Analysis of Interacting Nucleic Acids in Dilute Solutions

Motivated by the growing demand for analysis tools for diverse natural and engineered DNA and RNA systems, we develop a general theory and set of computational algorithms to perform thermodynamic analysis of dilute reactive solutions and then apply these techniques to interacting nucleic acids. The theory correctly accounts for the effects of indistinguishability in partition function calculations for complexes of interacting strands. With partition functions in hand, the unique complex concentrations corresponding to thermodynamic equilibrium are obtained by solving a convex programming problem. Partition function and concentration information can then be used to calculate equilibrium base-pairing observables corresponding to experimentally measurable properties. The underlying physics and mathematical formulation of these problems lead to an interesting blend of approaches, including ideas from graph theory, group theory, dynamic programming, combinatorics, convex optimization, and Lagrange duality. To make these analysis tools available to researchers worldwide, we present NUPACK, a web-based software suite for thermodynamic analysis of nucleic acids. Its efficacy is demonstrated in example calculations and the results are shown to be in agreement with experiment. Finally, the thermodynamic properties of a DNA-based triggered self-assembly device [1] are analyzed using NUPACK and extensions of its tools. The computational results complement experimental studies, exposing novel properties about the system and dictating further research.</p

Thermodynamic Analysis of Interacting Nucleic Acid Strands

Motivated by the analysis of natural and engineered DNA and RNA systems, we present the first algorithm for calculating the partition function of an unpseudoknotted complex of multiple interacting nucleic acid strands. This dynamic program is based on a rigorous extension of secondary structure models to the multistranded case, addressing representation and distinguishability issues that do not arise for single-stranded structures. We then derive the form of the partition function for a fixed volume containing a dilute solution of nucleic acid complexes. This expression can be evaluated explicitly for small numbers of strands, allowing the calculation of the equilibrium population distribution for each species of complex. Alternatively, for large systems (e.g., a test tube), we show that the unique complex concentrations corresponding to thermodynamic equilibrium can be obtained by solving a convex programming problem. Partition function and concentration information can then be used to calculate equilibrium base-pairing observables. The underlying physics and mathematical formulation of these problems lead to an interesting blend of approaches, including ideas from graph theory, group theory, dynamic programming, combinatorics, convex optimization, and Lagrange duality

Regulation of the formin cappuccino is critical for polarity of Drosophilao ocytes

The Drosophila formin Cappuccino (Capu) creates an actin mesh‐like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late‐oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu's function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN‐expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N‐terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development

Regulation of the formin cappuccino is critical for polarity of Drosophilao ocytes

The Drosophila formin Cappuccino (Capu) creates an actin mesh‐like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late‐oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu's function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN‐expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N‐terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development

Pulsatory Patterns in Active Fluids

We show that pulsatory patterns arise in thin active films in which two chemical species regulate active stress. The regulating species diffuse within the film and are advected by self-generated flows resulting from active stress gradients. Spontaneous pulsatory patterns emerge when the following conditions are met: (i) the fast-diffusing species up-regulates and the slow-diffusing species down-regulates active stress, or (ii) the active stress up-regulator turns over faster compared to the active stress down-regulator. Our study, motivated by pulsatory patterns in the actomyosin cortex in cells and tissues, provides a simple generic mechanism for oscillatory patterns in active fluids

The application of nitrido manganese reagents to the synthesis of protected N-methyl D-fucosamine

A synthesis of protected D-fucosamine and N-methyl fucosamine from D-galactose is described. The synthesis is carried out in ten steps and includes the amination reaction of D-fucal with an easily prepared Mn≡N reagent

Filament Assembly by Spire: Key Residues and Concerted Actin Binding

The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers

Filament Assembly by Spire: Key Residues and Concerted Actin Binding

The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers