Supramolecular DNA-based catalysis in organic solvents.

The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these "supraG4zymes" enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments.

Late-Stage Modification of Aminoglycoside Antibiotics Overcomes Bacterial Resistance Mediated by APH(3') Kinases.

The continuous emergence of antimicrobial resistance is causing a threat to patients infected by multidrug-resistant pathogens. In particular, the clinical use of aminoglycoside antibiotics, broad-spectrum antibacterials of last resort, is limited due to rising bacterial resistance. One of the major resistance mechanisms in Gram-positive and Gram-negative bacteria is phosphorylation of these amino sugars at the 3'-position by O-phosphotransferases [APH(3')s]. Structural alteration of these antibiotics at the 3'-position would be an obvious strategy to tackle this resistance mechanism. However, the access to such derivatives requires cumbersome multi-step synthesis, which is not appealing for pharma industry in this low-return-on-investment market. To overcome this obstacle and combat bacterial resistance mediated by APH(3')s, we introduce a novel regioselective modification of aminoglycosides in the 3'-position via palladium-catalyzed oxidation. To underline the effectiveness of our method for structural modification of aminoglycosides, we have developed two novel antibiotic candidates overcoming APH(3')s-mediated resistance employing only four synthetic steps.

Highly Stiff and Stretchable DNA Liquid Crystalline Organogels with Super Plasticity, Ultrafast Self-Healing, and Magnetic Response Behaviors.

DNA-based gels are attractive materials as they allow intuitive rational design, respond to external physicochemical stimuli, and show great potential for biomedical applications. However, their relatively poor mechanical properties currently limit their technological application considerably as the latter requires mechanical integrity and tunability. With this work, a DNA organogel is reported that gels through supramolecular interactions, which induce mesophase ordering, and that exhibits exceptional stretchability, deformability, plasticity, and biocompatibility. Moreover, the nature of the supramolecular bond enables complete self-healing within 3 s. Most importantly, the DNA-based liquid crystalline organogels exhibit impressive ultimate tensile strengths above 1 MPa, stiffness higher than 20 MPa, and toughness up to 18 MJ m-3 , rendering these materials the strongest among reported DNA networks. In addition, the facile access is demonstrated to composite DNA materials by blending magnetic nanoparticles with the organogel matrix giving access to magnetic field induced actuation. It is believed that these findings contribute significantly to the advancement of DNA gels for their use in smart materials and biomedical applications.

Carrier-free micellar CpG interacting with cell membrane for enhanced immunological treatment of HIV-1.

Unmethylated CpG motifs activate toll-like receptor 9 (TLR9), leading to sequence- and species-specific immune stimulation. Here, we engineered a CpG oligodeoxyribonucleotide (ODN) with multiple hydrophobic moieties, so-called lipid-modified uracil, which resulted in a facile micelle formation of the stimulant. The self-assembled CpG nanostructure (U4CpG) containing the ODN 2216 sequence was characterized by various spectroscopic and microscopic methods together with molecular dynamics simulations. Next, we evaluated the nano-immunostimulant for enhancement of anti-HIV immunity. U4CpG treatment induced activation of plasmacytoid dendritic cells (pDCs) and natural killer (NK) cells in healthy human peripheral blood, which produced type I interferons (IFNs) and IFN-γ in human peripheral blood mononuclear cells (PBMCs). Moreover, we validated the activation and promotion efficacy of U4CpG in patient-derived blood cells, and HIV-1 spread was significantly suppressed by a low dosage of the immunostimulant. Furthermore, U4CpG-treated PBMC cultured medium elicited transcription of latent HIV-1 in U1 cells indicating that U4CpG reversed HIV-1 latency. Thus, the functions of U4CpG in eradicating HIV-1 by enhancing immunity and reversing latency make the material a potential candidate for clinical studies dealing with viral infection.

Fullerenes Enhance Self-Assembly and Electron Injection of Photosystem I in Biophotovoltaic Devices.

This paper describes the fabrication of microfluidic devices with a focus on controlling the orientation of photosystem I (PSI) complexes, which directly affects the performance of biophotovoltaic devices by maximizing the efficiency of the extraction of electron/hole pairs from the complexes. The surface chemistry of the electrode on which the complexes assemble plays a critical role in their orientation. We compared the degree of orientation on self-assembled monolayers of phenyl-C61-butyric acid and a custom peptide on nanostructured gold electrodes. Biophotovoltaic devices fabricated with the C61 fulleroid exhibit significantly improved performance and reproducibility compared to those utilizing the peptide, yielding a 1.6-fold increase in efficiency. In addition, the C61-based devices were more stable under continuous illumination. Our findings show that fulleroids, which are well-known acceptor materials in organic photovoltaic devices, facilitate the extraction of electrons from PSI complexes without sacrificing control over the orientation of the complexes, highlighting this combination of traditional organic semiconductors with biomolecules as a viable approach to coopting natural photosynthetic systems for use in solar cells.

Mechanochemical bond scission for the activation of drugs.

