Supporting Information: Tuning the electromechanical properties of single DNA molecular junctions

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1 Supporting Information: Tuning the electromechanical properties of single DNA molecular junctions Christopher Bruot, Limin Xiang, Julio L. Palma, Yueqi Li and Nongjian Tao Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, AZ Experimental methods DNA sample preparation: The oligonucleotide samples used in this study were ordered HLPC purified from Alpha DNA. For free-end DNA molecules, the oligonucleotides were ordered with a thiol linker consisting of a three (six) carbon spacer attached to the phosphate-backbone at the 3 (5 ) side. Closed-end DNA molecules were ordered with a thiol-modified thymine base within the sequence in order to create a hairpin structure. After receiving the oligonucleotides, all samples were suspended, at a concentration of 100 μm, in 18MΩ DI water and stored at -20C. Prior to break junction measurements, the target molecule was added to phosphate buffer (ph=7.0) for a final concentration of 10μM. The DNA samples were then annealed by heating to 95 C and cooling gradually to 4C over 4 hours. The samples were kept at 4C until the conductance measurements were performed. All break junction measurements were carried out in phosphate buffer solution with ~10μM target DNA molecule, at room temperature. STM break junction measurements: Gold STM substrates were prepared in house by thermal evaporation of 1300Å of gold ( % purity, Alfa Aesar) onto freshly cleaved mica slides. Evaporation was done in vacuum (~10-8 Torr) and annealed at 300C for 3 hours to produce Au(111) surfaces. The resulting S1

2 substrates were stored under vacuum until prior to STM measurements. Immediately before the sample was added, the substrates were flame annealed with a hydrogen flame to clean and anneal the surface. Gold STM tips were mechanically cut from gold wire (0.25 mm) and coated with Apiezon wax to insulate from leakage current during measurements in aqueous environment. Break junction experiments were performed using a Molecular Imaging STM head and scanner, along with a Digital Instruments Nanoscope IIIA controller, to collect current information and control the bias and tip position. For all experiments, the current amplifier gain was 10nA/V and z-axis piezo sensitivity was 3.9nm/V. Custom LabView (National Instruments) programs were used to control the STM tip movement, bias voltage and measure current during break junction experiments. During break junction measurements the STM tip was moved at a ramp rate of 5 V/s, equivalent to a stretching rate of ~20nm/s. The stretching length was measured by processing the resulting conductance versus tip displacement decay curves with another home built LabView program, following an algorithm discussed previously 1. Gaussian fittings for all histograms were carried out using Origin 8.0 (OriginLab). DNA structure: Closed-end vs. Free-end In order to confirm the double helix structure of the closed-end and free-end DNA sequences measured in the present experiments, we performed gel electrophoresis, circular dichroism and melting temperature experiments on the DNA samples in the same phosphate buffer environment as during STM break junction measurements. In addition, we compare the gel electrophoresis mobility and circular dichroism spectra of 10 base pair free-end DNA and closed-end DNA, which are expected to form double helical structures, with a 22 bases random sequence which does not form any secondary structures (see discussion below). Polyacrylamide Gel Electrophoresis: S2

3 The double stranded DNA structure of all molecules measured was confirmed using native polyacrylamide gel electrophoresis (PAGE). Electrophoretic experiments were performed at 20 C using a voltage of 200V for ~3 hours with 50pmol of each sample and 10% native PAGE gels in 1xTAE Mg 2+ buffer. Ethidium bromide (EB) was used to stain the gels, which were then scanned in a Biorad Gel Doc XR+ system for sample visualization. Figure S1 shows the PAGE measurements for all DNA sequences which were measured with the STM break junction. To show that the closed-end sequence forms a double strand hairpin structure during measurement the PAGE mobility of identical sequence 10 base pair free-end and closed-end DNA molecules was compared with a randomly sequenced 22 base pair single strand DNA molecule. The 22 bp single strand sequence also included the 3 carbon spacers in order to mimic the closed-end single strand sequence. The results of the PAGE measurements along with drawings of the molecular sequences are shown in figure S2. From this gel we see that the 10 base pair closed-end DNA is between the 10 base pair free-end and the 22 bases ssdna. This suggests that the closed-end DNA does indeed form a double helix structure, but has a different mobility than free-end DNA which is unsurprising considering the fact that closed-end DNA has hairpin structures. S3

