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JoVE Journal Biochemistry
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Biochemistry

Published: February 7, 2021 doi: 10.3791/62112
Automatic Translation
1,2, 1, 2, 2, 1,3
1Department of Chemistry, Tufts University, 2Department of Physics, Wesleyan University, 3Wyss Institute at Harvard University

Materials

Name Company Catalog Number Comments
3-aminopropyltriethoxysilane (APTS) Sigma Aldrich 440140-100ML Store in dessicator box
5 Minute Epoxy Devcon 20845 use for sealing the microfluidic device
acetone Pharmco 329000ACS use for cleaning coverslips
bath sonicator Fisher Scientific CPXH Model 2800 catalog number 15-337-410
Beaker, glass, 100 mL
Benchtop centrifuge
Biotin-PEG-Succinimidyl Valerate (MW 5,000) Laysan Bio BIO-SVA-5K Succinimidyl valerate has a longer half-life than succinimidyl carbonate
biotinylated oligonucleotide Integrated DNA Technologies custom - see protocol for design considerations Request 5' Biotin modification and HPLC purification
Bovine Serum Albumin (BSA) VWR 0903-5G prepare a 10 mg/mL solution (aq) and heat to 95 °C before using
Centri-Spin-10 Size Exclusion Spin Columns Princeton Separations CS-100 or CS-101 used to purify thiolated oligonucleotides after reducing the disulfide bond
Centrifuge tubes 1.5. mL
Coverslips, 1-inch square glass
coverslip holders
diamond point wheel Dremel 7134 use for drilling holes in quartz flow cell topper
dithiothreitol (DTT) Thermo Scientific A39255 No-Weigh Format, 7.7 mg/vial
drill press rotary tool workstation stand Dremel 220-01 facilitates quartz drilling
EcoRV (REase used to generate example data) New England Biolabs R0195T or R0195M Use 100,000 units/mL stock to avoid adding excess glycerol Check REBASE for suppliers of other REases
ethanol various CAS 64-17-5 denatured or 95% are acceptable, use for cleaning coverslips
Ethylenediaminetetraacetic acid (EDTA) Sigma Aldrich EDS BioUltra, anhydrous, store in dessicator box
Flea Micro Spinbar Fisherbrand 14-513-65 3 mm x 10 mm size to fit beneath coverslip rack
fluorescein Acros Organics 17324 use to make experimental buffers
gravity convection oven Binder 9010-0131
handheld rotary multitool Dremel 8220 use for drilling holes in quartz flow cell topper
ImagEM X2 EM-CCD Camera Hamamatsu C9100-23B air cooling is adequate for this experiment, use HCImage software or similar to control
Imaging spacer, double-sided, adhesive
Jar, glass with screw cap, (approximately 50 mm diameter by 50 mm high)
magnesium chloride hexahydrate Fisher Bioreagents BP214-500 use to make experimental buffer with magnesium
MATLAB software Data analysis
metal tweezers Fisher Brand 16-100-110
methoxy-PEG-Succinimidyl Valerate (MW 5,000) Laysan Bio M-SVA-5K Both PEGs should have the same NHS ester so that the rate of reaction is consistent
microcentrifuge Eppendorf 5424
multiposition magnetic stirrer VWR 12621-022
N-cyclohexyl-2-aminoethanesulfonic acid (CHES) Acros Organics AC20818 CAS 103-47-9, use to make CHES buffer
orbital shaker and heater for microcentrifuge tubes Q Instruments 1808-0506 with 1808-1061 adaptor for 24 x 2.0 mL or 15 x 0.5 mL tubes
Parafilm
PE60 Polyethylene tubing (inner diameter 0.76 mm, outer diameter 1.22 mm) Intramedic 6258917 22 G blunt needles are a good fit for this tubing size
Phosphate-Buffered Saline (PBS) 10x Sigma Aldrich P7059 Use at 1x strength
potassium hydroxide VWR Chemicals BDH BDH9262 use a 1 M solution to clean coverslips
Qdot 655 ITK Amino (PEG) Quantum Dots Invitrogen Q21521MP
Quartz Slide, 1 inch square, 1 mm thick Electron Microscopy Sciences 72250-10 holes must be drilled in the corners for inlet and outlet tubing insertion
reinforced plastic tweezers Rubis K35a use for handling coverslips and building microfluidic device
SecureSeal Adhesive Sheets Grace Biolabs SA-S-1L cut to form spacer for microfluidic device
Single channel syringe pump for microfluidics New Era Pump Systems NE-1002X-US fitted with a 50 mL syringe and a 22 G blunt needle
Slide-a-Lyzer MINI Dialysis Devices, 10 kDa MWCO, 0.1 mL Thermo Scientific 69570 or 69572 used for buffer exchange during quantum dot coupling to DNA
sodium bicarbonate EMD Millipore SX0320 use to make buffer for surface functionalization; 100 mM, pH 8
sodium chloride Macron 7581-12 use to make experimental buffers
Sodium phosphate dibasic solution (BioUltra, 0.5 M in water) Sigma Aldrich 94046 use to make 100 mM sodium phosphate buffer
Sodium phosphate monobasic solution (BioUltra, 5M in water) Sigma Aldrich 74092 use to adjust pH of 100 mM sodium phosphate buffer
Streptavidin from Streptomyces avidinii Sigma Aldrich S4762 dissolve at 1 mg/mL and store 25 mL aliqouts at -20 ?
Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) Thermo Scientific A39268 No-Weigh Format, 2 mg/vial
Syringe fitted with blunt 21 G needle
Syringe pump
thiolated oligonucleotide Integrated DNA Technologies custom - see protocol for design considerations Request 5' Thiol Modifier C6 S-S and HPLC purificaiton
TIRF imaging system with 488 nm laser illumination various custom built
Tris -HCl Research Products International T60050 use to make experimental buffers
Tris base JT Baker 4101 use to make experimental buffers
Tween-20 Sigma P7949 use to make blocking buffer
Ultrapure water
vortex mixer VWR 10153-842
Wash-N-Dry Coverslip Rack Electron Microscopy Sciences 70366-16 used for surface functionalization of coverslips

