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 oligonucleotides in purified water and follow the further resuspension steps given in the corresponding manual from the company. Adjust the amount of water to obtain a final concentration of 200 &#181;M.</li>
	<li>Determine concentrations of the single DNA strands to verify the values given by the distributor (e.g., via UV-Vis spectroscopy at 260 nm) since the exact stoichiometry is crucial for the assembly process. Calculate the concentration of the oligonucleotides, taking into account the specific molar weight and the extinction coefficient for each single-stranded DNA oligonucleotide. 
	NOTE: The extinction coefficient values inherently vary for the different sequences and are stated in the documentation of commercially purchased DNA. They can also be calculated according to the nearest-neighbor model using a number of freely available online tools. To comply with the linear range of the spectrometer, consider diluting the sample used for concentration determination.</li>
	<li>Using a micropipette, mix the DNA strands-each at the same concentration-in a container suitable for the thermocycler (e.g., a 200 &#181;L PCR tube) to form the desired nHTs (final buffer conditions: 1xTE (10 mM Tris and 1 mM EDTA, pH 8) and 12.5 mM MgCl2). 
	NOTE: Final DNA nHT samples consist of n- 1 strands, U1&#8722; Un&#8722;1, and one Tn strand. The concentration per strand can vary from sub-micromolar to more than 10 &#181;M, depending on the needs of the user (e.g., &#60;1 &#181;m per strand for the subsequent dilution to observe single filaments or higher concentrations for the examination of dense network mechanics).</li>
	<li>Hybridize the nHTs in a thermocycler (denaturation for 10 min at 90 &#176;C, with a subsequent quick drop to 65 &#176;C; slow temperature decrease from 65 &#176;C to 55 &#176;C, with complementary base pairing in 20 temperature steps of -0.5 K for 30 min each; and a quick drop from 55 &#176;C to 20 &#176;C); see Figure 2A. 
	NOTE: The slow decrease steps from 65 &#176;C can be lengthened to increase the average tube length; however, the subsequent quick drop to 20 &#176;C is crucial to avoid the aggregation of polymerized tubes.</li>
	<li>Store the hybridized nHTs for up to 3 weeks at 4 &#176;C without detectable degradation. 
	NOTE: For fluorescence microscopy, labeled nHTs are hybridized by (partly) substituting U1 with the modified U1-Cy3 (Table 1). For longer observation times, the addition of established anti-bleaching agents is advisable.</li>
	<li>Carefully dilute the sample to the desired final concentration (e.g., 4 &#181;M for networks or 20 nM for single-filament observations) using the sample buffer, if necessary. To minimize possible error sources, assemble samples at the desired final concentration.</li>
</ol>
2. Shear Rheology
<ol>
	<li>Choose an appropriate geometry (plate-plate or cone-plate) for the dynamic shear rheometer. For small volumes (i.e., below 200 &#181;L), use the cone-plate geometry.</li>
	<li>Load the sample on the dynamic shear rheometer.</li>
	<li>Passivate the air-water interface of the sample and environment using the following steps to prevent potential evaporation effects.
	<ol>
		<li>Surround the sample with 2.5 mL of the sample buffer bath.</li>
		<li>Add a surfactant just below the micelle concentration.</li>
		<li>Use a Hamilton syringe to surround the sample with a lipid to avoid direct contact of the sample with air.</li>
	</ol>
	</li>
	<li>Seal the sample chamber with a cap equipped with wet sponges and, if possible, with an additional water bath (not in contact with the sample) to further suppress evaporation.</li>
	<li>Start the measurement using the rheometer-specific software at room temperature; alternatively, perform the measurement at different temperatures, such as 37 &#176;C (if physiological conditions are needed). 
	NOTE: Cooling the sample is often accompanied by the condensation of water vapor from the surrounding air, while heating leads to enhanced evaporation. Both effects can uncontrollably change the concentration of the sample, so all measurements in a series should be performed at a constant temperature.</li>