4 Figure S1: PAGE measurements of DNA samples. (1) Free-end 3-3, (2) Free-end 5-5, (3) Free-end 5-3, (4) 10 base pair Closed-end, (5) 14 base pair Closed-end, (6) 18 base pair Closed-end, (7) 22 base pair Closed-end. S4

5 Figure S2: (a)comparison of PAGE mobility for (1) free-end DNA, (2) ssdna and (3) closedend DNA. (b)drawings of the molecular structures and sequences. Red lines in (2) and (3) represent C3 spacer inserted to enable the hairpin structure. Circular dichroism measurements The native, biological form structure of duplex DNA samples was confirmed for both free-end and closed-end molecules using circular dichroism (CD). CD measurements were performed using a Jasco (Easton, MD) J-815 Spectropolarimeter from 320 nm to 220 nm with a scanning rate of 50 nm/min. The spectra were compiled by averaging the results from 5 scans, taken in 100mM Na + phosphate buffer solution with 5 um of dsdna at room temperature to replicate the environment during STM break junction experiments. Figure S3 show CD spectra S5

6 for the 10 base pair free-end DNA, closed-end DNA and ssdna structures described in Figure S2. The spectra for both the free-end and closed-end molecules have a peak feature around 260 nm and a dip feature around 240 nm. These spectra are in good agreement with previously reported B-form structure for molecules with similar sequences 2 4. As opposed to the structures which form double strand DNA, the ssdna shows a peak around 280nm and a dip shifted toward 250nm. The closed-end DNA has a shoulder around 280nm, which is due to the nonpaired bases in the loops of the hairpin structure. However, the spectra show that the majority of closed-end DNA have formed a hairpin secondary structure. Figure S3: Circular dichroism measurements of 22 bases ssdna,10 base pair (a) closed-end DNA and (b) free-end DNA. Melting temperature Melting temperature experiments were performed in a Varian Cary 300 Bio UV spectrophotometer with a Peltier thermal controller to determine melting temperature. 20 um S6

7 dsdna were prepared with 120mM Na + phosphate buffer and annealed as for STM-BJ measurements, then heated at a rate of 0.2 C/min from 10 C to 80 C with the absorbance at 260 nm recorded in 60s intervals. Figure S4 shows the melting temperature curve for 10 base pair long closed-end DNA. Fitting the melting temperature curves to a two state thermodynamic model incorporating the intra-molecular nature of the hairpin, yielded a melting temperature of 85±9 C, well above the experimental temperatures of 22 C. Figure S4: Melting temperature curve for 10 base pair closed-end DNA sample. Electromechanical response of closed-end DNA 2-dimensional histograms of logarithm of conductance vs. tip displacement for all closedend DNA molecules are shown in Figure S5. The electromechanical response of these molecules was extracted by fitting the conductance at each tip displacement on the molecular plateau to a Gaussian function. The resulting Gaussian fitted peak values were than fit with a linear function, which gives the slope of ln(conductance) vs tip displacement. The extracted electromechanical responses are listed in Table S1. S7

8 Figure S5: 2-D histograms for (a-d) 10, 14, 18, and 22 base pair closed-end DNA molecules. Table S1: Electromechanical response of closed-end DNA. Slope of natural log of conductance versus tip displacement. Stretching length vs. molecular length fitting S8

9 Figure S6 shows, in more detail, the linear fitting of stretching length to molecular length for closed-end DNA molecules studied here. The red line shown in the figure is a fitting to the spring model, Eq. 2, in the text. Figure S6: Measured Stretching length vs. molecular length for all closed-end DNA molecules measured. Red line is a fitting with spring model discussed in main text. References: (1) Bruot, C.; Xiang, L.; Palma, J. L.; Tao, N. ACS Nano 2015, 9 (1), (2) Kypr, J.; Kejnovská, I.; Renčiuk, D.; Vorlíčková, M. Nucleic Acids Res. 2009, 37, (3) Vorlickova, M.; Kejnovska, I.; Bednarova, K.; Renciuk, D.; Kypr, J. Chirality 2012, 24, (4) Ng, H.-L.; Dickerson, R. E. Nucleic Acids Res. 2002, 30, S9