University of Groningen
Applications of DNA hybrids in biobased medicine and materials
Liu, Qing
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Publication date: 2018
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Liu, Q. (2018). Applications of DNA hybrids in biobased medicine and materials. University of Groningen.
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Summary
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Aside from the role as a carrier for genetic information, DNA has also gained tremendous interest in material science owing to its versatile properties, such as outstanding specificity and programmability. On one hand, great efforts have been devoted to exploiting pristine DNA, utilizing its appealing intrinsic features: stiffness (50 nm in persistence length), short structural repeats (~3.6 nm in helical pitch) and nano-scaled size (2 nm in diameter). Thus, a large plethora of 1D-, 2D- and 3D DNA nanostructures with various sizes, geometries and shapes is readily accessible and the subsequent applications are developing further and further. On the other hand, a number of methodologies have been employed to modify DNA, chemically and physically, which generates some properties and structures that pristine DNA does not bear. Among these, hydrophobic modification of DNA is appealing because it can impart special characteristics on DNA, which are not achieved with another material combination.
In Chapter 2, a lipid-modified DNA system (lipid-DNA) was established for the catalytic oxidization of dopamine to aminochrome. 1-Dodecyne was introduced to an uracil base which was subsequently incorporated into two DNA aptamers, yielding a lipidated dopamine-binding aptamer (lipoDBA) and a lipidated hemin-binding G-quadruplex (lipoGQ), respectively. Mixing of lipoDBA and lipoGQ generated DNA micelles. The association of substrate and catalyst with lipidated aptamers, respectively, led to the concentration of the substrate at a close spatial position to the catalytic sites, resulting in an accelerated oxidation process. We found that the DNA micelles showed best enhancement when the molar ratio between lipoGQ and lipoDBA was 4 : 6. Besides, di-lipidated G-quadruplexes (2lipoGQ) showed better catalytic performance than tetra-lipidated G-quadruplexes
(4lipoGQ), which can be attributed to the retarded flexibilities of both lipidated
aptamers in the 4lipoGQ containing system. Additionally, the catalyzed H2O2
-mediated oxidation of dopamine to aminochrome was also observed if the lipoGQ catalytic units were substituted with an artificial catechol oxidase-mimicking lipidated dinuclear Cu(II)-complex.
To further expand the application of lipid-DNA, in Chapter 3 we exploited the possibility of using lipid-DNA as a tool for ophthalmic drug delivery. Three aptamer sequences were selected for the specific binding of three ocular drugs: kanamycin B, brimonidine and travoprost. All three aptamers were extended with a domain containing four alkyl-modified 2’-deoxyuridine nucleotides as hydrophobic modification. The obtained lipid-DNA self-assembled into micelles, as demonstrated by DLS measurements. ITC results revealed that the binding ability of the aptamer part of all lipid-DNA was not compromised compared to pristine aptamer. It was demonstrated that the lipid modification resulted in the corneal adherence of the
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aptameric NPs both in vitro and in vivo while it did not contribute to toxicity of the amphiphiles. The drug release was proven in vitro, showing a similar effect as free drugs. Moreover, the efficiency of the drug carrier was proven in vitro. Better bacterial killing was found on porcine cornea for the nucleic acid carrier system compared to the free drug. It can be envisioned that the NPs offer an additional drug loading modality as the lipid core of the micelle can accommodate water insoluble medication, which can further enhance their performance in drug delivery by introducing an additional load for co-therapy.
In Chapter 4, the hybridization property of a DNA hairpin was evaluated photochemically after a molecular motor was incorporated into the middle of the backbone. Both cis and trans isomers were prepared and subsequently incorporated into a 16-mer strand of self-complimentary DNA via solid phase synthesis. The hairpin structure of both isomers modified DNA was confirmed by PAGE gel electrophoresis. The melting temperature analysis revealed that the Tm of
8T-trans-8A was 65 °C while the that of 8T-cis-8T-trans-8A was 59 °C, of which the measured ΔTm of
6 °C (for an 8 bp hairpin) represents a very promising value. It ranks this investigation among the most successful attempts to influence DNA hybridization through the incorporation of a photoswitchable backbone linker. Besides, it was found that the isomerization process of the motor was not hindered in non-hybridization conditions without degradation, however, it was slightly slowed down by the biomolecule. When the motor-DNA was hybridized prior to UV irradiation, motor-DNA was again readily photoisomerized without the occurrence of side reactions but thermal helix inversion (THI) was hindered by the hybridized DNA strands. When the sample was heated above the Tm, THI occurred in a similar
manner in aqueous buffer as in water. This study marks the first time that a molecular motor has been used to control the secondary structure of DNA, and in fact one of the first examples of a molecular motor being applied under physiological conditions, demonstrating the ability to regulate a key biological process such as DNA hybridization.
Aside from the chemically hydrophobic modification of DNA, the physical modification of DNA was also investigated. In Chapter 5, the DNA-surfactant complex in a gel state was studied. We found that hydrophilic DNA precipitated from aqueous phase after complexation with positively charged surfactants. When immersed in organic phases, DNA-surfactant complex turned into a gel state (organogels) with a nematic liquid crystalline mesophase, as demonstrated by POM and SAXS. The gel state was confirmed by dynamic mechanical analysis (DMA) and it was found that the elasticity of the DNA-surfactant organogels positively
Summary
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correlated to the lengths of the employed dsDNA strands. The obtained DNA organogels exhibited remarkable extensibility, deformability, stiffness, toughness, and plasticity. For instance, the organogels showed excellent elongation with an extensibility beyond 3·104% before fracture and the largely stretched sample can
partially self-recover. Moreover, mechanical analysis revealed a tensile strength of 1-3MPa, Young’s moduli of above 20MPa and a toughness of 18 MJ/m3, which are of
the same magnitude to covalently crosslinked polymer gels. Additionally, the supramolecular nature of the network allowed rapid self-healing within 5 seconds hereby recovering its mechanical properties. To the best of our knowledge, this is the first example of gels that can be healed in such a short timeframe. Finally, magnetic NPs were incorporated into these tough DNA-LC organogels without compromising the formation of the nematic mesophase. This endows the organogels with the ability to shape-respond to external magnetic fields and exemplarily demonstrates the facile preparation of DNA organogel composites.
The findings in the different chapters illustrate that the functionality of DNA systems can largely increase when combined with other moieties. Structure formation can be manipulated by the blending with hydrophobic moieties. In the one case, this leads to nanoparticles with adhering properties to special tissue. In another case, it influences the bulk order and mechanical properties of DNA organogels. Finally, merging of DNA with complex molecular motors leads to dynamic systems where the energy of light controls key biological functionalities as non-invasive external power source.
