The authors would like to thank: Dr. Frank Jiang, Dr. David Lin, Dr. Bernard Yurke and Dr. Uday Chippada for their contributions on developing the DNA gel technology; Dr. Norell Hadzimichalis, Smit Shah, Kimberly Peterman, Robert Arter for their comments and edits of this manuscript; funding sources including the New Jersey Commission on Spinal Cord Research (Grant #07A-019-SCR1, N.A.L.) and New Jersey Neuroscience Institute (M.L.P.); and publishers of Tissue Engineering, Part A for permission to reprint Figures 2 and 4 and Biomaterials for permission to reprint Figure 3.

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The authors have nothing to disclose.

The ability of DNA gels to soften or stiffen before and after cell adhesion makes them an ideal model to study the role of dynamic tissue stiffness on cell function. All three designs have been used in mechanical and biological studies. However, all three designs have similar elasticities at various crosslink percentages, indicating crosslink length does not influence DNA gel elasticity (Table 2). In contrast, acrylamide concentration affects elasticity. These designs may differ in crosslinking kinetics ...

Static and dynamic substrates are two categories of biomaterials that were developed to study the effects of tissue elasticity or stiffness on cell function. Static substrates are unable to change their physical properties after they are fabricated and/or once cells are plated. Polyacrylamide (PA) gels were the first two-dimensional, static substrates that were synthesized for mechanobiology investigations 5,17. PA gels are easy to prepare, inexpensive, versatile, and can be fabricated with a broad range of elastic moduli. Although these technical advantages make PA gels a commonly applied substrate, static substrates are not indicative of the dynamic nature of the extracellular matrix (ECM) and surrounding cellular environment in vivo. For example, the ECM undergoes stiffness alterations as a result of injury, development, or disease. Dynamic substrates are therefore favored as tissue-mimicking substrate models in mechanobiology studies 22,24,25.
Numerous synthetic, natural, two-dimensional, three-dimensional, static, and dynamic biomaterials have been developed to mimic tissue stiffness 1,3,6,16,23,26. Some dynamic substrates require heat, UV, electrical current, ions, and pH changes to alter their mechanical properties 2,4,7,8,12,15,16, but these stimuli can restrict the hydrogel&#8217;s bio-application. DNA-crosslinked polyacrylamide hydrogels (DNA gels) are dynamic two-dimensional elastic substrates. DNA crosslinks allow for temporal, spatial, and reversible modulation of DNA gel stiffness by addition of single-stranded DNA (ssDNA) to media or buffer 9-11,13,14,18,21. Unlike the aforementioned dynamic gels where stimuli are applied for modulation of elasticity, the DNA gels rely on the diffusion of applied ssDNA for the alteration of elasticity. Therefore, the upper gel surface, where cells are grown, is the first area modulated because the rate of elasticity modulation is dependent on the gel thickness.
DNA gels are similar to their PA gel counterparts in that they have a polyacrylamide backbone, however the bis-acrylamide crosslinks are replaced with crosslinks composed of DNA (Figure 1). Two ssDNAs (SA1 and SA2) hybridize with a crosslinker strand (L2) to make up the DNA crosslinks of the gel. SA1 and SA2 have distinct sequences that both contain an Acrydite modification at the 5&#180; end for effective incorporation into the PA network. For preparation of the gels, SA1 and SA2 are individually polymerized into a PA backbone and, subsequently, the polymerized SA1 and SA2 are mixed together. L2, the crosslinker, is added to the SA1 and SA2 mixture. The L2 base sequence is complementary to both SA1 and SA2 sequences and L2 hybridizes with SA1 plus SA2 to form the DNA crosslinks. Initial, DNA gel elasticity is determined by both L2 concentrations and crosslinking (Tables 1 and 2). DNA gels containing equal stoichiometric amounts of L2, SA1, and SA2 are the stiffest gels because SA1 and SA2 are 100% crosslinked by L2 (designated as 100% gels). Lower concentrations of L2 result in a lower percentage of DNA crosslinking and, therefore, softer DNA gels. Gels as low as 50% crosslinked (designated as 50% gels) have been constructed 9-11.
