Name: dna nanotubes as a versatile tool to study semiflexible polymers
Uploaded: 2017-10-25T14:00:00
Description: Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

We acknowledge funding by DFG (1116/17-1) and the Leipzig School of Natural Sciences &#34;BuildMoNa&#34; (GSC 185). This work has been supported through the Fraunhofer Attract project 601 683. T. H. acknowledges funding from the European Social Fund (ESF-100077106).

1. Preparation of n HTs
NOTE: Here, n denotes the number of different single DNA strands involved in the formation of the helix tubes of a certain size. For n=8, eight different single DNA strands make up a unit ring.
<ol>
	<li>Purchase DNA sequences (HPLC purity grade or higher) from a suitable DNA synthesis service or perform high-quality synthesis and purification (exemplary sequences given in Table 1).</li>
	<li>Resuspend lyophilized oligonucle...

To obtain properly formed networks, assembling the DNA nanotubes is a crucial step. Errors during the synthesis process negatively impact the tube quality; therefore, it is recommended that HPLC or a more stringent process be used to purify the oligonucleotides. Since the formation of discrete rather than aggregated DNA nanotubes, as well as their length distribution, depends upon the equimolar stoichiometry of the n constituent oligonucleotides within the set, it is necessary to remeasure the concentrations of ...

Due to the complex behaviors enabled by their unique mechanical properties, semiflexible polymers are fundamental building blocks of living matter. In contrast to flexible polymers, semiflexible polymers adopt an outstretched configuration due to their non-vanishing backbone stiffness while still remaining subject to strong thermal fluctuations1. Thus, purely stochastic models cannot be applied to their behaviors, as with the extremes of fully flexible or rigid polymers. The so-called worm-like chain model2,3,4 was developed to quantify this stiffness via the lp, which is the decay constant of the tangent-tangent correlation along the filament4. If lp is comparable to the contour length (lc) of the filament, the polymer is considered semiflexible1. Analogous to the poles of a tent, their arrangements in networks or bundles stabilizes the entire collective system at low volume fractions, leading to unusual viscoelastic properties5,6,7,8,9. These structures provide high elasticities at large mesh sizes10, maintaining mechanical integrity while still facilitating diffusive and active transport processes. This property is especially suitable for biological systems such as the cytoskeleton or the extracellular matrix, but it is also widely used in food engineering1,11,12.
Going beyond their significance to living matter, it is crucial to comprehensively examine the physical properties of these structures in order to have the tools to develop biomimetic materials or novel hydrogels. In terms of semiflexible polymers, this implies the systematic determination of the collective properties of networks resulting from single-filament properties such as lp and the development of a descriptive theoretical framework. In pioneering studies, the cellular biopolymer actin was established as a model system for semiflexible polymers and is still widely considered the gold standard5,13,14,15,16,17. However, exhaustive studies are limited with this system since they are bound to the inherent properties of this protein. Various theoretical approaches have aimed at building a description of the non-trivial mechanical behaviors at the single-filament level and have led to notably different scaling predictions for the dependence of the linear elastic plateau shear modulus, G0 (i.e., the &#34;elasticity&#34; of the network), with respect to concentration (c) and lp6,7,13,14,15,18,19,20,21,22,23. While the concentration scaling is readily accessible in experiments with actin-based or other model systems and while theoretical predictions have been rigorously verified13,16,24,25, the scaling with respect to lp has remained experimentally inaccessible. This, however, is a major limitation since lp is also an independent variable that is the defining quantity of semiflexible polymers.
This central, natural limitation imposed by the fixed lp of actin or other biologically-derived polymers such as collagen has recently been resolved by employing tile-based DNA tubes, which are tunable in their mechanical properties9,26,27,28. Slight variations in the architectures of the tubes (e.g., different numbers of constituent DNA strands within the unit ring) yield distinct values for lp, which can be evaluated via fluorescence microscopy, either by analyzing one fluctuating tube or by evaluating the curved configurations of several adhered tubes, as described previously9,28. These analyses revealed that lp values of the different tube populations vary over more than one order of magnitude and that different evaluation techniques yield consistent results9,28.
Surprisingly, the overall scaling of the linear elastic plateau shear modulus G0 with respect to the concentration and lp has been reported to be inconsistent with all previous theoretical approaches9, in particular demonstrating a much stronger than predicted dependence upon lp. These findings emphasize the value of a new model system to study the central properties of semiflexible polymers. Employing n-helix DNA tubes dramatically broadens the scope of these investigations. Not only can lp be freely varied without changing the basic material, but the inherent programmable nature of DNA can enable the systematic examination of additional elements, such as crosslinks or kinetic switching processes. Additionally, these tubes are soluble in water and, in contrast to most proteins, stable in adequate pH and ionic conditions for several weeks, without detectable degradation9.
To assemble these tubes, a discrete set of DNA oligonucleotides is used, each of which contains two domains that share complementary base sequences to two neighboring strands (due to the specific sequences, a single strand cannot form structures such as hair pins). The complementary sequences hybridize in a cyclic manner, forming enclosed, half-overlapping rings of n interconnected double-helical segments (Figure 1A and B). These rings form at a discrete diameter (Figure 1C), and their half-overlapping configuration exposes axial sticky ends complementary to the sticky ends of another ring. This selective addition of matching oligonucleotides triggers a stacking of the rings, leading to the effective polymerization of filamentous DNA helix tubes of size n (nHT). Their contour lengths typically measure several microns in length, and their length distribution is comparable to that of actin filaments9,26,27,28. It has been shown for similar DNA nanotubes that they indeed exhibit polymerization kinetics similar to those of actin filaments and microtubules29. Depending on the number n of individual DNA strands making up the basic ring structure, the nHT architecture, as well as its circumference and diameter, can be controllably varied. Using more DNA strands increases the circumference of the rings/tubes, and the corresponding architectural change shifts the mechanical properties to higher lp values (Figure 1C), corresponding to a higher rigidity. On the mesoscopic scale, these larger lp values translate into less bent conformations due to the higher stiffness (Figure 1D and E).

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			<td style="width:123px;"></td>
			<td style="width:180px;">For sequences see Table 1</td>
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			<td style="width:180px;">For sequence see Table 1</td>
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			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
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			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
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			<td style="width:196px;">Applied Biosystems</td>
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			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Rheometer</td>
			<td style="width:196px;">TA Instruments</td>
			<td style="width:123px;">ARES</td>
			<td style="width:180px;"></td>
		</tr>
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			<td height="42" style="height:42px;width:169px;">SYBR&#174; Green I nucleic acid gel stain</td>
			<td style="width:196px;">Thermo Fisher Scientific Inc.</td>
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			<td style="width:196px;">Sigma-Aldrich Co.</td>
			<td style="width:123px;">X-100</td>
			<td style="width:180px;">Suppresses evaporation of sample at air-water interface</td>
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</table>

dna nanotubes as a versatile tool to study semiflexible polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

The assembly of DNA nanotubes via a temperature ramp (Figure 2) is a very reliable method to form these artificial semiflexible polymers. These polymers have comparable characteristics to their naturally occurring counterparts, such as actin filaments, but provide a much broader experimental framework since their mechanical properties can be controllably altered9,27. Like biopolymers, they can be arra...

Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

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.

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