The overall goal of this new DNA-based model system is to understand the fundamental properties of a class of semiflexible polymers, which also describes the behavior of a vital class of biopolymers. This new method allows us to study key questions in the field of soft metaphysics, specifically, it allows to explore the behavior of semiflexible filaments and their arrangements into networks. The main advantage of this technique is that the mechanical properties of intravisive filaments are experimentally accessible and can be precisely tuned.
Semiflexible polymers are defined by the special properties of the filaments which were previously only treated in the theoretical framework due to the lack of a tunable experimental systems. Though this method provides insight into the defining quantity of semiflexible polymers, it can also be applied as a new hydrogel to study cell migration in matrices of different stiffness's without changing the under laying mesh size. The smallest repetitive unit in the nanotube can be thought of as a unit ring.
Several of these unit rings stack during hybridization. This leads to the formation of elongated nanotube's. To begin, re-suspend lyophilized oligonucleotides in purified water and follow the further re-suspension steps given in the corresponding manual from the company.
Adjust the amount of water to obtain a final concentration of 200 micromolar. Determine the concentration's of the single DNA strands to verify the values given by the distributor since the exact stoichiometry is crucial for the assembly process. Using a micropipette, mix the DNA strands each of the same concentration in a container suitable for the thermocycler to form the desired DNA and helix tubes.
Hybridize the DNA and helix tubes in a thermocycler using the parameters found in the text protocol. Store the hybridized DNA and helix tubes for up to 3 weeks at 4 degrees celsius without detectable degradation. Before performing rheology, carefully dilute the sample to the desired final concentration using the sample buffer if necessary.
Choose an appropriate geometry for the dynamic shear rheometer. Then, load the sample on the rheometer. To prevent potential evaporation effects, passivate the air water interface of the sample and environment through 1 of 3 techniques.
Either surround the sample with 2.5 milliliters of the sample buffer bath. Or, add a surfactant just below the micelle concentration to the sample. Alternatively, use the hamilton syringe to surround the sample with lipid to avoid direct contact of the sample with air.
Seal the sample chamber using a cap equipped with wet sponges. And, if possible use an additional water bath to further suppress evaporation. Start the measurement using the rheometer specific software at room temperature allowing the sample to equilibrate for 2 hours at room temperature.
The equilibration time is crucial to allow the thermoflect system to a complete isotropic randomized state since this is the only confirmation allowing comparison between the different measurements. Following measurement, process the data as described in the text protocol. Prepare 8 aliquots of 12 microliters for each sample using 1 to 4 micromolar DNA per strand, 12.5 millimolar magnesium chloride, and 1x nucleic acid gel stain.
Also, make a negative control with no DNA, but include the nucleic acid gel stain. Transfer the aliquots to a real time PCR plate and seal with adhesive film. Centrifuge the plate at 100 times G for 1 minute at room temperature to remove the gas bubbles.
It's critical to remove the gas bubbles because it can expand during measurements and the samples cannot be analyzed. Following centrifugation, load the sealed plate into a real time quantitative PCR system. Program an annealing program to denature the DNA at ninety degree celsius for 10 minutes and drop the temperature quickly to seventy two degrees celsius.
Then the temperature should decrease slowly by 0.5 degrees celsius every thirty minutes. After the signal has decayed, accelerate the temperature ramp. Make sure that the lid of the cycler heats to at least one hundred degrees celsius to prevent the condensation of evaporated sample on the adhesive film.
During the assembly, excite the samples with an argon ion laser at four hundred eighty eight nanometers and detect the fluorescence at five hundred fifty nanometers when using a CY3 dye. For other dyes, choose an appropriate excitation emission combination. Measure the fluorescence signal as often as possible to increase the overall signal quality using the built in camera.
Representative raw data of a rheological measurement are shown revealing the elasticity of the network. Shown here is the time sweep during equilibration time. This plot reveals the strain dependent behavior of the network.
In this plot, the frequency dependent behavior of the network is evident. At this point the values at a specific frequency are chosen. The values for the specific DNA helix tube population were plotted against the concentration.
This process was repeated for all the DNA and helix tubes. The slope was then extracted in a log logged plot yielding the power law exponent. Finally, the values were replotted against the persistent length for each measured concentration.
These graphs allow extraction of the scaling of the elastic plateau modulus with respect to the concentration and persistence length. After development, this technique paved the way for researchers in the field of soft matter and polymer physics to explore the effect of the filament stiffness for the class semiflexible polymers. Following this procedure, additional effects such as the recitation of the entangled networks, crossing networks in general, and structure formation process can be addressed.
After watching this video you should have a good understanding on how to hybridize DNA nanotubes and how to measure the rheological properties of their networks.