<img alt="Figure 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> 
Figure 1.&#160;DNA gel crosslinking and uncrosslinking schematic 9-11,13,14,18,21.&#160; Step 1: SA1 (red) and SA2 (blue) are individually polymerized into a polyacrylamide backbone (black). After polymerization, SA1 and SA2 polymerized solutions are mixed together. Step 2: L2 (green) is added and hybridizes with SA1 plus SA2 to form the crosslinks of the gel. Step 3: R2 hybridizes with the toehold of L2. Step 4: Toehold hybridization of R2 propels the unzipping of L2 from SA1 and SA2.
Unlike PA gels, DNA gels can stiffen and soften after synthesis. For that reason, cells grown on DNA gels can be subjected to dynamic stiffness alterations. To stiffen cell-adherent gels, L2 can be added to the culture media of low percentage gels to increase the percentage of crosslinks. To soften cell-adherent gels, L2 can be removed to decrease the percentage of crosslinks 10,13,21. L2 has an additional toehold sequence at the 3&#180; end to allow L2 to uncrosslink from SA1 and SA2 (Table 1). Removal of L2 is accomplished by hybridization of a reversal strand called R2. R2 is complementary to the full length of L2 and hybridizes first with the L2 toehold. Toehold hybridization propels the unzipping of L2 from SA1 and SA2, which eliminates the crosslink and reduces the gel stiffness.
In this report and video, step-by-step instructions are provided for the preparation of stiffening and softening DNA gels. While 100% and 80% gel preparations are described, this protocol can be tailored to create DNA gels of other initial and final crosslinked percentages. In general, 100% and 80% gels are prepared, immobilized onto glass cover slips, functionalized, and seeded with cells. L2 is added to the media of 80% gels and R2 is added to the media of 100% gels, 48 hr after plating. The addition of L2 to media stiffens 80% gels to 100% crosslinked, whereas the addition of R2 to media softens 100% gels to 80% crosslinked. Stiffened gels are designated as 80&#8594;100% gels and softened gels are designated as 100&#8594;80% gels in the text. For control or static gels, ssDNA consisting of Ts or As is delivered to another set of 100% and 80% gels. After a minimum of two days following elasticity modulation, cells can be processed and analyzed.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody align="center">
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">DNA crosslink</td>
			<td rowspan="2"># of bases</td>
			<td>Sequence (Toehold)</td>
			<td rowspan="2">Modification</td>
			<td rowspan="2">Melting temperature (Tm, &#176;C)</td>
			<td rowspan="2">Comments</td>
		</tr>
		<tr>
			<td>5&#39;&#8594;3&#39;</td>
		</tr>
		<tr>
			<td rowspan="4">Design 1</td>
			<td>SA1</td>
			<td>10</td>
			<td>GCA CCT TTG C</td>
			<td>5&#39; Acrydite</td>
			<td>34.9</td>
			<td></td>
		</tr>
		<tr>
			<td>SA2</td>
			<td>10</td>
			<td>GTC AGA ATG A</td>
			<td>5&#39; Acrydite</td>
			<td>23.6</td>
			<td></td>
		</tr>
		<tr>
			<td>L2</td>
			<td>30</td>
			<td>TCA TTC TGA CGC AAA GGT GCG CTA CAC TTG</td>
			<td></td>
			<td>56</td>
			<td>A 10 bp toehold sequence is included.&#160;</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>30</td>
			<td>CAA GTG TAG CGC ACC TTT GCG TCA GAA TGA</td>
			<td></td>
			<td></td>
			<td>R2 is complementary to L2</td>
		</tr>
		<tr>
			<td rowspan="4">Design 2</td>
			<td>SA1</td>
			<td>14</td>
			<td>CGT GGC ATA GGA CT</td>
			<td>5&#39; Acrydite</td>
			<td>46.9</td>
			<td></td>
		</tr>
		<tr>
			<td>SA2</td>
			<td>14</td>
			<td>GTT TCC CAA TCA GA</td>
			<td>5&#39; Acrydite</td>
