Abstract
We report a surface passivation method based on dichlorodimethylsilane (DDS)–Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS–Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.
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Acknowledgements
We thank B. Leslie, S. Syed, T. Ngo, F. Tritschler and Q. Xu for their helpful comments in data analyses and manuscript preparation; J. Guan, C. Yu and S. Granick for the helpful discussions and for generously letting us use the oxygen plasma cleaning machine and Piranha fume hood; T. Le of the H. Kim laboratory at Georgia Institute of Technology and H.R. Koh of the S. Myong laboratory at the University of Illinois for performing independent tests of our protocols; and W. Cheng (University of Michigan) for providing the Alexa 594–labeled protein G B1 domain sample. This work was supported by a US National Science Foundation grant (PHY 1430124 to T.H.) and US National Institutes of Health grants (GM065367 and AG042332 to T.H. and GM60819 to S.A.W.). T.H. is also supported by the Howard Hughes Medical Institute.
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B.H. and T.H. designed the project; B.H., K.Y.H., R.Z., H.K. and X.S. performed the experiments; S.C.A. and S.A.W. provided the S4-rRNA complex and three other ribosomal protein samples; B.H., K.Y.H., R.Z., H.K., X.S., A.J., D.S., V.A. and T.H. analyzed the data; and B.H., K.Y.H., R.Z., H.K., X.S. and T.H. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Nonspecific binding of 10 nM Cy5-labeled DinB on nine different surfaces, measured by the number of DinB spots over an imaging area of 2,500 μm2.
Free DinB was washed out using 50 μl imaging buffer after incubation. Numbers on the x-axis indicate different surfaces: 1) PEG; 2) PEG-siloxane-Tween-20 (ref. 12); 3) PEG-siloxane-F-127; 4) siloxane-Tween-20; 5) siloxane-F-127; 6) PEG-DDS-Tween-20; 7) PEG-DDS-F-127; 8) DT20*; 9) DDS-F-127 (ref. 13). Error bars indicate s.d. DT20 was chosen for its best passivation performance as well as lowest preparation time and agent cost (see online Methods).
Supplementary Figure 2 The background fluorescence spot counts in both Cy3 and Cy5 channels on the PEG and DT20 surfaces.
The laser power was adjusted for visualization of single organic fluorophores. The appearing spots are from the impurities on the surface and their intensity is comparable to single organic fluorophores. The scale bars indicate 9 μm.
Supplementary Figure 3 The higher surface biotin density on the PEG surface and its effect on nonspecific binding.
(a) Fluorescence images of biotin-bound Cy5-labeled NeutrAvidin on both surfaces. To achieve the similar surface density of NeutrAvidin, a 5 times higher concentration of Cy5-labeled NeutrAvidin was used for the DT20 surface than for the PEG surface, indicating the PEG surface has a higher surface density of biotin groups than the DT20 surface. The scale bars indicate 9 μm. (b) Passivation capacity of the PEG surface at different surface NeutrAvidin densities, measured by the average number of spots over an imaging area of 2500 μm2 with 50 nM DinB. The 1 × NeutrAvidin stock contains 0.2 mg/mL NeutrAvidin in T50 buffer (pH 8.0). Error bars indicate s.d.
Supplementary Figure 4 Fluorescence images of 50 nM Cy5-labeled DinB nonspecifically bound on the DDS-BSA and DT20 surfaces.
Here the DDS-BSA surface refers to a DDS-coated surface that was non-specifically passivated by BSA molecules. The only difference between these two surfaces is that the DT20 surface was subjected to an additional Tween-20 passivation treatment. Other conditions, such as the BSA incubation time and concentration were the same. It suggests that the passivation effect is due to Tween-20 rather than the BSA molecules on the surface. The scale bars indicate 9 μm.
Supplementary Figure 5 Fluorescence images of nonspecifically bound Cy5-labeled DinB in the same area of the DT20 surface without embedded BSA or NeutrAvidin.
100 nM DinB was injected in the flow chamber and washed away after 3 min incubation. And then image (a) was recorded. The laser was kept on until all the fluorescent spots in image (a) were photobleached. Another 100 nM DinB was injected and washed away after 3 min incubation. The same area could be imaged for the second time, as shown in image (b). Images (a) and (b) are false-colored in green and red respectively and their overlay is shown in image (c). Only a few colocalized spots marked by white circles were observed. The scale bars indicate 9 μm.