	<li>Allow the sample to equilibrate for 2 h at room temperature. The time evolution may be monitored with a time sweep (one data point per minute; strain &#947; = 5%; frequency f = 1 Hz).</li>
	<li>Measure the mechanical properties with the desired test protocols. Start with a series of frequency sweeps (f-sweeps) because they leave the sample intact and allow for more measurements. 
	NOTE: Additionally, short f-sweeps should be carried out before and after longer measurements (such as long f-sweeps with a much higher resolution) to verify that the sample remained unchanged throughout the full measurement protocol.
	<ol>
		<li>Perform a short f-sweep (e.g., &#947; = 5%; f = 0.01 Hz to 30 Hz; 5 data points per decade).</li>
		<li>Perform a long f-sweep (e.g., &#947; = 5%; f = 0.001 Hz to 30 Hz; 21 data points per decade); the low-frequency regime can be omitted if not necessary to reduce measurement time.</li>
		<li>Perform a short f-sweep.</li>
		<li>Perform a &#947;-sweep (e.g., f = 1 Hz; &#947; = 0.0125% to 100%; 20 data points per decade). 
		NOTE: Use the short f-sweep first, as it is usually inconsistent with previous f-sweeps because networks usually rupture during &#947;-sweep or detach from the plates. Alternatively, use &#947;-ramp (e.g., = 0.025 s-1, t = 60 s); however, the ramps usually require changing the motor mode, so subsequent short f-sweeps would be falsified.</li>
	</ol>
	</li>
	<li>Bin and/or smooth the raw data with a Gaussian kernel.</li>
</ol>
3. Fluorescence-assisted Analysis of DNA Annealing
<ol>
	<li>Prepare 8 aliquots of 12 &#181;L for each sample using 1-4 &#181;M DNA per strand, 12.5 mM MgCl2 in 1x TE buffer, and 1x nucleic acid gel stain from a 10,000x DMSO stock. Make a negative control with no DNA, but include nucleic acid gel stain.</li>
	<li>Transfer the aliquots to a real-time PCR plate and seal with adhesive film (e.g., a 96-well reaction plate). 
	NOTE: The high temperatures used in the thermal annealing protocols will evaporate samples. Thus, make sure that the wells are tightly sealed with adhesive film to prevent water evaporation and concentration increase, particularly during long-term experiments.</li>
	<li>Centrifuge the plate at 100 x g for 1 min at room temperature to remove the gas bubbles; if the gas bubbles expand, the wells cannot be analyzed.</li>
	<li>Load the sealed plate into a real-time quantitative PCR system.</li>
	<li>Run an annealing program. Denature the DNA at 90 &#176;C for 10 min. Drop the temperature quickly to 72 &#176;C. Then, decrease the temperature slowly by 0.5 &#176;C every 30 min. After the signal has decayed, accelerate the temperature ramp. 
	NOTE: 72 &#176;C is sufficiently above the real hybridization temperature; thus, the signal is recorded over a broad temperature range suitable for determining the necessary temperature range.</li>
	<li>Make sure that the lid of the cycler heats to at least 100 &#176;C to prevent the condensation of evaporated sample on the adhesive film.</li>
	<li>During assembly, excite the samples with an argon-ion laser at 488 nm and detect the fluorescence at 550 nm when using a nucleic acid gel stain (or spectrally similar). 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. 
	NOTE: Here, measurements were taken every 9 s.</li>
	<li>If preset melting and annealing programs are applied, use the provided software to analyze the data. If not, subtract the previous mean value from the mean value for each temperature step to obtain the relative change of the fluorescent signal.</li>
</ol>
4. AFM Imaging
<ol>
	<li>Cleave mica (e.g., mica &#34;V1&#34;) by attaching commercially available adhesive tape and ripping it off; the uppermost layer of the mica should be removed.</li>
	<li>Using a micropipette, deposit pre-formed nHT in Mg2+ containing folding buffer on the freshly cleaved mica and let it settle for 10 min.</li>
	<li>Spin the samples in short 3-s intervals at 2,000 x g on a small table centrifuge to remove the remaining liquid. Several nHTs should still be attached to the mica surface.</li>
	<li>Use a cantilever tip with a high stiffness (e.g., 54 Nm-1) and determine its resonance frequency with the internal AFM software.</li>
	<li>Record the height profile with the AFM in air in tapping mode at low scan rates (e.g., 1 line/s) to avoid damaging or displacing the nHTs.</li>
</ol>