Pharmaceutical drug therapy is often hindered by issues caused by poor drug selectivity, including unwanted side effects and drug resistance. Spatial and temporal control over drug activation in response to stimuli is a promising strategy to attenuate and circumvent these problems. Here we use ultrasound to activate drugs from inactive macromolecules or nano-assemblies through the controlled scission of mechanochemically labile covalent bonds and weak non-covalent bonds. We show that a polymer with a disulfide motif at the centre of the main chain releases an alkaloid-based anticancer drug from its ß-carbonate linker by a force-induced intramolecular 5-exo-trig cyclization. Second, aminoglycoside antibiotics complexed by a multi-aptamer RNA structure are activated by the mechanochemical opening and scission of the nucleic acid backbone. Lastly, nanoparticle-polymer and nanoparticle-nanoparticle assemblies held together by hydrogen bonds between the peptide antibiotic vancomycin and its complementary peptide target are activated by force-induced scission of hydrogen bonds. This work demonstrates the potential of ultrasound to activate mechanoresponsive prodrug systems.

Controlling Optical and Catalytic Activity of Genetically Engineered Proteins by Ultrasound.

Ultrasound (US) produces cavitation-induced mechanical forces stretching and breaking polymer chains in solution. This type of polymer mechanochemistry is widely used for synthetic polymers, but not biomacromolecules, even though US is biocompatible and commonly used for medical therapy as well as in vivo imaging. The ability to control protein activity by US would thus be a major stepping-stone for these disciplines. Here, we provide the first examples of selective protein activation and deactivation by means of US. Using GFP as a model system, we engineer US sensitivity into proteins by design. The incorporation of long and highly charged domains enables the efficient transfer of force to the protein structure. We then use this principle to activate the catalytic activity of trypsin by inducing the release of its inhibitor. We expect that this concept to switch "on" and "off" protein activity by US will serve as a blueprint to remotely control other bioactive molecules.

DNA hybridization as a general method to enhance the cellular uptake of nanostructures.

The biomedical application of nanoparticles (NPs) for diagnosis and therapy is considerably stalled by their inefficient cellular internalization. Many strategies to overcome this obstacle have been developed but are not generally applicable to different NP systems, consequently underlining the need for a universal method that enhances NP entry into cells. Here we describe a method to increase NP cellular uptake via strand hybridization between DNA-functionalized NPs and cells that bear the respective complementary sequence incorporated into the membrane. By this, the NPs bind efficiently to the cellular surface enhancing internalization of three completely different NP types: DNA tetrahedrons, gold (Au) NPs, and polystyrene (PS) NPs. We show that our approach is a simple and generalizable strategy that can be applied to virtually every functionalizable NP system.

Modular and Versatile Trans-Encoded Genetic Switches.

Current bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans-encoded and based on tRNA-mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA.

Fast, Efficient, and Targeted Liposome Delivery Mediated by DNA Hybridization.

Safety and efficacy, two significant parameters in drug administration, can be improved by site-specific delivery approaches. Here a fast, efficient, and targeted liposome delivery system steered by a DNA hybridization recognition mechanism is presented. For this purpose, lipid-terminated DNA is inserted in both liposome and cell membranes by simple mixing of the components. Cellular accumulation of cargo encapsulated in the liposomal core is substantially enhanced when the DNA sequence on the cell is complementary to that on the liposome. Additionally, in mixed cell populations, liposomes discriminate targets by their complementary DNA sequences. Exposure of cells to low temperature and endocytosis inhibitors suggests a caveolae-dependent endocytosis uptake pathway. Mechanistically, hybridization between DNA strands spatially traps liposomes and cell membranes in close proximity, consequently increases the local liposome concentration, and thereby enhances cellular uptake of liposomes and their payload. This programmable delivery system might contribute to new applications in molecular biology and drug delivery.

Electrostatically PEGylated DNA enables salt-free hybridization in water.

Chemically modified nucleic acids have long served as a very important class of bio-hybrid structures. In particular, the modification with PEG has advanced the scope and performance of oligonucleotides in materials science, catalysis and therapeutics. Most of the applications involving pristine or modified DNA rely on the potential of DNA to form a double-stranded structure. However, a substantial requirement for metal-cations to achieve hybridization has restricted the range of applications. To extend the applicability of DNA in salt-free or low ionic strength aqueous medium, we introduce noncovalent DNA-PEG constructs that allow canonical base-pairing between individually PEGylated complementary strands resulting in a double-stranded structure in salt-free aqueous medium. This method relies on grafting of amino-terminated PEG polymers electrostatically onto the backbone of DNA, which results in the formation of a PEG-envelope. The specific charge interaction of PEG molecules with DNA, absolute absence of metal ions within the PEGylated DNA molecules and formation of a double helix that is significantly more stable than the duplex in an ionic buffer have been unequivocally demonstrated using multiple independent characterization techniques.

Self-Regenerating Soft Biophotovoltaic Devices.

