Dhiraj Bhatia

A synthetic icosahedral DNA-based host-cargo complex for functional in vivo imaging.

The encapsulation of molecular cargo within well-defined supramolecular architectures is highly challenging. Synthetic hosts are desirable because of their well-defined nature and addressability. Encapsulation of biomacromolecules within synthetic hosts is especially challenging because of the formers large size, sensitive nature, retention of functionality post-encapsulation and demonstration of control over the cargo. Here we encapsulate a fluorescent biopolymer that functions as a pH reporter within synthetic, DNA-based icosahedral host without molecular recognition between host and cargo. Only those cells bearing receptors for the DNA casing of the host-cargo complex engulf it. We show that the encapsulated cargo is therefore uptaken cell specifically in Caenorhabditis elegans. Retention of functionality of the encapsulated cargo is quantitatively demonstrated by spatially mapping pH changes associated with endosomal maturation within the coelomocytes of C. elegans. This is the first demonstration of functionality and emergent behaviour of a synthetic host-cargo complex in vivo.

Controlled release of encapsulated cargo from a DNA icosahedron using a chemical trigger.

Controlled Release of Encapsulated Cargo from a DNA Icosahedron using a Chemical Trigger DNA Trojan horse : A DNA icosahedron (black, see scheme) held together with aptamers (red) was used to encapsulate molecular cargo like fluorescent dextran (green). In the presence of a molecular trigger (gray hexagons), the aptamers fold back leading to opening of the icosahedron and simultaneous release of the encapsulated cargo. Angewandte Chemie

Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins

Summary Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways

Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be achieved by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real time imaging of three different endocytic ligands - folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single particle tracking of Gal3 or STxB-functionalized, QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs that bear a unique stoichiometry of endocytic ligands represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics.

Synthetic, biofunctional nucleic acid-based molecular devices

Structural DNA nanotechnology seeks to create architectures of highly precise dimensions using the physical property that short lengths of DNA behave as rigid rods and the chemical property of Watson-Crick base-pairing that acts as a specific molecular glue with which such rigid rods may be joined. Thus DNA has been used as a molecular scale construction material to make molecular devices that can be broadly classified under two categories (i) rigid scaffolds and (ii) switchable architectures. This review details the growing impact of such synthetic nucleic acid based molecular devices in biology and biotechnology. Notably, a significant trend is emerging that integrates morphology-rich nucleic acid motifs and alternative molecular glues into DNA and RNA architectures to achieve biological functionality.

A method to study in vivo stability of DNA nanostructures.

DNA nanostructures are rationally designed, synthetic, nanoscale assemblies obtained from one or more DNA sequences by their self-assembly. Due to the molecularly programmable as well as modular nature of DNA, such designer DNA architectures have great potential for in cellulo and in vivo applications. However, demonstrations of functionality in living systems necessitates a method to assess the in vivo stability of the relevant nanostructures. Here, we outline a method to quantitatively assay the stability and lifetime of various DNA nanostructures in vivo. This exploits the property of intact DNA nanostructures being uptaken by the coelomocytes of the multicellular model organism Caenorhabditis elegans. These studies reveal that the present fluorescence based assay in coelomocytes of C. elegans is an useful in vivo test bed for measuring DNA nanostructure stability.

Gene delivery: Designer DNA give RNAi more spine

Precisely engineered DNA nanostructures can be used to deliver small interfering RNA molecules into cells and tumours to suppress genes.

A Method to Encapsulate Molecular Cargo Within DNA Icosahedra

DNA self-assembly has yielded various polyhedra based on platonic solids. DNA polyhedra can act as nanocapsules by entrapping various molecular entities from solution and could possibly find use in targeted delivery within living systems. A key requirement for encapsulation is that the polyhedron should have maximal encapsulation volume while maintaining minimum pore size. It is well known that platonic solids possess maximal encapsulation volumes. We therefore constructed an icosahedron from DNA using a modular self-assembly strategy. We describe a method to determine the functionality of DNA polyhedra as nanocapsules by encapsulating different cargo such as gold nanoparticles and functional biomolecules like FITC dextran from solution within DNA icosahedra.

Icosahedral DNA Nanocapsules by Modular Assembly

The construction of well-defined 3D architectures is one of the greatest challenges of self-assembly. Nanofabrication through molecular self-assembly has resulted in the formation of DNA polyhedra with the connectivities of cubes,tetrahedra, octahedra,dodecahedra,and buckminsterfullerene. DNA polyhedra could also function as nanocapsules and thereby enable the targeted delivery of entities encapsulated from solution. Key to realizing this envisaged function is the construction of complex polyhedra that maximize encapsulation volumes while preserving small pore size. Polyhedra based on platonic solids are most promising in this regard, as they maximize encapsulation volumes. We therefore constructed the most complex DNA-based platonic solid, namely, an icosahedron, through a unique modular assembly strategy and demonstrated this functional aspect for DNA polyhedra by encapsulating gold nanoparticles from solution.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Designer Nucleic Acid-Based Devices in Nanomedicine

Structural DNA nanotechnology utilizes key properties of DNA such as its persistence length and base pairing specificity to build molecularly identical architectures on the nanoscale. Of particular interest are the family of well-defined three-dimensional architectures including various polyhedra, boxes, tubes, and DNA-based dendrimers. Such scaffolded DNA architectures have recently been explored as nanoscale containers for functional molecules and as molecular breadboards to site specifically display the latter.