Content
2012
Controlling The Mobility Of Oligonucleotides In The Nanochannels Of Mesoporous Silica
27.12.2012
Advanced Functional Materials,
2012,
DOI: 10.1002/adfm.201101365,
22, 106–112
published on 27.12.2012
Advanced Functional Materials, online article
Advanced Functional Materials, online article
Oligonucleotides used in gene therapy and silencing are fragile compounds that degrade easily in biological environments. Porous biocompatible carrier particles may provide a useful strategy to deliver these therapeutics to their target sites. Development of appropriate delivery vehicles, however, requires a better understanding of the oligonucleotide-host interactions and the oligonucleotide dynamics inside carrier particles. We investigated template-free SBA-15 type mesoporous silica particles and report their loading characteristics with siRNA depending on the surface functionalization of their porous network. We show that the siRNA uptake capability of the particles can be controlled by the composition of the functional groups. Fluorescence recovery after photobleaching measurements revealed size-dependent mobility of siRNA and double-stranded DNA oligonucleotides within the functionalized silica particles and provided evidence for the stability of the oligonucleotides inside the pores. Hence, our study demonstrates the potential of mesoporous silica particles as a means for alternative gene delivery in nanomedicine.
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Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature
14.12.2012
Science,
2012,
DOI: 10.1126/science.1229919,
Vol. 338 no. 6113 pp. 1458-1461
published on 14.12.2012
Science, online article
Science, online article
We demonstrate that, at constant temperature, hundreds of DNA strands can cooperatively fold a long template DNA strand within minutes into complex nanoscale objects. Folding occurred out of equilibrium along nucleation-driven pathways at temperatures that could be influenced by the choice of sequences, strand lengths, and chain topology. Unfolding occurred in apparent equilibrium at higher temperatures than those for folding. Folding at optimized constant temperatures enabled the rapid production of three-dimensional DNA objects with yields that approached 100%. The results point to similarities with protein folding in spite of chemical and structural differences. The possibility for rapid and high-yield assembly will enable DNA nanotechnology for practical applications.
Cryo-EM structure of a 3D DNA-origami object
19.11.2012
A key goal for nanotechnology is to design synthetic objects that may ultimately achieve functionalities known today only from natural macromolecular complexes. Molecular self-assembly with DNA has shown potential for creating user-defined 3D scaffolds, but the level of attainable positional accuracy has been unclear. Here we report the cryo-EM structure and a full pseudoatomic model of a discrete DNA object that is almost twice the size of a prokaryotic ribosome. The structure provides a variety of stable, previously undescribed DNA topologies for future use in nanotechnology and experimental evidence that discrete 3D DNA scaffolds allow the positioning of user-defined structural motifs with an accuracy that is similar to that observed in natural macromolecules. Thereby, our results indicate an attractive route to fabricate nanoscale devices that achieve complex functionalities by DNA-templated design steered by structural feedback.
Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures
16.11.2012
Science,
2012,
DOI: 10.1126/science.1225624,
Vol. 338 no. 6109 pp. 932-936
published on 16.11.2012
We created nanometer-scale transmembrane channels in lipid bilayers by means of self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrated and spanned a lipid membrane, as well as a barrel-shaped cap that adhered to the membrane, in part via 26 cholesterol moieties. In single-channel electrophysiological measurements, we found similarities to the response of natural ion channels, such as conductances on the order of 1 nanosiemens and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. Single-molecule translocation experiments show that the synthetic channels can be used to discriminate single DNA molecules.
Enhanced Bioactivity of Internally Functionalized Cationic Dendrimers with PEG Cores
09.11.2012
Biomacromolecules,
2012,
DOI: 10.1021/bm301384y,
published on 09.11.2012
Biomacromolecules, online article
Biomacromolecules, online article
Hybrid dendritic-linear block copolymers based on a 4-arm poly(ethylene glycol) (PEG) core were synthesized using an accelerated AB2/CD2 dendritic growth approach through orthogonal amine/epoxy and thiol-yne chemistries. The biological activity of these 4-arm and the corresponding 2-arm hybrid dendrimers revealed an enhanced, dendritic effect with an exponential increase in cell internalization concomitant with increasing amine end groups and low cytotoxicity. Furthermore, the ability of these hybrid dendrimers to induce endosomal escape combined with their facile and efficient synthesis makes them attractive platforms for gene transfection. The 4-arm-based dendrimer showed significantly improved DNA binding and gene transfection capabilities in comparison with the 2-arm derivative. These results combined with the MD simulation indicate a significant effect of both the topology of the PEG core and the multivalency of these hybrid macromolecules on their DNA binding and delivery capablities.
