helical-organization-blood-coagulation-factor-viii-on-lipid

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 ...

University of Texas Medical Branch

university of texas medical branch

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.

Building programmable jigsaw puzzles with 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.

Controlled spacing of cationic gold nanoparticles by nanocrown RNA.

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.

Self-assembly of DNA arrays into multilayer stacks.

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.

Cell-targeted self-assembled DNA nanostructures.

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.

Structure of Trypanosoma brucei flagellum accounts for its bihelical motion.

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.

Article	Year
Building programmable jigsaw puzzles with RNA. Science (New York, N.Y.)\| PubMed ID: 15604402	2004
Controlled spacing of cationic gold nanoparticles by nanocrown RNA. Journal of the American Chemical Society\| PubMed ID: 16117496	2005
Self-assembly of DNA arrays into multilayer stacks. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 19177652	2009
Cell-targeted self-assembled DNA nanostructures. Journal of the American Chemical Society\| PubMed ID: 19754205	2009
Structure of Trypanosoma brucei flagellum accounts for its bihelical motion. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21690369	2011
In Silico, In Vitro, and In Vivo Studies Indicate the Potential Use of Bolaamphiphiles for Therapeutic siRNAs Delivery. Molecular therapy. Nucleic acids\| PubMed ID: 23511334	2013

Alexey Y. Koyfman

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