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Introduction

Protocol

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Acknowledgements

Materials

References

Biochemistry

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Published: February 7th, 2021

DOI:

10.3791/62112

1Department of Chemistry, Tufts University, 2Department of Physics, Wesleyan University, 3Wyss Institute at Harvard University

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT↓ATC (where ↓ is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Restriction endonucleases (REases) are enzymes that effect sequence-specific double-strand breaks in DNA. The discovery of REases in the 1970s led to the development of recombinant DNA technology, and these enzymes are now indispensable laboratory tools for genetic modification and manipulation1. Type II REases are the most widely used enzymes in this class as they cleave DNA at a fixed location either within or near their recognition sequence. However, there is a great deal of variation among the Type II REases, and they are divided into several subtypes based on particular enzymatic properties rather than being classified according to their e....

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1. General information

  1. Oligonucleotide design
    NOTE: The 60 base-pair (bp)-long DNA substrate is formed from a pair of complementary oligonucleotides with a duplex melting temperature of 75 °C in 100 mM NaCl.
    1. Order one oligonucleotide synthesized with a single 5' biotin modification and the other with a 5' thiol modification (with a six-carbon spacer). Place the recognition site in the center of the duplex region.
      NOTE: The oligonucleotide sequences for use with EcoRV ar.......

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The flow cell is directly coupled to a high numerical aperture oil-immersion 60x magnification objective on an inverted microscope equipped with laser illumination for through-objective TIRF imaging (Figure 5A). After introducing the DNA substrate and washing away excess DNA and quantum dots, there are typically thousands of individual quantum dots in a field of view (Figure 5B). These quantum dots are stably attached to the glass surface, and they do not underg.......

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The DNA substrate for this assay is labeled with a quantum dot using a two-step reaction scheme using sulfo-SMCC. This bifunctional crosslinker consists of an NHS ester moiety that can react with a primary amine, and a maleimide moiety that can react with a sulfhydryl group20. The thiolated oligonucleotides used to prepare the substrate are shipped in their oxidized form. It is important to reduce and purify them, as described, before proceeding with the coupling procedure, or the efficiency of th.......

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This work was supported by Award Number K12GM074869 to CME from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

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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

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