Magnesium-free self-assembly of multi-layer DNA objects
02.10.2012
Nature Communications,
2012,
doi:10.1038/ncomms2095,
3, Article number: 1103
published on 02.10.2012
Nature Communications, online article
Nature Communications, online article
Molecular self-assembly with DNA offers a route for building user-defined nanoscale objects, but an apparent requirement for magnesium in solution has limited the range of conditions for which practical utility of such objects may be achieved. Here we report conditions for assembling templated multi-layer DNA objects in the presence of monovalent ions, showing that neither divalent cations in general or magnesium in particular are essential ingredients for the successful assembly of such objects. The percentage of DNA strands in an object that do not form thermally stable double-helical DNA domains (Tm>45 °C) with the template molecule correlated with the sodium requirements for obtaining folded objects. Minimizing the fraction of such weakly binding strands by rational design choices enhanced the yield of folding. The results support the view that DNA-based nanodevices may be designed and produced for a variety of target environments.
Release pathways of interferon α2a molecules from lipid twin screw extrudates revealed by single molecule fluorescence microscopy
10.09.2012
Journal of Controlled Release,
2012,
doi:10.1016/j.jconrel.2012.07.014,
Volume 162, Issue 2, Pages 295–302
published on 10.09.2012
Journal of Controlled Release, online article
Journal of Controlled Release, online article
The pathways of interferon α2a release from a triglyceride based implant system were studied by single molecule fluorescence microscopy. The protein was labeled with a stable fluorescent dye ATTO647N, freeze-dried and embedded into the lipid matrix via twin-screw extrusion. The implant system consisted of a pore-forming agent (water soluble PEG 6000) and two types of triglycerides with different melting ranges which allowed the production of the implants at moderate temperatures and without the use of organic solvents. Single molecule microscopy and single particle tracking of labeled proteins contained in these implants revealed that two populations of diffusing proteins were present. Moreover, proteins were not only released via water-filled pores (created by dissolution of the pore-former), but surprisingly also through diffusion in a phase of molten lipid. Diffusion coefficients of IFNα 2a derived by tracking of individual protein molecules within the implant system were similar to diffusion coefficients obtained from control measurements in pure molten lipid and highly concentrated solutions of PEG 6000. In conclusion, tracking of individual protein molecules was successfully used to elucidate the release pathways of proteins from a relevant lipid based implant system.
Amyloid-Like Structures Formed by Azobenzene Peptides: Light-Triggered Disassembly
12.07.2012
Spectroscopy,
2012,
doi:10.1155/2012/108959,
Volume 27 (2012), Issue 5-6, Pages 387-391
published on 12.07.2012
Spectroscopy, online article
Spectroscopy, online article
The light-driven disassembly process of amyloid-like structures formed by azobenzene model peptides is studied by time-resolved mid-IR spectroscopy from nanoseconds to minutes. The investigated peptide consists of two amino acid strands connected by the azobenzene switch. The peptides aggregate to amyloid-like structures when the azobenzene chromophore is in the trans-conformation. Illumination, resulting in a trans- to cis-isomerization of the azobenzene, leads to disaggregation of the aggregated structures. After optical excitation and isomerization of the azobenzene, one finds absorption changes which recover to a large extent on the time scale of few nanoseconds. These early absorption transients are assigned to the relaxation of vibrational excess energy (heat) or to structural rearrangements of isomerized azobenzene and the aggregated surroundings. It is only on the time scale of minutes that spectral signatures appear which are characteristic for the disassembly of the aggregated structure.
Dynamics of ultraviolet-induced DNA lesions: Dewar formation guided by pre-tension induced by the backbone
12.06.2012
New Journal of Physics,
2012,
doi:10.1088/1367-2630/14/6/065006,
14
published on 12.06.2012
New Journal of Physics, online article
New Journal of Physics, online article
The photophysical and photochemical processes driving the formation of the ultraviolet (UV)-induced DNA Dewar lesion from the T(6-4)T dimer are investigated by time-resolved spectroscopy and quantum chemical modelling. Time-resolved absorption and emission spectroscopy in the UV revealed a biexponential decay of the electronically excited state (S1) with time constants in the 100 ps and 1 ns range. From the S1 state the system forms the Dewar lesion (proven by time-resolved infrared spectroscopy), the triplet state of the T(6-4)T dimer and the ground state of the original T(6-4)T dimer. The decay process from the excited singlet is activated and thus temperature dependent. Quantum chemical modelling is used to describe the reaction path via a minimum on the excited electronic potential energy surface in close proximity to a triplet state. The transition to the Dewar isomer competes with internal conversion and with triplet formation. Only if the backbone between the two thymines is closed, is the Dewar isomer formed with a significant yield. The simulations reveal that the tension built up by the backbone is required for guiding the reaction to the conical intersection leading to the Dewar isomer.
