Saikat Chakraborty

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.

The poly dA helix: a new structural motif for high performance DNA-based molecular switches

We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA15) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH+-H+A base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA15. The pH-triggered transition between the two defined helical forms of dA15 is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA15 represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.

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

Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing

MicroRNAs control gene expression either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. How this regulation is achieved and maintained is still largely unknown. MiRNAs that reside on clustered or polycistronic transcripts represent a more complex case where individual miRNAs from a cluster are processed with different efficiencies despite being cotranscribed. To shed light on the regulatory mechanisms that might be operating in these cases, we considered the long polycistronic primary miRNA transcript pri-miR-17-92a that contains six miRNAs with diverse functions. The six miRNA domains on this cluster are differentially processed to produce varying amounts of resultant mature miRNAs in different tissues. How this is achieved is not known. We show, using various biochemical and biophysical methods coupled with mutational studies, that pri-miR-17-92a adopts a specific three-dimensional architecture that poses a kinetic barrier to its own processing. This tertiary structure could create suboptimal protein recognition sites on the pri-miRNA cluster due to higher-order structure formation.

The RNA2–PNA2 hybrid i-motif—a novel RNA-based building block

We report the formation of a hybrid RNA2-PNA2 i-motif comprised of two RNA and two PNA strands based on the sequence specific self assembly of RNA, with potential as a building block for structural RNA nanotechnology.

Kinetic hybrid i-motifs: Intercepting DNA with RNA to form a DNA2–RNA2 i-motif

We have constructed and characterized a long-lived hybrid DNA(2)-RNA(2) i-motif that is kinetically formed by mixing equivalent amount of C-rich RNA (R) and C-rich DNA (D). Circular dichroism shows that these hybrids are distinct from their parent DNA(4) or RNA(4) i-motif. pH dependent CD and UV thermal melting experiments showed that the complexes were maximally stable at pH 4.5, the pK(a) of cytosine, consistent with the complex being held by CH(+)-C base pairs. Fluorescence studies confirmed their tetrameric nature and established the relative strand polarities of the RNA and DNA strands in the complex. These showed that in a hybrid D(2)R(2) i-motif two DNA strands occupy one narrow groove and the two RNA strands occupy the other. This suggests that even the sugar-sugar interactions are highly specific. Interestingly, this hybrid slowly disproportionates into DNA(4) i-motifs and ssRNA which would be valuable to study intermediates in DNA(4) i-motif formation.

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.

The Predictive Power of Synthetic Nucleic Acid Technologies in RNA Biology

CONSPECTUS: The impact of nucleic acid nanotechnology in terms of transforming motifs from biology in synthetic and translational ways is widely appreciated. But it is also emerging that the thinking and vision behind nucleic acids as construction material has broader implications, not just in nanotechnology or even synthetic biology, but can feed back into our understanding of biology itself. Physicists have treated nucleic acids as polymers and connected physical principles to biology by abstracting out the molecular interactions. In contrast, biologists delineate molecular players and pathways related to nucleic acids and how they may be networked. But in vitro nucleic acid nanotechnology has provided a valuable framework for nucleic acids by connecting its biomolecular interactions with its materials properties and thereby superarchitecture ultramanipulation that on multiple occasions has pre-empted the elucidation of how living cells themselves are exploiting these same structural concepts. This Account seeks to showcase the larger implications of certain architectural principles that have arisen from the field of structural DNA/RNA nanotechnology in biology. Here we draw connections between these principles and particular molecular phenomena within living systems that have fed in to our understanding of how the cell uses nucleic acids as construction material to achieve different functions. We illustrate this by considering a few exciting and emerging examples in biology in the context of both switchable systems and scaffolding type systems. Due to the scope of this Account, we will focus our discussion on examples of the RNA scaffold as summarized. In the context of switchable RNA architectures, the synthetic demonstration of small molecules blocking RNA translation preceded the discovery of riboswitches. In another example, it was after the description of aptazymes that the first allosteric ribozyme, glmS, was discovered. In the context of RNA architectures as structural scaffolds, there are clear parallels between DNA origami and the recently emerging molecular mechanism of heterochromatin formation by Xist RNA. Further, following the construction of well-defined 2D DNA-protein architectures, the striking observation of remarkably sculpted 2D RNA-protein hydrogel sheets in Caenorhabditis elegans speaks to the in vivo relevance of designer nucleic acid architectures. It is noteworthy that discoveries of properties in synthetic space seem to precede the uncovering of similar phenomena in vivo.

A structural map of oncomiR-1 at single-nucleotide resolution

Abstract The miR-17–92a cluster, also known as ‘oncomiR-1’, is an RNA transcript that plays a pivotal regulatory role in cellular processes, including the cell cycle, proliferation and apoptosis. Its dysregulation underlies the development of several cancers. Oncomir-1 comprises six constituent miRNAs, each processed with different efficiencies as a function of both developmental time and tissue type. The structural mechanisms that regulate such differential processing are unknown, and this has impeded our understanding of the dysregulation of oncomiR-1 in pathophysiology. By probing the sensitivity of each nucleotide in oncomiR-1 to reactive small molecules, we present a secondary structural map of this RNA at single-nucleotide resolution. The secondary structure and solvent accessible regions of oncomiR-1 reveal that most of its primary microRNA domains are suboptimal substrates for Drosha-DGCR8, and therefore resistant to microprocessing. The structure indicates that the binding of trans-acting factors is required to remodel the tertiary organization and unmask cryptic primary microRNA domains to facilitate their processing into pre-microRNAs.

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.