Supplementary material 1: DNA tracing

Transcription

1 Supplementary material 1: DNA tracing Figure S1:Typical AFM image showing DNA molecules relaxed when deposited with Mg 2+ DNA molecules that appear to have a higher or larger end (indicated by a red arrow on the molecule b), molecules that are only partially inside the image frame (molecule d) or have crossings (molecule c) were not traced and used in the data analysis. Only the molecules that are fully inside the frame such as the molecule (a) were included in the statistics. Supplementary material 2: 251 bp DNA contour length in ultra-high vacuum Figure S2 shows the comparison of contour lengths of 149 DNA molecules deposited using APTES (dark grey bars) and 125 molecules deposited with Mg 2+ (dashed white bars) imaged in ultra-high vacuum (UHV) at 10-9 mbar. Gauss fits of both contour length distributions are also shown. When DNA is deposited with APTES the fit is centered on 82 nm, in excellent agreement with the expected length of B- form DNA. There is not a single APTES-deposited DNA molecule that has its contour length at or close to the A-form DNA contour length (58 nm for 251 bp DNA (20)). The contour length histogram for the 2mM Mg 2+ deposition shows a much broader distribution with the Gauss fit centered at 78 nm. Most of the Mg 2+ deposited molecules are significantly shorter than the predicted B-DNA length would be, some having the contour lengths close or equal to the A-DNA length. S1

2 Figure S2: Upper left AFM image shows 251 bp DNA molecules deposited using APTES; lower left image shows a typical image for the Mg 2+ deposition (100nm scale bar). Histograms are based on lengths measured for 149 DNA molecules deposited on APTES (grey bars), and 125 DNA molecules deposited using Mg 2+ (dashed bars). The graph also shows Gauss fits for the two distributions. The central value of the DNA length on APTES is at 82 nm, which is in excellent agreement with the theoretical B-DNA conformation. The center of the length distribution fit for Mg 2+ is significantly lower at 78 nm, indicating a partial conformational change towards the A-form DNA. Our data shows that even in UHV, at a pressure of 10-9 mbar, where the dehydration of DNA should be nearly complete, the B- to A-form conformational transition that is reported for partial dehydrations upon drying in air, can still be completely inhibited if silane is used, or takes place in an incomplete way if divalent cations are used to precipitate DNA on a surface. This data is in good agreement with data acquired in air, and thus shows that DNA remains in physiological B-form conformation on silane because of its strong binding to the substrate, rather than due to residual water layer covering DNA. DNA deposition for UHV experiments All samples were prepared at room temperature at constant humidity (20-22%). For APTES deposited DNA, the mica surface was first treated with APTES solution. A 10 to 15 μl droplet of 0.05% APTES solution was deposited on freshly cleaved mica for one minute and then rinsed 3 times with 1 ml of ultrapure water. Finally the surface was dried using a gentle flow of compressed nitrogen. The initial DNA solution (the same as the one used for experiments in air) was diluted using ultrapure water to a concentration of 0.5ng/μL. A 20 μl droplet of the solution was deposited on the APTES treated mica surface for 1min.The sample was then rinsed two times with 1 ml of ultrapure water, and dried using a gentle nitrogen flow. S2

3 For Mg 2+ mediated deposition, the DNA solution was first diluted using ultrapure water to a final concentration of between 0.5 and 1 ng/μl. Afterwards MgCl 2 was added to a final concentration of 2 mm. A 20 μl droplet of the solution was then deposited on the freshly cleaved mica and incubated for 1 minute. Finally, the sample was gently rinsed two times with 1 ml of ultrapure water and then gently dried using a compressed nitrogen flow (99.999% nitrogen purity), and rapidly imaged. AFM probes for UHV We have used two types of AFM probes in our experiments to make sure that different tip radii and the resulting tip convolution would not affect the contour length measurement. The first type were ultrasharp diamond-like carbon (DLC) whisker AFM tips (NT-MDT, Russia) whose typical radius is below 3 nm. The typical resonant frequency of DLC tips is 325 khz and a typical spring constant is 40 N/m. A second type was 10 nm radius silicon NSC35 series probes (MikroMasch, Europe). Both tips have varying spring constant values ranging from 5 to 15 N/m and resonant frequencies ranging between khz. UHV AFM instrument and settings Measurements in UHV were done with a home built non-contact AFM operating at room temperature. Imaging was done without annealing to avoid any risk of denaturing DNA and at a pressure of 10-9 mbar. In UHV, the imaging was performed in the non-contact mode where small and negative frequency shift values were used as z-position feedback set point (typically Δf was set to -2 Hz). The oscillation amplitude of the tip was always kept constant and equal to 10 nm using a separate feedback loop. Any eventual electrostatic tip-sample contact potential difference was systematically compensated before measurements by applying an appropriate bias voltage that was determined using the CPD compensation module of Nanonis SPM control software ( the software that was also used to control the overall operation of the UHV operated AFM. S3