Tuning Nanoparticle Uptake: Live-Cell Imaging Reveals Two Distinct Endocytosis Mechanisms Mediated by Natural and Artificial EGFR Targeting Ligand
28.05.2012
Therapeutic nanoparticles can be directed to cancer cells by incorporating selective targeting ligands. Here, we investigate the epidermal growth factor receptor (EGFR)-mediated endocytosis of gene carriers (polyplexes) either targeted with natural EGF or GE11, a short synthetic EGFRbinding peptide. Highly sensitive live-cell fluorescence microcopy with single particle resolution unraveled the existence of two different uptake mechanisms; EGF triggers accelerated nanoparticle endocytosis due to its dual active role in receptor binding and signaling activation. For GE11, an alternative EGFR signaling independent, actin-driven pathway is presented.
ONIOM approach for non-adiabatic on-the-fly molecular dynamics demonstrated for the backbone controlled Dewar valence isomerization
25.05.2012
Journal of Chemical Physics,
2012,
http://dx.doi.org/10.1063/1.4720090,
Volume 136; Issue 20; Pages 10
published on 25.05.2012
Journal of Chemical Physics, online article
Journal of Chemical Physics, online article
Non-adiabatic on-the-fly molecular dynamics (NA-O-MD) simulations require the electronic wavefunction, energy gradients, and derivative coupling vectors in every timestep. Thus, they are commonly restricted to the excited state dynamics of molecules with up to ≈20 atoms. We discuss an approximation that combines the ONIOM(QM:QM) method with NA-O-MD simulations to allow calculations for larger molecules. As a proof of principle we present the excited state dynamics of a (6-4)-lesion containing dinucleotide (63 atoms), and especially the importance to include the confinement effects of the DNA backbone. The method is able to include electron correlation on a high level of theory and offers an attractive alternative to QM:MM approaches for moderate sized systems with unknown force fields.
Cascaded Photoinduced Drug Delivery to Cells from Multifunctional Core–Shell Mesoporous Silica
02.05.2012
Advanced Healthcare Materials,
2012,
DOI: 10.1002/adhm.201100033,
Volume 1, Issue 3, pages 316–320
published on 02.05.2012
Advanced Healthcare Materials, online article
Advanced Healthcare Materials, online article
Different bioactive molecules are released into living cells from lipid-covered mesoporous silica nanoparticles. The release is triggered by light, as the particles feature covalently attached photosensitizers as membrane-opening agents. It is demonstrated that the particles achieve endosomal escape and that they release their cargo into the cytosol.
DNA Origami Gatekeepers for Solid-State Nanopores
04.04.2012
Angewandte Chemie,
2012,
DOI: 10.1002/anie.201200688,
published on 04.04.2012
Angewandte Chemie, online article
Angewandte Chemie, online article
Molecular self-assembly with DNA enables the construction of soluble objects with nanometer to micrometer scale absolute dimensions and custom chemical features, including crystals, patterns, bricks, boxes, and curved shapes, that can open novel paths to scientific discovery. Herein, we report on DNA nanoplates for nanopore-based sensing approaches. Nanopores in biological or solid-state membranes offer great potential for label-free single-molecule sensing applications. Biological nanopores, such as alpha-hemolysin, can be customized within the limits of protein engineering. Artificial nanopores in solid-state membranes can be made with user-defined dimensions, but chemical modifications require substantial effort. A challenge
in the field is to gain control over both the geometrical
and chemical specifications of nanopores. We hypothesized that using DNA-based nanoplates as covers for solid-state nanopores could provide a route for meeting this challenge.