DOWNLOAD MATERIALS LIST

References

  1. Roberts, R. J. How restriction enzymes became the workhorses of molecular biology. Proceedings of the National Academy of Sciences of the United States of America. 102 (17), 5905-5908 (2005).
  2. Loenen, W. A., Dryden, D. T., Raleigh, E. A., Wilson, G. G., Murray, N. E. Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Research. 42 (1), 3-19 (2014).
  3. Roberts, R. J., Vincze, T., Posfai, J., Macelis, D. REBASE--a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Research. 43, Database issue 298-299 (2015).
  4. Bonnet, I., et al. Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA. Nucleic Acids Research. 36 (12), 4118-4127 (2008).
  5. Biebricher, A., Wende, W., Escude, C., Pingoud, A., Desbiolles, P. Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers. Biophysical Journal. 96 (8), 50-52 (2009).
  6. Blainey, P. C., et al. Nonspecifically bound proteins spin while diffusing along DNA. Nature Structural & Molecular Biology. 16 (12), 1224-1229 (2009).
  7. Huang, C. -F., et al. Direct visualization of DNA recognition by restriction endonuclease EcoRI. Journal of Experimental & Clinical Medicine. 5 (1), 25-29 (2013).
  8. Palma, M., et al. Selective biomolecular nanoarrays for parallel single-molecule investigations. Journal of American Chemical Society. 133 (20), 7656-7659 (2011).
  9. Reinhard, B. M., Sheikholeslami, S., Mastroianni, A., Alivisatos, A. P., Liphardt, J. Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes. Proceedings of the National Academy of Sciences of the United States of America. 104 (8), 2667-2672 (2007).
  10. van den Broek, B., Noom, M. C., Wuite, G. J. DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway. Nucleic Acids Research. 33 (8), 2676-2684 (2005).
  11. Gambino, S., et al. A single molecule assay for measuring site-specific DNA cleavage. Analytical Biochemistry. 495, 3-5 (2016).
  12. Floyd, D. L., Harrison, S. C., van Oijen, A. M. Analysis of kinetic intermediates in single-particle dwell-time distributions. Biophysical Journal. 99 (2), 360-366 (2010).
  13. Loparo, J. J., van Oijen, A. Single-molecule enzymology. Handbook of Single-Molecule Biophysics. Hinterdorfer, P., Oijen, A. , Springer. New York, NY. 165-182 (2009).
  14. Taylor, J. D., Badcoe, I. G., Clarke, A. R., Halford, S. E. EcoRV restriction endonuclease binds all DNA sequences with equal affinity. Biochemistry. 30 (36), 8743-8753 (1991).
  15. Pingoud, A., Jeltsch, A. Structure and function of type II restriction endonucleases. Nucleic Acids Research. 29 (18), 3705-3727 (2001).
  16. Tanner, N. A., Loparo, J. J., van Oijen, A. M. Visualizing single-molecule DNA replication with fluorescence microscopy. Journal of Visualized Experiments: JoVE. (32), e1529 (2009).
  17. Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C., van Oijen, A. M. Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay. Journal of Visualized Experiments: JoVE. (37), e1689 (2010).
  18. Baldwin, G. S., Vipond, I. B., Halford, S. E. Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease. Biochemistry. 34 (2), 705-714 (1995).
  19. Winkler, F. K., et al. The crystal-structure of Ecorv endonuclease and of Its complexes with cognate and non-cognate DNA fragments. EMBO Journal. 12 (5), 1781-1795 (1993).
  20. Sioss, J. A., Stoermer, R. L., Sha, M. Y., Keating, C. D. Silica-coated, Au/Ag striped nanowires for bioanalysis. Langmuir. 23 (22), 11334-11341 (2007).
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Cite this Article

Etson, C. M., Todorov, P., Shatery… More

Etson, C. M., Todorov, P., Shatery Nejad, N., Shrestha, N., Walt, D. R. Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage. J. Vis. Exp. (168), e62112, doi:10.3791/62112 (2021).

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