The authors have nothing to disclose.

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).

<table><tbody><tr><td>AFM cantilever ACTA</td><td>AppNano</td><td></td><td></td></tr><tr><td>AFM - NanoWizard 3</td><td>JPK Instruments</td><td></td><td></td></tr><tr><td>CCD camera</td><td>Andor</td><td>iXon DV887</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DNA oligonucleotides</td><td>Biomers.net</td><td></td><td>For sequences see Table 1</td></tr><tr><td>DNA Cy3-labeled oligonucleotides</td><td>Biomers.net</td><td></td><td>For sequence see Table 1</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E-9884</td><td></td></tr><tr><td>Epi-fluorescence micro-scope</td><td>Leica</td><td>DM-IRB</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M-8266</td><td></td></tr><tr><td>Mica &quot;V1&quot;, 12 mm round</td><td>Plano GmbH</td><td>50-12</td><td></td></tr><tr><td>MicroAmp&amp;reg; Fast Optical 96-Well Reaction Plate</td><td>Thermo Fisher Scientific Inc.</td><td>4346907</td><td></td></tr><tr><td>MicroAmp&amp;reg; Optical Adhesive Film</td><td>Thermo Fisher Scientific Inc.</td><td>4306311</td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific Inc.</td><td></td><td></td></tr><tr><td>100x objective</td><td>Leica5</td><td>506168</td><td></td></tr><tr><td>Purified water</td><td>Merk Millipore - Milli-Q &amp;amp; Elix</td><td></td><td></td></tr><tr><td>Sapphire PCR tubes</td><td>Greiner Bio-One</td><td>683271</td><td></td></tr><tr><td>TProfessional Standard PCR Thermocycler</td><td>Core Life Sciences Inc.</td><td>070- Standard</td><td></td></tr><tr><td>7900HT Fast Real-Time PCR System</td><td>Applied Biosystems</td><td>4351405</td><td></td></tr><tr><td>Rheometer</td><td>TA Instruments</td><td>ARES</td><td></td></tr><tr><td>SYBR&amp;reg; Green I nucleic acid gel stain</td><td>Thermo Fisher Scientific Inc.</td><td>S7567</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T4661</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich Co.</td><td>X-100</td><td>Suppresses evaporation of sample at air-water interface</td></tr></tbody></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 ...

dna-nanotubes-as-a-versatile-tool-to-study-semiflexible-polymers

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.8K Views.  Fraunhofer Institute for Cell Therapy and Immunology. 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.

Video: DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Scalable Nanohelices for Predictive Studies and Enhanced 3D Visualization

Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications.&#160; For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately.&#160; To study the effect of local structure on the properties of these complex geometries one must develop realistic models.&#160; To date, software packages are rather limited in creating atomistic helical models.&#160; This work focuses on producing atomistic models of silica glass (SiO2) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of &#8220;bulk&#8221; silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented.&#160; The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix.&#160; With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions.&#160; The second method involves a more robust code which allows flexibility in modeling nanohelical structures.&#160; This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models.&#160; Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created.&#160; An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material.&#160; In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures.&#160; One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.

Accurate modeling of nanohelical structures is important for predictive simulation studies leading to novel nanotechnology applications.&#160; Currently, software packages and codes are limited in creating atomistic helical models.&#160; We present two procedures designed to create atomistic nanohelical models for simulations, and a graphical interface to enhance research through visualization.

Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing ...

Engineering

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Chemistry

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Biochemistry

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...