Light-Switchable Hemithioindigo–Hemistilbene-Containing Peptides: Ultrafast Spectroscopy of the Z → E Isomerization of the Chromophore and the Structural Dynamics of the Peptide Moiety
16.03.2012
The Journal of Physical Chemistry B,
2012,
DOI: 10.1021/jp300982a,
116 (14), pp 4181–4191
published on 16.03.2012
The Journal of Physical Chemistry B, online article
The Journal of Physical Chemistry B, online article
Two hemithioindigo–hemistilbene (HTI) derivatives, designed to operate as structural switches in peptides, as well as two HTI peptides are characterized by ultrafast spectroscopy in the visible and the infrared. The two HTI switches follow the reaction scheme published for other HTI compounds with a picosecond excited state reaction (τ1 ≈ 6 ps) and isomerization from Z to E with τ2 = 13 and 51 ps. As compared to the isolated chromophores, the isomerization reaction is slowed down in the chromopeptides to τ2 = 24 and 69 ps. For the smaller peptide containing 6 amino acids, the structural changes of the peptide moiety observed via the IR spectrum in the amide I band follow the isomerization of the molecular switch closely. In the larger cyclic chromopeptide, containing 20 amino acids and mimicking a β-hairpin structure in the Z-form of the chromophore, the peptide moiety also changes its structure during isomerization of the chromophore. However, the IR spectrum at the end of the observation period of 3 ns deviates significantly from the stationary difference spectrum. These signatures indicate that strong additional structural changes, e.g., breaking of interchain hydrogen bonds, also occur on longer time scales.
Direct Visualization of Dye and Oligonucleotide Diffusion in Silica Filaments with Collinear Mesopores
14.03.2012
Nano Letters,
2012,
DOI: 10.1021/nl2039474,
12 (3), pp 1354–1361
published on 14.03.2012
Nano Letters, online aticle
Nano Letters, online aticle
The diffusion dynamics of terrylene diimide (TDI) dye molecules and dye-labeled double-strand DNA were studied in micrometer long silica filaments containing collinear, oriented mesopores using single molecule fluorescence microscopy. TDI was used as a stable and hydrophobic probe molecule for single molecule structural analysis. We used template-free mesoporous silica filaments with 4 nm pore diameter and chemical functionalization with one or two types of trialkoxysilane groups to enhance the affinity between the host system and the guest molecules. Insights about the mesoporous structure as well as the translational and orientational diffusion dynamics of the guest molecules observed along micrometer long trajectories could be obtained. Additionally, the stability of DNA oligomers (15 base pairs, bp, about 5.3 nm long) within the mesopores was examined, showing no degradation of the oligonucleotide upon incorporation into the mesopores. Diffusion of both guest molecules could be controlled by exposure to vapors of water or chloroform; the latter both induced a reversible on–off control of the translational movement of the molecules.
Mechanism of UV-Induced Formation of Dewar Lesions in DNA
09.01.2012
Angewandte Chemie,
2011,
DOI: 10.1002/anie.201106231,
Volume 51, Issue 2, pages 408–411,
published on 09.01.2012
Angewandte Chemie, online article
Angewandte Chemie, online article
Living organisms exposed to sunlight are constantly challenged
by the formation of UV-induced lesions in DNA, which induce cell death and cause mutations. UVirradiation of TpT and TpC sequences leads to the formation of two primary lesions, namely cyclobutane-pyrimidine dimers (CPD)and lesions, as depicted in Scheme 1. These lesions possess an additional absorption at lmax=320 nm and rearrange to give Dewar valence isomers.
Heat shock protein 90’s mechanochemical cycle is dominated by thermal fluctuations
03.01.2012
PNAS,
2011,
doi: 10.1073/pnas.1107930108,
vol. 109, 161-166
published on 03.01.2012
PNAS, online article
PNAS, online article
The molecular chaperone and heat shock protein 90 (Hsp90) exists mainly as a homodimer in the cytoplasm. Each monomer has an ATPase in its N-terminal domain and undergoes large conformational changes during Hsp90’s mechanochemical cycle. The threecolor single-molecule assay and data analysis presented in the following allows one to observe at the same time nucleotide binding and the conformational changes in Hsp90. Surprisingly, and completely unlike the prior investigated systems, nucleotides can bind to the N-terminally open and closed state without strictly forcing the protein into a specific conformation. Both the transitions between the conformational states and the nucleotide binding/ unbinding are mainly thermally driven. Furthermore, the two ATP binding sites show negative cooperativity; i.e., nucleotides do not bind independently to the two monomers.We thus reveal a picture
of how nucleotide binding and conformational changes are connectedin the molecular chaperone Hsp90, which has far-ranging
conseque nces for its function and is distinct from previously investigated motor proteins.
