Biomod/2012/UT/Nanowranglers/Methods

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Computational methods

  1. Sequence Design
    Sequences of the DNA strands were designed using the program CircDesigNA
    (http://cssb.utexas.edu/circdesigna)
    Helical structures of DNA were generated by GIDEON, a program for designing and analyzing complex DNA structures [Cite Birac et al., 2006].
  2. Simulation
    Simulation of kinetics was generated using software KinTek [Cite Johnson, 1996] (Edition v3.0)
    http://www.kintek-corp.com/KGExplorer/DownloadSoftware.php)

Experimental methods

  1. Synthesis and Purification of DNA
    Synthesis:
    Individual DNA strands were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA).
    Purification:
    All unmodified DNA strands, and reporter strand labeled with FAM were purified by denaturing polyacrylamide gel electrophoresis.
    All other modified DNA strands were purified using HPLC by IDT without further treatment.
    Concentrations of DNA solutions were obtained by measuring ultraviolet light absorption at 260 nm using Nanodrop.
  2. Making/Running 12% Denaturing Gel
    Making gel:
    In a 50 mL conical tube, mix 30 mL of 20% Acrylamide in 1X TBE, 20 mL of 1X TBE Denaturing PAGE dilution buffer
    Add 500 μL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
    Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
    Running gel:
    Prepare sample by mixing DNA sample with equal volume of 2X Denaturing Dye
    Denature sample by heating at 90 °C for 5 minutes
    Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
    Clear lanes of excess urea by pipetting
    Pre-run gel at 450 V for 10-20 minutes
    Load samples, run gel at 450 V with a fan
  3. Making/Running 12% or 5% Native Gel
    Making gel:
    For a 12% gel: In a 50 mL conical tube, mix 15 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 25 mL of sterilized deionized water (sd H2O)
    For a 5% gel: In a 50 mL conical tube, mix 6.25 mL of 40% Acyrlamide, 5 mL of 100% Glycerol, 5 mL of autoclaved 10X TBE, 33.75 mL of sterilized deionized water (sd H2O)
    Add 500 µL of 10% APS and 50 µL of TEMED, mix thoroughly by inverting
    Immediately pour gel solution into glass plate assembly, apply a comb, wait for gel to solidify
    Running gel:
    Prepare sample by mixing DNA sample with 1/5 volume of 6X Orange Dye
    Attach gel assembly to PAGE rig, fill top and bottom chambers with 1X TBE
    Clear lanes of excess urea by pipetting
    Pre-run gel at 300 V for 10-20 minutes
    Load samples, run gel at 300 V with a fan
  4. Visualizing/Excising Gel
    Visualizing gel:
    Add 5 µL of 10,000X SYBR Gold Nucleic Acid Gel Stain, 50 mL of 1X TBE to glass dish
    Remove gel from glass plates into dish, let incubate on rotator for 15 minutes
    Scan for Fluorescence Intensity of Storm Imager
    Excising gel:
    Print actual size image of gel
    Move gel on top of saran-wrapped PAGE glass plate, place over image
    Use new razor to cut DNA band, place into 1.7 mL tube
  5. DNA Elution following Denaturing or Native PAGE
    Following Denaturing PAGE:
    Crush isolated DNA with plunger rod until gel is fine
    Suspend gel in 500 µL of 1X TBE
    Place tube on shaking incubator at 80 °C on high for 15 minutes
    Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
    Concentrate DNA by ethanol precipitating flow-through
    Following Native PAGE:
    Crush isolated DNA with plunger rod until gel is fine
    Suspend gel in 1 mL of 1X TBE
    Elute DNA overnight at 37 °C
    Spin down at 14,000 rpm for 2 minutes, transfer supernatant to filter-based centrifuge tube
