# Mechanical Properties of DNA Hydrogels: Towards Highly Programmable Biomaterials
## 2 Structural DNA Hydrogel Design
### Entangled and Crosslinked  
Entangled structures are commonly composed of rolling chain amplification (RCA) produced DNA concatemers [48; 51; 52] or DNA from salmon testes, which may be covalently crosslinked by ethylene glycol diglycidyl ether (EDGE) and oxidized alginate (OA) [53; 54; 55]. The source of DNA material is an important factor as synthetically synthesized DNA can cost a few hundred to a few thousand dollars for a few milligrams required for milliliter scale DNA hydrogel synthesis [56]; typically requiring 2-4 milligrams (mg) of DNA per 100 \(\upmu\)L, or 2-4 percent weight/volume (%w/v) of hydrogel. However, RCA is able to produce a few hundred micrograms of DNA per milliliter of overnight reaction [57; 58; 59], though it is currently limited to short tandem repeats (~100 nucleotides [nt]) without control over the repeat number. Multiprimed chain amplification (MCA) is the subsequent priming of the concateneric strand produced by RCA (Figure 1a); this approach allows for a significant amount of DNA to be produced with a multitude of complementary domains enabling partial hybridization between multiple strands ranging from a few hundred to thousands of nucleotides in length. [PERSON] et al. demonstrated the ability of MCA to produce enough material to assemble a DNA hydrogel by entanglement which formed spheroidal clusters or \"nests\" measuring close to 1 micron in diameter (Figure 1a) [51]. This DNA hydrogel yielded a storage modulus (G') ranging from 5-7 Pascals (Pa) at frequencies between 0.1-100 Hertz (Hz). [PERSON] et al. found similar mechanical properties when using the same technique to produce a DNA hydrogel seeded with silver nanoclusters. The all-DNA MCA-based gel yielded a storage modulus ranging from 5-7 Pa over the course of 3 min and when heated up to 75 \({}^{\circ}\)C [52]. When silver nanoclusters were seeded onto cytosine-rich domains the storage modulus increased to G' = 12-20 Pa and exhibited similar temperature independent rheological properties between 40 \({}^{\circ}\)C and 75 \({}^{\circ}\)C. [PERSON] et al. introduced a different approach to RCA-based hydrogels by synthesizing two independent concateneric ssDNA scaffolds, thus voiding the MCA-step (Figure 1b). A set of complimentary short ssDNA strands were used to crosslink the independent scaffolds and control the strength of the linker by altering the sequence and length of the hybridizing regions. A fluorescence resonance energy transfer (FRET) dye pair was incorporated within the crosslinker to monitor the dissociation of the linkage and visualize strain in the material. This approach yielded temperature-dependent properties with diminishing storage moduli from 400, 160, and 6 Pa which melted between 60-80 \({}^{\circ}\)C for DNA concentrations of 1.4, 0.7, and 0.36% \(w/v\), respectively [48]. Interestingly the 0.7% \(w/v\) DNA hydrogel showed an increase in its storage modulus by an order of magnitude, from \(\sim\)10 to \(\sim\)100 Pa by simply heating the hydrogel to 80 \({}^{\circ}\)C and cooling it to 10 \({}^{\circ}\)C over approximately 15 min. A second heating and cooling ramp showed no further increase in the mechanical properties. Melting the DNA hydrogel and cooling is thought to allow for reorganization of the internal structure by releasing entrapped complementary binding regions during heating and increasing the number of crosslinks upon cooling. An alternative approach to entangled DNA hydrogels is to use biologically sourced DNA, commonly acquired from salmon testes. [PERSON] et al. were able to form a hydrogel by wet spinning DNA in a room temperature ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4), which showed a fiber swelling of 600% from a diameter of \(\sim\)100 \(\upmu\)m in deionized water and diminished to 150% upon addition of 150 mM NaCl. The salt-induced deswelling is due to the neutralization of the negatively charged DNA backbone, thus diminishing the electrostatic repulsion between DNA strands [60].