Supplementary Figure 6 Fluorescence images of nonspecifically bound Rep-Cy5 on the DT20 and PEG surfaces at 5 nM concentration.
The scale bars indicate 9 μm.
Supplementary Figure 7 STED images of surface-bound proteins on the PEG and DT20 surfaces.
(a,b) STED images of the surface-bound streptavidin-Alexa 594 on both surfaces. (a) Fluorescence images of the surface (DT20) immobilized streptavidin-Alexa 594 by confocal and STED microscope before the injection of 100 nM fluorescently labeled molecules. (b) Non-specific binding tests of the DT20 and PEG surfaces after washing out 100 nM streptavidin-Alexa 594 by STED microscope. (c) Non-specific binding tests of the DT20 and PEG surfaces in the presence of 50 nM Alexa 594-labeled protein G B1 domain (PG-A594) by confocal and STED microscope.
Supplementary Figure 8 Fluorescence images of nonspecifically bound Cy3-labeled ssDNA (34 nt) at 160 nM and pH 8.0.
The scale bar indicates 9 μm.
Supplementary Figure 9 Nonspecific binding of 500 nM Abberior Star 635–labeled ssDNA (18 nt) at different MgCl2 concentrations on the DT20 surface.
Error bars indicate s.d.
Supplementary Figure 10 Typical fluorescence intensity and FRET efficiency traces of HJ dynamics at 40 mM Mg2+ on the PEG and DT20 surfaces.
The transitions between the two FRET efficiency levels represent the transitions of HJ between its two stacked states.
Supplementary Figure 11 FRET histograms of single HJs at different Mg2+ concentrations.
The three peaks in the histograms correspond to, from low to high in the FRET efficiency, the Cy3 (donor) only molecules and the two stacked conformations of dual labeled HJ. The peak positions are denoted as X1, X2 and X3 while the ratio between peak 2 and 3 is denoted as Y2/Y3. The peak positions and the ratio between the low and high FRET peaks are identical between the two surfaces.
Supplementary Figure 12 The dynamics of the ribosomal protein S4-rRNA complex.
(a) The schematics showing the FRET changes caused by the docking and undocking dynamics of the ribosomal protein S4-rRNA complex. (b) FRET histograms of the ribosomal protein S4-rRNA complex dynamics at 20 mM MgCl2. The average dwell times on the high FRET state are 4.6 s for the PEG surface and 5.5 s for the DT20 surface while the average dwell time on the low FRET state is 1.5 s for both surfaces. Close agreements on the histograms and average dwell times indicate the unaltered dynamics on the DT20 surface.
Supplementary Figure 13 The dissociation curve of biotinylated BSA on the DT20 surface.
Fluorophore-labeled biotinylated DNA was immobilized through NeutrAvidin, which was, in turn, immobilized through biotinylated BSA molecules on the surface. Any free fluorophore-labeled DNA was washed away after this immobilization step. Average surface spot counts over an imaging area of 2500 μm2 were recorded at different time points up to 2 hours. To avoid the photobleaching issue, imaging buffer was used and the same area was never exposed more than once. The dissociation of biotinylated BSA from the surface can be monitored by the decrease in the surface spot counts of fluorophore-labeled DNA. Error bars indicate s.d.
Supplementary Figure 14 The durability of the Tween-20 passivation layer.
Non-specific binding tests of 500 nM Cy3-labeled ssDNA (34 nt) over an imaging area of 2500 μm2 were conducted at different time points after the Tween-20 passivation treatment was done. Up to 5 hours after passivation, the Tween-20 layer is still effective. Error bars indicate s.d.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–14 and Supplementary Protocol (PDF 2826 kb)
DT20 Cy3 blank channel
Cy3 background of a blank channel on the DT20 surface with imaging buffer (AVI 19206 kb)
500 nM Atto 647N–labeled ssDNA (18 nt)
Fluorescence images of nonspecifically bound Atto 647N–labeled ssDNA (18 nt) on the DT20 surface (AVI 19206 kb)
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Hua, B., Han, K., Zhou, R. et al. An improved surface passivation method for single-molecule studies. Nat Methods 11, 1233–1236 (2014). https://doi.org/10.1038/nmeth.3143
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DOI: https://doi.org/10.1038/nmeth.3143
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