    Add flow-through to concentrator filter until concentrated down to approximately 50 µL
  6. Ethanol Precipitation
    Add 2.5 V of 100% ethanol, 0.1 V of 3 M NaAc (pH 5.2), 2 µL of glycogen to initial volume of solution
    Vortex, place in -80 °C freezer for 15 minutes
    Spin down at 13,000 rpm for 15 minutes at 4 °C
    Remove supernatant from pellet, wash pellet by adding 1 mL of 70% ethanol
    Spin down at 13,000 rpm for 5 minutes, discard supernatant
    Dry using speed vac for 10-20 minutes
    Resuspend DNA in sterilized deionized water
  7. Forming/Checking Hairpins
    Denature strands by heating to 90 °C for 1 minute then slowly decreasing the temperature to 37 °C at 0.1 °C s-1
    Incubate strands at 37 °C
    Transfer 20 µL of each tube to new tubes
    Add 1/5 V of 6X Orange DNA Loading Dye to each tube
    Load 20 µL of each samples into 12% native gel
    Run gel at 300 V with a fan
    Visualize DNA
  8. Walker System Assembly
    Substrate 1 (S1) Assembly:
    Combine hairpin 02aA, track strand 01Tb, hairpin 03bB
    Anneal by heating system at 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
    Walker (W) Assembly:
    Combine W1-BHQ1, W2-BHQ1
    Anneal by heating mixture to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
    S1-W Assembly:
    Incubate Substrate 1 (S1) and Walker (W) at 37 °C for 3 hours
    Substrate 2 Assembly:
    Combine hairpin 04cA, track strand 01Ta, hairpin 05dB-JOE, hairpin 06eA-TMR, 07f-FAM
    Anneal by heating system to 95 °C for 5 minutes, then cooling to 37 °C at 0.24 °C min-1
    Final System (S1-W-S2) Assembly:
    Incubate S2 and S1-W at 37 °C for 3 hours
  9. Real-time fluorescence measurement
    CHA Experiment:
    Prepare stock solution of RepF:RepQ complex (1:2) by annealing 20 μM RepF, 20 μM RepQ at a 1:2 volume ratio in 1XTMgK buffer
    Fold F1, F2 separately in 1XTMgK buffer by heating to 90 °C for 1 min then slowly decreasing the temperature to 37 °C at a rate of 0.1 °C s-1
    Prepare all samples in 1XTMgK buffer, pre-warm to 37 °C for 15–30 min before mixing
    Prevent loss due to adsorption to plastic by supplementing with 10 μM (dT21)
    Start reaction by adding F1
    Use multichannel pipet to transfer 17 μL of reaction mixtures to a 384-well plate
    Repeat
    Immediately transfer plate to TECAN Safire plate reader for fluorescence measurements
    Set excitation, emission wavelengths to 495 nm (5-nm bandwith), 520 nm (5-nm bandwith), respectively.
    All kinetic measurements are carried out at 37 °C
    Autonomous Walker Experiments:
    Assemble walker using method 8 and with 100 nM track and 85 nM walker
    Use TECAN Safire plate reader to measure fluorescence by setting excitation and emission wavelengths to specified values, with 5-nm bandwidths
    For FAM: 495 nm and 520 nm
    For JOE: 529 nm and 555 nm
    For TAMRA: 559 nm and 583 nm
    Measure with temperature controller set to 37 °C
  10. Buffers Used
    10X TBE:
    Tris base (pH 8.0, F.W. 121.1): 107.8 g (890 mM)
    EDTA (F.W. 372.2): 7.44 g (20 mM)
    Boric acid (F.W. 61.83): 55.03 g (890 mM)
    Adjust to 1 L with sd H2O
    Filter with .0.2 μM filter
    10 X TMgK:
    Tris (pH 8.0): 9.86 g Tris-HCl (F.W. 157.6) + 4.532 g Tris base (F.W. 121.1), adjust to 100 mL with sd H2O (1 M)
    MgCl2 6H2O (F.W. 203.3): 20.33 g, adjust to 100 mL with sd H2O (1 M)
    KCl (F.W. 74.55): 7.455 g, adjust to 100 mL with sd H2O (1 M)
    Using solutions:
    Tris (pH 8.0, 1 M): 10 mL (100 mM)
    MgCl2 (1 M): 4 mL (40 mM)
    KCl (1 M): 15 mL (150 mM)
    sd H2O: to 100 mL
    Filter with .2 uM fliter
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