Undergraduate DNA nanotechnology research group from the University of Texas at Austin

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Computational methods

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 [4].
Simulation
Simulation of kinetics was generated using software KinTek [26] (Student Edition v3.0)

(http://www.kintek-corp.com/KGExplorer/DownloadSoftware.php)

Experimental methods

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
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
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
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
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
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
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
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
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
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

10X 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

Biomod/2012/UT/Nanowranglers/Methods

Computational methods

Experimental methods

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