Integration of functional peptides into nucleic acid-based nanostructures.

In many applications such as diagnostics and therapy development, small peptide fragments consisting of only a few amino acids are often attractive alternatives to bulky proteins. This is due to factors such as the ease of scalable chemical synthesis and numerous methods for their discovery. One drawback of using peptides is that their activity can often be negatively impacted by the lack of a rigid, 3D stabilizing structure provided by the rest of the protein. In many cases, this can be alleviated by different methods of rational templating onto nanomaterials, which provides additional possibilities to use concepts of multivalence or rational nano-engineering to enhance or even create new types of function or structure. In recent years, nanostructures made from the self-assembly of DNA strands have been used as scaffolds to create functional arrangements of peptides, often leading to greatly enhanced biological activity or new material properties. This review will give an overview of nano-templating approaches based on the combination of DNA nanotechnology and peptides. This will include both bioengineering strategies to control interactions with cells or other biological systems, as well as examples where the combination of DNA and peptides has been leveraged for the rational design of new functional materials.

Constraint Release for Reptating Filaments in Semiflexible Networks Depends on Background Fluctuations.

Entangled semiflexible polymer networks are usually described by the tube model, although this concept has not been able to explain all experimental observations. One of its major shortcomings is neglecting the thermal fluctuations of the polymers surrounding the examined test filament, such that disentanglement effects are not captured. In this study, we present experimental evidence that correlated constraint release which has been predicted theoretically occurs in entangled, but not in crosslinked semiflexible polymer networks. By tracking single semiflexible DNA nanotubes embedded both in entangled and crosslinked F-actin networks, we observed different reptation dynamics in both systems, emphasizing the need for a revision of the classical tube theory for entangled polymer solutions.

Measuring structural parameters of crosslinked and entangled semiflexible polymer networks with single-filament tracing.

Single-filament tracing has been a valuable tool to directly determine geometrical and mechanical properties of entangled polymer networks. However, systematically verifying how the stiffness of the tracer filament or its molecular interactions with the surrounding network impacts the measurement of these parameters has not been possible with the established experimental systems. Here we use mechanically programmable DNA nanotubes embedded in crosslinked and entangled F-actin networks, as well as in synthetic DNA networks, in order to measure fundamental, structural network properties like tube width and mesh size with respect to the stiffness of the tracers. While we confirm some predictions derived from models based purely on steric interactions, our results indicate that these models should be expanded to account for additional interfilament interactions, thereby describing the behavior of real polymer networks.

Cells in Slow Motion: Apparent Undercooling Increases Glassy Behavior at Physiological Temperatures.

Solvent conditions are unexpectedly sufficient to drastically and reversibly slow down cells. In vitro on the molecular level, protein-solvent interactions drastically change in the presence of heavy water (D2 O) and its stronger hydrogen bonds. Adding D2 O to the cell medium of living cells increases the molecular intracellular viscosity. While cell morphology and phenotype remain unchanged, cellular dynamics transform into slow motion in a changeable manner. This is exemplified in the slowdown of cell proliferation and migration, which is caused by a reversible gelation of the cytoplasm. In analogy to the time-temperature superposition principle, where temperature is replaced by D2 O, an increase in viscosity slows down the effective time. Actin networks, crucial structures in the cytoplasm, switch from a power-law-like viscoelastic to a more rubber-like elastic behavior. The resulting intracellular resistance and dissipation impair cell movement. Since cells are highly adaptive non-equilibrium systems, they usually respond irreversibly from a thermodynamic perspective. D2 O induced changes, however, are fully reversible and their effects are independent of signaling as well as expression. The stronger hydrogen bonds lead to glass-like, drawn-out intramolecular dynamics, which may facilitate longer storage times of biological matter, for instance, during transport of organ transplants.

Charge-Shifting Polycations Based on N,N-(dimethylamino)ethyl Acrylate for Improving Cytocompatibility During DNA Delivery.

Synthetic polycations are studied extensively as DNA delivery agents because of their ease of production, good chemical stability, and low cost relative to viral vectors. This report describes the synthesis of charge-shifting polycations based on N,N-(dimethylamino)ethyl acrylate (DMAEA) and 3-aminopropylmethacryamide (APM), called PAD copolymers, and their use for in vitro DNA delivery into HeLa cells. PAD copolymers of varying compositions were prepared by RAFT polymerization to yield polymers of controlled molecular weights with low dispersities. Model hydrolysis studies were carried out to assess the rate of charge-shifting of the polycations by loss of the cationic dimethylaminoethanol side chains. They showed reduction in the net cationic charge by about 10-50% depending on composition after 2 days at pH 7, forming polyampholytes comprising permanent cationic groups, residual DMAEA, as well as anionic acrylic acid groups. HeLa cells exposed for 4 h to PAD copolymers with the greatest charge-shifting ability showed comparable or higher viability at high concentrations, relative to the noncharge shifting polycations PAPM and polyethyleneimine (PEI) 2 days post-exposure. Cell uptake efficiency of PAD/60bp-Cy3 DNA polyplexes at 2.5:1 N/P ratio was very high (>95%) for all compositions, exceeding the uptake efficiency of PEI polyplexes of equivalent composition. These results suggest that these PAD copolymers, and in particular PAD80 containing 80 mol % DMAEA, have suitable rates of charge-shifting hydrolysis for DNA delivery, as PAD80 showed reduced cytotoxicity at high concentrations, while still retaining high uptake efficiencies. In addition, the polyampholytes formed during DMAEA hydrolysis in PAD copolymers can offer enhanced long-term cytocompatibility.