Hari K. K. Subramanian

The Label-Free Unambiguous Detection and Symbolic Display of Single Nucleotide Polymorphisms on DNA Origami

Single nucleotide polymorphisms (SNPs) are the most common genetic variation in the human genome. Kinetic methods based on branch migration have proved successful for detecting SNPs because a mispair inhibits the progress of branch migration in the direction of the mispair. We have combined the effectiveness of kinetic methods with atomic force microscopy of DNA origami patterns to produce a direct visual readout of the target nucleotide contained in the probe sequence. The origami contains graphical representations of the four nucleotide alphabetic characters, A, T, G and C, and the symbol containing the test nucleotide identity vanishes in the presence of the probe. The system also works with pairs of probes, corresponding to heterozygous diploid genomes.

Nucleic Acid Nanostructures for Chemical and Biological Sensing

The nanoscale features of DNA have made it a useful molecule for bottom-up construction of nanomaterials, for example, two- and three-dimensional lattices, nanomachines, and nanodevices. One of the emerging applications of such DNA-based nanostructures is in chemical and biological sensing, where they have proven to be cost-effective, sensitive and have shown promise as point-of-care diagnostic tools. DNA is an ideal molecule for sensing not only because of its specificity but also because it is robust and can function under a broad range of biologically relevant temperatures and conditions. DNA nanostructure-based sensors provide biocompatibility and highly specific detection based on the molecular recognition properties of DNA. They can be used for the detection of single nucleotide polymorphism and to sense pH both in solution and in cells. They have also been used to detect clinically relevant tumor biomarkers. In this review, recent advances in DNA-based biosensors for pH, nucleic acids, tumor biomarkers and cancer cell detection are introduced. Some challenges that lie ahead for such biosensors to effectively compete with established technologies are also discussed.

Programmable RNA microstructures for coordinated delivery of siRNAs

RNA is a natural multifunctional polymer, and is an essential component in both complex pathways and structures within the cellular environment. For this reason, artificial self-assembling RNA nanostructures are emerging as a powerful tool with broad applications in drug delivery and metabolic pathway regulation. To date, coordinated delivery of functional molecules via programmable RNA assemblies has been primarily done using nanosize RNA scaffolds. However, larger scaffolds could expand existing capabilities for spatial arrangement of ligands, and enable the controlled delivery of highly concentrated molecular loads. Here, we investigate whether micron-size RNA scaffolds can be assembled and further functionalized with different cargos (e.g. various siRNAs and fluorescent tags) for their synchronized delivery to diseased cells. Since known design approaches to build large RNA scaffolds are still underdeveloped, we apply a tiling method widely used in DNA nanotechnology. DNA tiles have been extensively used to build a variety of scalable and modular structures that are easily decorated with other ligands. Here, we adapt a double crossover (DX) DNA tile motif to design de novo DX RNA tiles that assemble and form lattices via programmed sticky end interactions. We optimize assembly protocols to guarantee high yield of RNA lattices. The resulting constructs are robust and modular with respect to the presence of distinct siRNAs and fluorophores. RNA tiles and lattices are successfully transfected in either human breast cancer or prostate cancer cells, where they efficiently knockdown the expression of target genes. Blood serum stability assays indicate that RNA lattices are more resilient to nuclease degradation when compared to individual tiles, thus making them better suited for therapeutic purposes. Overall, because of its design simplicity, we anticipate that this approach will be utilized for a wide range of applications in therapeutic RNA nanotechnology.

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

Abstract Rational design of nucleic acid molecules yields self-assembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. Here we demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 μm in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowly annealed, and a one-pot transcription and anneal procedure. We identify the tile nick position as a structural requirement for lattice formation. Our results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.

pH-Driven Reversible Self-Assembly of Micron-Scale DNA Scaffolds

Inspired by cytoskeletal scaffolds that sense and respond dynamically to environmental changes and chemical inputs with a unique capacity for reconfiguration, we propose a strategy that allows the dynamic and reversible control of the growth and breakage of micron-scale synthetic DNA structures upon pH changes. We do so by rationally designing a pH-responsive system composed of synthetic DNA strands that act as pH sensors, regulators, and structural elements. Sensor strands can dynamically respond to pH changes and route regulatory strands to direct the self-assembly of structural elements into tubular structures. This example represents the first demonstration of the reversible assembly and disassembly of micron-scale DNA scaffolds using an external chemical input other than DNA. The capacity to reversibly modulate nanostructure size may promote the development of smart devices for catalysis or drug-delivery applications.

A Coarse-Grained Model of DNA Nanotube Population Growth

We derive a coarse-grained model that captures the temporal evolution of DNA nanotube length distribution during growth experiments. The model takes into account nucleation, polymerization, joining, and fragmentation processes in the nanotube population. The continuous length distribution is segmented, and the behavior of nanotubes in each length bin is modeled using ordinary differential equations. The binning choice determines the level of coarse graining. This model can handle time varying concentration of tiles, and we foresee that it will be useful to model dynamic behaviors in other types of biomolecular polymers.

Dynamic Control of Aptamer–Ligand Activity Using Strand Displacement Reactions

Nucleic acid aptamers are an expandable toolkit of sensors and regulators. To employ aptamer regulators within nonequilibrium molecular networks, the aptamer-ligand interactions should be tunable over time, so that functions within a given system can be activated or suppressed on demand. This is accomplished through complementary sequences to aptamers, which achieve programmable aptamer-ligand dissociation by displacing the aptamer from the ligand. We demonstrate the effectiveness of our simple approach on light-up aptamers as well as on aptamers inhibiting viral RNA polymerases, dynamically controlling the functionality of the aptamer-ligand complex. Mathematical models allow us to obtain estimates for the aptamer displacement kinetics. Our results suggest that aptamers, paired with their complement, could be used to build dynamic nucleic acid networks with direct control over a variety of aptamer-controllable enzymes and their downstream pathways.

Engineering DNA nanotubes for resilience in an E. coli TXTL system

Abstract Deoxyribonucleic acid (DNA) nanotechnology is a growing field with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription–translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

A coarse-grained model captures the temporal evolution of DNA nanotube length distributions

We derive a coarse-grained model that captures the temporal evolution of DNA nanotube length distribution during growth experiments. The model takes into account nucleation, polymerization, joining, and fragmentation processes in the nanotube population. The continuous length distribution is segmented, and the time evolution of the nanotube concentration in each length bin is modeled using ordinary differential equations. The binning choice determines the level of coarse graining. This model can handle time varying concentration of tiles, and we foresee that it will be useful to model dynamic behaviors in other types of biomolecular polymers.