This paper describes the fabrication of soft, stretchable biophotovoltaic devices that generate photocurrent from photosystem I (PSI) complexes that are self-assembled onto Au electrodes with a preferred orientation. Charge is collected by the direct injection of electrons into the Au electrode and the transport of holes through a redox couple to liquid eutectic gallium-indium (EGaIn) electrodes that are confined to microfluidic pseudochannels by arrays of posts. The pseudochannels are defined in a single fabrication step that leverages the non-Newtonian rheology of EGaIn. This strategy is extended to the fabrication of reticulated electrodes that are inherently stretchable. A simple shadow evaporation technique is used to increase the surface area of the Au electrodes by a factor of approximately 106 compared to planar electrodes. The power conversion efficiency of the biophotovoltaic devices decreases over time, presumably as the PSI complexes denature and/or detach from the Au electrodes. However, by circulating a solution of active PSI complexes the devices self-regenerate by mass action/self-assembly. These devices leverage simple fabrication techniques to produce complex function and prove that photovoltaic devices comprising PSI can retain the ability to regenerate, one of the most important functions of photosynthetic organisms.

Accelerating chemical reactions by molecular sledding.

The speed-up of covalent bond formation was achieved between a sulfhydryl group and a 2-bromopropionic acid derivative by utilizing sliding peptide-modified substrates. Moreover, a new type of DNA cleaving reagent was developed, consisting of pVIc covalently coupled to verteporfin. This peptide-porphyrin conjugate allowed targeting of DNA and resulted in increased photodegradation of double-stranded nucleic acids.

Orientation and Incorporation of Photosystem I in Bioelectronics Devices Enabled by Phage Display.

Interfacing proteins with electrode surfaces is important for the field of bioelectronics. Here, a general concept based on phage display is presented to evolve small peptide binders for immobilizing and orienting large protein complexes on semiconducting substrates. Employing this method, photosystem I is incorporated into solid-state biophotovoltaic cells.

Tuning Ice Nucleation with Supercharged Polypeptides.

Supercharged unfolded polypeptides (SUPs) are exploited for controlling ice nucleation via tuning the nature of charge and charge density of SUPs. The results show that positively charged SUPs facilitate ice nucleation, while negatively charged ones suppress it. Moreover, the charge density of the SUP backbone is another parameter to control it.

Filling the Green Gap of a Megadalton Photosystem I Complex by Conjugation of Organic Dyes.

Photosynthesis is Nature's major process for converting solar into chemical energy. One of the key players in this process is the multiprotein complex photosystem I (PSI) that through absorption of incident photons enables electron transfer, which makes this protein attractive for applications in bioinspired photoactive hybrid materials. However, the efficiency of PSI is still limited by its poor absorption in the green part of the solar spectrum. Inspired by the existence of natural phycobilisome light-harvesting antennae, we have widened the absorption spectrum of PSI by covalent attachment of synthetic dyes to the protein backbone. Steady-state and time-resolved photoluminescence reveal that energy transfer occurs from these dyes to PSI. It is shown by oxygen-consumption measurements that subsequent charge generation is substantially enhanced under broad and narrow band excitation. Ultimately, surface photovoltage (SPV) experiments prove the enhanced activity of dye-modified PSI even in the solid state.

Functional Exchangeability of Oxidase and Dehydrogenase Reactions in the Biosynthesis of Hydroxyphenylglycine, a Nonribosomal Peptide Building Block.

A key problem in the engineering of pathways for the production of pharmaceutical compounds is the limited diversity of biosynthetic enzymes, which restricts the attainability of suitable traits such as less harmful byproducts, enhanced expression features, or different cofactor requirements. A promising synthetic biology approach is to redesign the biosynthetic pathway by replacing the native enzymes by heterologous proteins from unrelated pathways. In this study, we applied this method to effectively re-engineer the biosynthesis of hydroxyphenylglycine (HPG), a building block for the calcium-dependent antibiotic of Streptomyces coelicolor, a nonribosomal peptide. A key step in HPG biosynthesis is the conversion of 4-hydroxymandelate to 4-hydroxyphenylglyoxylate, catalyzed by hydroxymandelate oxidase (HmO), with concomitant generation of H2O2. The same reaction can also be catalyzed by O2-independent mandelate dehydrogenase (MdlB), which is a catabolic enzyme involved in bacterial mandelate utilization. In this work, we engineered alternative HPG biosynthetic pathways by replacing the native HmO in S. coelicolor by both heterologous oxidases and MdlB dehydrogenases from various sources and confirmed the restoration of calcium-dependent antibiotic biosynthesis by biological and UHPLC-MS analysis. The alternative enzymes were isolated and kinetically characterized, confirming their divergent substrate specificities and catalytic mechanisms. These results demonstrate that heterologous enzymes with different physiological contexts can be used in a Streptomyces host to provide an expanded library of enzymatic reactions for a synthetic biology approach. This study thus broadens the options for the engineering of antibiotic production by using enzymes with different catalytic and structural features.