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In order to achieve a reversible loading and release of a drug or a protein the DNA nanostructure should be functionalized with responsive elements that can undergo reversible input-induced conformational changes or confer a specific function to the, otherwise inert, structure.
Different inputs can in principle be used in this regard such as small molecules, specific DNA sequences, pH, light or temperature 24 , A DNA bipyramid with an opening mechanism triggered by UV-light has been described 26 and a similar approach has been proposed to induce an isothermal pH-induced disassembly of a tetrahedron through the reversible formation and dissociation of a triple helix Triple helical motifs has been also used as functional units to address and precisely locate the assembly of nonrepetitive 2D DNA grids 28 and 3D crystals Another interesting example is a DNA icosahedron able to displace its molecular cargo upon controlled opening after cdGMP molecules binding The integration of functional responsive units in DNA nanostructures is, however, not straightforward.
For example, we recently integrated temperature-dependent hairpins into an octahedral DNA nanocage to allow the reversible encapsulation and release of a protein 12 , The responsive elements integrated into the DNA nanostructures showed a temperature-dependent behaviour that was significantly different to that observed in the isolated state in solution A computational analysis coupled to experiments permitted us to describe the behaviour of the responsive elements in the nanostructure and to ascribe the difference to the geometrical constraints imposed by the nanostructure 12 , Motivated by the above results here we propose a computational and experimental integrated approach to study the effect of engineering a functional responsive DNA-based element into a DNA nanostructure.
We have selected a well-known and characterized pH-dependent clamp-switch based on the formation and dissociation of a parallel DNA triple helix 31 , 32 , and we designed a novel octahedral cage decorated with two copies of this functional element.
The effect of the nanostructure environment on the pH-dependent behaviour of the responsive element has been investigated through accelerated molecular dynamics aMD simulation, fluorescence and gel electrophoresis. Results indicate that the presence of a spacer between the scaffold and the clamp-switch is required to retain the reversible pH-dependent mechanism observed for the isolated element in solution, confirming the importance of integrating simulative tools and experiments to rationally design functional, stimuli-responsive nanostructures.
The Watson-Crick W-C strand has been connected to the triplex forming strand through the sculpting module of the PyMol program 38 to generate the triplex clamp-switch.
The structure has been minimized using the UCSF Chimera program 39 to remove any clashes and unwanted interactions introduced by the modeling. In the case of LT-cage, two 7-thymidine spacers has been introduced Figure 1E , yellow lines between the double helical portion of the clamp and the cage scaffold.
The steric clashes introduced by the modelling procedure have been removed through the SYBYL anneal module and then a first minimization of the entire structures has been performed using the SYBYL maximin2 module.
The system topologies and the coordinates of the triple helix at the two pH conditions i. To simulate the pH 5.
Figure 1. A Truncated octahedral DNA cage. Thick lines indicate double helices, thin lines the 5T linkers.
B Top view of the truncated octahedral cage. C Top view of the T-cage, functionalized with two pH-dependent functional elements, at pH 5. E Model of the LT-cage, functionalized with two pH-dependent functional elements connected to the cage scaffold through seven-base spacers yellow. The sequence of the clamp-switch triplex functional element is shown at the bottom of the figure.
The green and black sequences represent the two strands interacting through the W-C interactions, the underlined sequence represents the 25 bases loop and the red sequence the third strand establishing at pH 5.
Equilibration and MD protocol The nanocages were subjected to two minimization runs. In the first one restraints of 5. No issues seen so far. The result works perfectly, elegantly and smoothly in the browser. But what I've found surprised me a lot. After having put it to test for several weeks, now QuarkXPress is in my toolbox again and…well, it works way better than InDesign. Burkhardt, publisher of the online magazine "free.
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Although, in woodchucks HBx was shown to be important for establishment of virus infection [ 34 , 35 ], the molecular mechanism of HBx activity and its possible influence on cell proliferation remains obscure. This was further substantiated by Haviv and co-workers [ 28 ]. Further, we showed that HBx interacts with single-stranded nucleic acids in vitro [ 36 ], the implications of which in DNA repair process remains to be investigated. TFIIH is a multiprotein complex of 10 polypeptides [ 37 ].
Apart from being an important factor of basal transcriptional machinery, TFIIH has been clearly shown to be an integral component of the DNA repair pathway [ 38 — 41 ].
Although, interaction of HBx with a probable cellular repair protein UV-DDB was earlier reported by Lee and co-workers [ 42 ], a functional role in DNA repair which may result in lethal or hepatocarcinogenic mutations is not understood. Aboussekhra and co-workers [ 43 , 44 ] have shown that the addition of UV-DDB during in vitro DNA repair reaction had a very modest effect on the repair synthesis. Because of the high degree of homology between yeast and mammalian NER machinery, we have chosen yeast nuclear extracts to investigate the biochemical role of HBx in NER in vitro.
Further, S. In this context, we used mutant yeast extracts with various genetic mutations to investigate the role of HBx in the NER pathways.
Methods Saccharomyces cerevisiae strains and plasmids The genotype of S. Two NER defective yeast strains rad 1 and rad51 were employed in this study. Plasmids pUC18 and pBR were used for repair synthesis assays and were purified as described [ 47 ].
In addition, pSBDR contains neomycin resistance marker for selection in eukaryotic cells. Saturated yeast cultures were washed in water and resuspended into 2 ml of sterile water. One set of plates was immediately irradiated under a germicidal lamp for various dosages of UV light and another set of control plates was not exposed to UV-irradiation.
Colonies were counted to determine the survival fraction.
Plates were incubated in dark for 2 weeks in the presence of 0. The number of G resistant clones per cells is plotted. Live cells were counted by staining with trypan blue after transfection and prior to G selection. Cells were harvested by centrifugation for 10 min, washed in water, and suspended at 0. Spheroblasts were pelleted by centrifugation as above and washed three times in the same buffer. Phenylmethanesulfonyl flouride was added 0.
Cells were washed again in 1M sorbitol and suspended at 0. Spheroplasts were lysed in a motor-driven homogenizer with 10 strokes. Nuclei were resuspended 0. In vitro DNA repair reaction The repair reaction contained, 0.
After overnight electrophoresis, the gel was photographed under near-UV transillumination with Polaroid film and an autoradiograph of the dried gel was obtained. Synthesis and purification of an oligonucleotide containing a single 1.
The oligonucleotides were visualized using a hand-held UV lamp nm after placing the appropriate region of the gel onto TLC plate.
The desired platinated oligonucleotide was excised, crushed and suspended in 1 ml H2O. The suspension was incubated overnight with agitation.