Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.

A highly fluorescent, non-perturbing, pentacyclic adenine analog was designed, synthesized, incorporated into DNA and photophysical evaluated.

Supramolecular self-assembly of fibrous components and liquid–liquid phase separation are at the extremes of the order-to-disorder spectrum. They collectively play key roles in cellular organization. It is still a major challenge to design systems where both highly ordered nanostructures and liquid–liquid phase-separated domains can coexist. We present a three-component assembly approach that generates fibrous domains that exclusively form inside globally disordered, liquid condensates. This is achieved by creating amphiphilic peptides that combine the features of fibrillar assembly (the amyloid domain LVFFA) and complex coacervation (oligo-arginine and adenosine triphosphate (ATP)) in one peptide, namely, LVFFAR9. When this hybrid peptide is mixed in different ratios with R9 and ATP, we find that conditions can be created where fibrous assembly is exclusively observed inside liquid coacervates. Through fluorescence and atomic force microscopy characterization, we investigate the dynamic evolution of ordered and disordered features over time. It was observed that the fibers nucleate and mature inside the droplets and that these fiber-containing liquid droplets can also undergo fusion, showing that the droplets remain liquid-like. Our work thus generates opportunities for the design of ordered structures within the confined environment of biomolecular condensates, which may be useful to create supramolecular materials in defined compartments and as model systems that can enhance understanding of ordering principles in biology.

Ultrasensitive detection of DNA is achieved via two-photon excitation of a fluorescent base analogue.

The ability to routinely detect fluorescent analogs of nucleobases at the single-molecule level would create a wealth of opportunities to study nucleic acids. We report the multiphoton-induced fluorescence and single-molecule detection of a dimethylaminesubstituted extended-6-aza-uridine (DMA th aU). We show that DMA th aU can exist in a highly fluorescent form, emitting strongly in the visible region (470-560 nm). Using pulse-shaped broadband Ti:sapphire laser excitation, DMA th aU undergoes two-photon (2P) absorption at low excitation powers, switching to threephoton (3P) absorption at high incident intensity. The assignment of a 3P process is supported by cubic response calculations. Under both 2P and 3P excitation, the single-molecule brightness was over an order of magnitude higher than reported previously for any fluorescent base analog, which facilitated the first singlemolecule detection of an emissive nucleoside with multiphoton excitation.

No abstract

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein which results in its abnormal aggregation in the nervous system. Huntingtin aggregates are linked to toxicity and neuronal dysfunction, but a comprehensive understanding of the aggregation mechanism in vivo remains elusive. Here, we examine the morphology of polyQ aggregates in Caenorhabditis elegans mechanosensory neurons as a function of age using confocal and fluorescence lifetime imaging microscopy. We find that aggregates in young worms are mostly spherical with homogenous intensity, but as the worm ages aggregates become substantially more heterogeneous. Most prominently, in older worms we observe an apparent core/shell morphology of polyQ assemblies with decreased intensity in the center. The fluorescence lifetime of polyQ is uniform across the aggregate indicating that the dimmed intensity in the assembly center is most likely not due to quenching or changes in local environment, but rather to displacement of fluorescent polyQ from the central region. This apparent core/shell architecture of polyQ aggregates in aging C. elegans neurons contributes to the diverse landscape of polyQ aggregation states implicated in Huntington's disease.

The stability and delivery efficiency of protein-based polyelectrolyte-complex micelles was evaluated for a panel of proteins with varying net charge and charge distribution.