples. Serum circulating DNA isolation kit was used to isolate and preconcentrate DNA components from human serum samples containing varying concentrations of target DNA. DNA nanosensors were then used to quantify target DNA molecules from preconcentrated samples. (a) Real-time monitoring of fluorescence increase for reaction mixtures as a function of time. (b) Quantification of targets using end-point fluorescence measured at 1 hr. Error bars represent one standard deviation from triplicated analyses. 10