DNA preservation in silk

Abstract

The structure of DNA is susceptible to alterations at high temperature, pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells as well as plasmid DNA was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA+filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA+PT-filter) as a route to practical utility. After air-drying and water extraction, 50–70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. The 6% silk/DNA+PT-filter samples provided improved stability in comparison to 3% silk/DNA+filter and DNA+filter for DNA preservation, with ~40% of the band intensity remaining at 37 °C after 40 days and ~10% after exposure to the UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/Borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluoresence protein (dsRed) in HEK293 cells. The high molecular weight and high content of crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA+PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

Engineering the Future of Silk Materials through Advanced Manufacturing

“…Furthermore, silk has also been widely used in flexible electronics, optical and photonic devices owing to the great flexibility and optical properties. Recent research also demonstrates that silk can be utilized for preservation of perishable products by coating them with an ultrathin silk film …”

Silk Fibroin as a Functional Biomaterial for Drug and Gene Delivery

“…Aside from a few exceptions such as growth factors, the majority of the stabilisation approaches rely on entrapping the drug within the SF-matrix or SF-particles in an equally distributed manner [22]. SF-based biomaterials are generally stable to changes in temperature [23], humidity [88], and pH [89]. Therefore, they have been widely studied for enhancing the stability of other materials, for example, encapsulation of antibiotics such as erythromycin, which has very low stability in water.…”

“…In addition to drug stabilisation, SF has also been investigated for DNA preservation in order to protect the DNA from the potential destabilising conditions such as temperature and UV radiation. In a recent study porous cellulose paper was coated with SF and used to preserve the DNA extracted from human dermal fibroblast cells [23]. The results showed that the DNA integrity was maintained for 40 days following 10 h of UV radiation at relatively high temperature (37–40 °C) [23].…”

“…In a recent study porous cellulose paper was coated with SF and used to preserve the DNA extracted from human dermal fibroblast cells [23]. The results showed that the DNA integrity was maintained for 40 days following 10 h of UV radiation at relatively high temperature (37–40 °C) [23].…”

“…Hence, loading the biological therapeutics into a compatible polymer can increase their stability and consequently their half-life [22]. Moreover, incorporating the APIs into a natural biocompatible protein such as SF has multiple advantages e.g., preserving the API [23], improving the mechanical properties of the formulation [6], modifying drug release kinetics [24,25,26,27], enhancing cell adhesion [16], and compatibility with blood components [25]. The versatility of SF protein processing and formulating methods allows the preparation of a wide range of drug carriers with different sizes and morphologies using unmodified or engineered SF (Figure 1).…”