Multifunctional RNA nanoparticles.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Structure of Trypanosoma brucei flagellum accounts for its bihelical motion.

Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crystallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme motion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography.

Cell-targeted self-assembled DNA nanostructures.

We present two strategies for attaching self-assembled DNA arrays to the surfaces of cells. Our first approach uses biotin-streptavidin interactions to bind DNA architectures to biotinylated cells. The second approach takes advantage of specific antibody-cell surface interactions, conjugated arrays and the subsequent binding to native epidermal growth factor receptors expressed on cancer cells. DNA array-cell surface interactions were visualized by fluorescence, confocal microscopy, and scanning electron microscopy. This novel application of DNA nanoarrays provides strategies to specifically label cell surfaces with micrometer-sized patches, bind cells onto a designed functionalized DNA scaffold, engineer cell/cell networks into microtissues, and deliver materials to cell surfaces.

Enhancing the Magnitude of Antibody Responses through Biomaterial Stereochemistry.

d-Amino acid analogs of peptides and proteins are attractive for applications in biotechnology and medicine due to their reduced proteolytic sensitivity. Here, we report that self-assembling peptide nanofibers composed of d-amino acids act as immune adjuvants, and investigate their ability to induce antibody responses in comparison to their l-amino acid counterparts. The model antigenic peptide OVA (chicken egg ovalbumin aa 323-339) from chicken egg ovalbumin, known to elicit antibody responses in mice, was linked to an l- or d-amino acid self-assembling peptide domain to generate enantiomeric nanofibers displaying the same epitope. The chiral nature of the fusion peptides was confirmed by circular dichrosim spectroscopy and transmission electron microscopy studies indicated that OVA-bearing enantiomers self-assembled into nanofibers with similar morphologies. In mice, d-amino acid peptide nanofibers displaying OVA elicited stronger antibody responses, equivalent levels of CD4+ T cell responses, and long-term antigen-presentation in vivo compared to l-amino acid nanofibers. Our findings indicate that self-assembling peptides composed of d-amino acids are strong immune adjuvants and that biomaterial stereochemistry can be used as a design tool to program adaptive immune responses for vaccine development.

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes.

Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.

In this study we have investigated a new class of cationic lipids--"bolaamphiphiles" or "bolas"--for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5µg/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production.

Helical organization of blood coagulation factor VIII on lipid nanotubes.

Cryo-electron microscopy (Cryo-EM)(1) is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment(2). Coagulation Factor VIII (FVIII)(3) is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting(4). Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete(5). Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa(6,7). In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

In Silico, In Vitro, and In Vivo Studies Indicate the Potential Use of Bolaamphiphiles for Therapeutic siRNAs Delivery.

Specific small interfering RNAs (siRNAs) designed to silence different oncogenic pathways can be used for cancer therapy. However, non-modified naked siRNAs have short half-lives in blood serum and encounter difficulties in crossing biological membranes due to their negative charge. These obstacles can be overcome by using siRNAs complexed with bolaamphiphiles, consisting of two positively charged head groups that flank an internal hydrophobic chain. Bolaamphiphiles have relatively low toxicities, long persistence in the blood stream, and most importantly, in aqueous conditions can form poly-cationic micelles thus, becoming amenable to association with siRNAs. Herein, two different bolaamphiphiles with acetylcholine head groups attached to an alkyl chain in two distinct configurations are compared for their abilities to complex with siRNAs and deliver them into cells inducing gene silencing. Our explicit solvent molecular dynamics (MD) simulations showed that bolaamphiphiles associate with siRNAs due to electrostatic, hydrogen bonding, and hydrophobic interactions. These in silico studies are supported by various in vitro and in cell culture experimental techniques as well as by some in vivo studies. Results demonstrate that depending on the application, the extent of siRNA chemical protection, delivery efficiency, and further intracellular release can be varied by simply changing the type of bolaamphiphile used.Molecular Therapy-Nucleic Acids (2013) 2, e80; doi:10.1038/mtna.2013.5; published online 19 March 2013.

Self-assembly of DNA arrays into multilayer stacks.

We describe the self-assembly of multilayer hexagonal DNA arrays through highly regular interlayer packing. Slow cooling of a mixture of three single-stranded DNA sequences with various Mg2+ concentrations leads to the self-assembly of diverse multilayer architectures. The self-assembled aggregates were deposited onto mica surfaces and examined with atomic force microscopy. The size of the two-dimensional arrays and subsequent stacking to form multilayer structures are highly dependent on Mg2+ concentration. DNA bilayers and multilayers of defined shape are favored in 2-5 mM Mg2+ with an average lateral size of 700 nm. Arrays are much larger (up to 20 microm across) in 10-15 mM Mg2+, although multiple layers still make up 20-60% of the observed structures. Domains within single layer architectures were identified using Moiré pattern analysis. Distinct structural phases within the multilayer assemblies include two layers translated by 17.5 nm and interlayer rotations of 20 degrees and 30 degrees. Three layer assemblies have cubic close packing and taller multilayer architectures of 2D DNA sheets were also identified.

Controlled spacing of cationic gold nanoparticles by nanocrown RNA.

Using atomic force microscopy, we describe the linear arrangement of cationic gold nanoparticles directed by programmable self-assembling RNA ladders and demonstrate that the regular spacing of nanoparticles is controlled by the RNA architecture acting as nanocrown scaffoldings. Thus, precise positioning of molecular components can be accomplished with RNA not only through electrostatic but also via size and shape recognitions.

Building programmable jigsaw puzzles with RNA.

One challenge in supramolecular chemistry is the design of versatile, self-assembling building blocks to attain total control of arrangement of matter at a molecular level. We have achieved reliable prediction and design of the three-dimensional structure of artificial RNA building blocks to generate molecular jigsaw puzzle units called tectosquares. They can be programmed with control over their geometry, topology, directionality, and addressability to algorithmically self-assemble into a variety of complex nanoscopic fabrics with predefined periodic and aperiodic patterns and finite dimensions. This work emphasizes the modular and hierarchical characteristics of RNA by showing that small RNA structural motifs can code the precise topology of large molecular architectures. It demonstrates that fully addressable materials based on RNA can be synthesized and provides insights into self-assembly processes involving large populations of RNA molecules.