4 Supplementary material 3: DNA persistence length L p Figure S3: a) Correlation function of: 500bp DNA deposited on APTES (black squares),1mm MnCl2 (red circles) and 2mM MgCl2 (blue triangles)and 845 bp nicked DNA. b) Correlation function of 845 bp DNA deposited on APTES (black squares) and 2mM MgCl2 in non-nicked (red circles) and nicked (blue triangles) forms. We calculated the persistence length of the 500bp and 845bp DNA molecules, by directly fitting the bond correlation function. We used the 3D equivalent of the eq. 1 in order to fit the data for the APTES deposited molecules, since APTES resulted in kinetic trapping of DNA conformations (10,15,16). For the 500bp molecules deposited with Mn 2+ or Mg 2+ ions the persistence length was comparable (L p~60nm) and slightly higher than the corresponding persistence length of APTES deposited molecules (Table S1). The Persistence length of 845bp DNA was almost identical for nicked and non-nicked DNA and was L p~50nm. (Table 2 in the main text). Table S1: 500bp DNA persistence length 500 bp DNA Number of molecules Persistence Length [nm] APTES ± 5 1mM MnCl ± 5 2mM MgCl ± 5 S4

5 Supplementary material 4: Equilibration time vs. conformational transition: 500 bp DNA Figure S4: Typical AFM images of 500bp DNA imaged in air together with corresponding histograms of contour lengths as a function of relaxation time (upper left AFM image represents DNA deposited on APTES modified mica; upper right image shows DNA deposited using Mg 2+ and rinsed and dried after 10 minutes on mica; lower left AFM image shows DNA deposited using Mg 2+ and rinsed and dried after 17 minutes; lower right image shows the same molecules deposited on Mg 2+ and rinsed and dried after 32 minutes). 100nm scale bar. Histograms are based on lengths measured for 123 DNA molecules deposited on APTES (black bars), 120 molecules deposited using Mg 2+ and rinsed after 10 minutes (white bars), 105 molecules deposited using Mg 2+ and rinsed after 17 minutes (dashed bars), and 110 molecules deposited using Mg 2+ and rinsed after 32 minutes (grey bars). The graph also shows Gauss fits for all the distributions. The central value of the fit is again at 163 nm for DNA length on APTES, in excellent agreement with the B-DNA conformation for 500bp DNA. The centers of length distributions fits for Mg 2+ deposited DNA are at 153 nm for the 10 minutes deposition, 147 nm for the 17 minutes deposition, and 143 nm for the 32 minutes deposition, indicating that the conformational change towards the A-form DNA increases with increased equilibration time. S5

6 Supplementary material 5: 500bp DNA height histograms Figure S5: Height histograms for 500bp DNA molecules deposited with APTES and Mg+ for 17min and 32min Height histograms for traced 500bp molecules show that 32min Mg+ deposited DNA is higher than 17min Mg+ deposited as well as APTES deposited DNA. The increase in the height of the DNA as the function of deposition time can be interpreted as bigger fractions of DNA undergoing B-to A form transition. S6

7 Supplementary material 6: Contour length of APTES deposited 500bp DNA in the presence of salts Figure S6: Typical AFM images of 500bp DNA deposited on 0.1% APTES for 3min with and without the presence of cations in solution. In order to check if the presence of salts in the DNA solution would influence the DNA contour length, we added 2mM MgCl 2 and 1mM MnCl 2 in the DNA solution before depositing them on 0.1% APTES treated mica. The average contour length of 500bp DNA stayed unaffected by the presence of salts in the solution and was the same as for the APTES deposited DNA (Table S2). Table S2: 500bp on 0.1% APTES Number of molecules Contour Length [nm] Control ± 8 + 2mM MgCl ± 5 + 1mM MnCl ± 5 S7

8 Supplementary material 7: End-to-end distance distribution for 500bp DNA Figure S7: Histograms of end-to-end shortest distance for the 500bp DNA imaged in air. Histograms are based on values measured on the same molecules as those whose contour length and typical AFM images are shown in Figure 2 in the article. The distribution of end-to-end distances for DNA deposited using APTES (grey bars) appears randomly spread over a very large range (10 nm to 140 nm) agreeing well with the 3D-to-2D projection deposition, while the end-to-end distributions of Mn 2+ (white bars) and Mg 2+ (dashed bars) deposited molecules is much narrower suggesting a relaxation on the surface following the deposition. In Figure S7 we have examined the polymer behavior of DNA as a function of different deposition methods by analyzing the end-to-end distance for the same molecules whose contour length is presented in Figure 2 in the article. Again, dark grey bars correspond to APTES deposited DNA, white bars to Mndeposited DNA, and dashed bars to Mg 2+ -deposited DNA. This time, the end-to-end distance of APTES deposited DNA (dark grey bars) varies between 10 nm and 140 nm, and no peak value can be singled out. On the other hand both Mg (dashed bars) and Mn (white bars) deposited DNA molecules show a more constant end-to-end distance value whose average value is 116 nm and 123 nm respectively for APTES deposited DNA the average value is only 67 nm. The end-to-end distance histograms are in good agreement with previous studies (10,16): lesser end-to-end distance variability that is observed for Mgand Mn 2+ -deposited DNA agrees well with DNA molecules being able to relax into a 2D equilibrium configuration, while APTES-deposited molecules remain in a variety of trapped configurations and cannot further evolve. The smaller value of the end-to-end distance of APTES-deposited DNA agrees well with a smaller contour length exponent for 3D-to-2D projected polymer, while the larger average end-to-end distance for Mg 2+ deposited DNA agrees well with the larger contour length exponent of a 2D polymer (10,38,39). S8

9 Supplementary material 8: Effect of nicked sites on conformational transition Figure S8: Typical AFM images of 845bp DNA deposited with Mg 2+ together with the corresponding Histograms of contour length (upper left AFM image shows intact DNA molecules; lower left image shows nicked DNA). 100nm scale bar. Histograms are based on lengths measured for 100 DNA molecules that were nicked in three positions (darker grey bars) and for 100 molecules that were not nicked (lighter grey bars). The graph also shows Gauss fits for both distributions. The center of the fit of the nicked DNA s length distribution is at 256 ± 11 nm, while the fit center for the non-nicked DNA is at 269 ± 9 nm. When DNA is bound with Mg+, nicking induces a more pronounced transition from B- to A-form DNA, which is evidenced by shorter contour length values. S9

10 Supplementary material 9: Reproducibilty of TER spectra In order to demonstrate the reproducibility of the TER spectra, in Figure S9 we show three spectra collected from the same strand at position 8. Figure S9: 3 individual TER spectra collected from a single DNA strand at position 8 Figure S10 shows 10 different spectra collected at position 8 of 10 randomly chosen strands. Figure S10: TER spectra collected from 10 randomly chosen strands at position 8 S10

11 Supplementary material 10: Spatial resolution of TERS The spatial resolution of TERS depends on the experimental setup (tip, illumination geometry, substrate), as well as on the analyte. To measure the resolution of TERS for our experimental system we acquired 64 TER spectra over a 500 nm line, crossing three DNA strands, with a step size of 7.8 nm. Figure S11 shows the height profile (green plot) measured along the selected line and the corresponding total TER intensity in the spectral range from 1750 cm -1 to 580 cm -1 (black plot). The evident correspondence between the height and the TER intensity allows to estimate the lateral resolution of TERS, based on the intensity cross-section. Full widths at half maximum (FWHM) of the Gaussians fitted to the TERS intensity profiles of the DNA strands were in the range between 9.8 nm and 22.3 nm. A more rigorous estimation of the lateral resolution would require 2D hyperspectral imaging of individual strands, which is difficult and has not yet been reported in the literature. Our results confirm that the resolution is on the order of nm, which is critical for the adequate choice of the number of positions along a single DNA molecule. Figure S11: (A) AFM topography of 500 bp DNA fixed on mica. (B) Height profile extracted along the white line marked in figure A. (C) Total intensity of TERS in the spectral range 1750 cm cm -1, acquired along the same line with a step size of 7.8 nm (64 spectra were collected along a 500 nm line). The intensity and height profiles match very well. Lateral spatial resolution of TERS is estimated based on FWHM of Gaussians fitted to the peaks in the intensity profile. S11

12 Supplementary material 11: Carbon contamination Figure S12 shows a typical example of sample decomposition due to illumination by intense laser light. Figure S12: TER spectra collected from 500 bp DNA, after a continuous exposure of the tip to the laser light lasting 4.4, 8.6, 16 and 32 mins. Time-increase of spectral features typical to carbon contamination is clearly visible Supplementary material 12: Stretching and breathing modes of DNA bases in TER spectra Figure S13: Selected TER spectra collected from 500 bp DNA, with wavenumbers related to stretching and breathing modes of DNA bases marked S12