			<td>40.2</td>
			<td></td>
		</tr>
		<tr>
			<td>L2</td>
			<td>40</td>
			<td>TCT GAT TGG GAA ACA GTC CTA TGC CAC GGT TAC CTT CAT C</td>
			<td></td>
			<td>65.9</td>
			<td>A 12 bp sequence toehold is included.&#160;</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>40</td>
			<td>GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG A</td>
			<td></td>
			<td>65.9</td>
			<td>R2 is complementary to L2</td>
		</tr>
		<tr>
			<td rowspan="3">Design 3</td>
			<td>SA1</td>
			<td>20</td>
			<td>ACG GAG GTG TAT GCA ATG TC</td>
			<td>5&#39; Acrydite</td>
			<td>55</td>
			<td></td>
		</tr>
		<tr>
			<td>SA2</td>
			<td>20</td>
			<td>CAT GCT TAG GGA CGA CTG GA</td>
			<td>5&#39; Acrydite</td>
			<td>56.6</td>
			<td></td>
		</tr>
		<tr>
			<td>L2</td>
			<td>40</td>
			<td>TCC AGT CGT CCC TAA GCA TGG ACA TTG CAT ACA CCT CCG T</td>
			<td></td>
			<td>68.8</td>
			<td>Toehold is not included.</td>
		</tr>
		<tr>
			<td rowspan="2">Control</td>
			<td rowspan="2">Control</td>
			<td rowspan="2">20-40</td>
			<td>AAA AAA (etc.) or</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>TTT TTT (etc.)</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Base sequences for ssDNA 9-11,13,14,18,21. Cellular and mechanical studies have utilized several different crosslink designs to generate DNA gels with a range of static and dynamic mechanical properties. The parameters modulated in crosslink design are base sequence and sequence length or crosslink length. Bold and italicized fonts illustrate base pairing between SA1 and L2 and between SA2 and L2, respectively.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody align="center">
		<tr>
			<td rowspan="2"></td>
			<td colspan="8">Design</td>
		</tr>
		<tr>
			<td colspan="3">1</td>
			<td colspan="3">2</td>
			<td colspan="2">3</td>
		</tr>
		<tr>
			<td>Acrylamide Concentration (%)</td>
			<td colspan="3">10</td>
			<td colspan="3">10</td>
			<td>10</td>
			<td>4</td>
		</tr>
		<tr>
			<td>SA1 plus SA2 hybridized to L2 (% crosslinked)</td>
			<td>50</td>
			<td>80</td>
			<td>100</td>
			<td>50</td>
			<td>80</td>
			<td>100</td>
			<td>100</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Elasticity (kPa, Mean&#177;SEM)</td>
			<td>6.6 &#177; 0.6</td>
			<td>17.1 &#177; 0.8</td>
			<td>29.8 &#177; 2.5</td>
			<td>5.85&#177; 0.62</td>
			<td>12.67 &#177; 1.33</td>
			<td>22.88 &#177; 2.77</td>
			<td>25.2 &#177; 0.5</td>
			<td>10.4 &#177; 0.6</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Young&#8217;s modulus (E) of DNA gels 9-11,13,14,18,21. Acrylamide concentration, crosslink percentage, and crosslink length can be modulated in DNA gels. Designs 1, 2, and 3 have 20, 28, and 40 bp crosslink lengths, respectively. 100% gels for all designs have similar moduli indicating crosslink length does not affect gel elasticity. However, variations in acrylamide concentration alter DNA gel elasticity.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Prior to our studies, cell-ECM interactions were observed on static compliant biomaterials or on irreversible and unidirectional dynamic substrates. These substrates do not accurately reflect the dynamic nature of the cellular microenvironment. Our work shifts the existing technical paradigms by providing a more physiologic model for studying cell-ECM interactions on softening and stiffening dynamic biomaterials. In previous studies, we analyzed the effects of dynamic substrates on cells by examining various cell morphol...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.3K Views.  JFK Medical Center. Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.