A rapid, multiplex digital PCR assay to detect gene variants and fusions in non-small cell lung cancer.

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1, and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target-signal encoding to improve the specificity of variant detection by reducing background noise by up to an order of magnitude. The assay reported an overall 100% positive percent agreement (PPA) and 98.5% negative percent agreement (NPA) compared with a sequencing-based assay in a cohort of 62 human formalin-fixed paraffin-embedded (FFPE) samples. In addition, the dPCR assay rescued actionable information in 10 samples that failed to sequence, highlighting the utility of a multiplexed dPCR assay as a potential reflex solution for challenging NSCLC samples.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Assembly of RNA Nanostructures from Double-Crossover Tiles.

Artificial self-assembling RNA scaffolds can be produced from many types of RNA motifs that are rationally designed. These scaffolds are of interest as nanoscale organizers, with applications in drug delivery and synthetic cells. Here we describe design strategies, production methods, and imaging of micrometer-sized RNA nanotubes and lattices that assemble from RNA tiles comprising multiple distinct strands.

Correction: A universal method for sensitive and cell-free detection of CRISPR-associated nucleases.

[This corrects the article DOI: 10.1039/C8SC03426E.].

A universal method for sensitive and cell-free detection of CRISPR-associated nucleases.

A multitude of biological applications for CRISPR-associated (Cas) nucleases have propelled the development of robust cell-based methods for quantitation of on- and off-target activities of these nucleases. However, emerging applications of these nucleases require cell-free methods that are simple, sensitive, cost effective, high throughput, multiplexable, and generalizable to all classes of Cas nucleases. Current methods for cell-free detection are cumbersome, expensive, or require sophisticated sequencing technologies, hindering their widespread application beyond the field of life sciences. Developing such cell-free assays is challenging for multiple reasons, including that Cas nucleases are single-turnover enzymes that must be present in large excess over their substrate and that different classes of Cas nucleases exhibit wildly different operating mechanisms. Here, we report the development of a cell-free method wherein Cas nuclease activity is amplified via an in vitro transcription reaction that produces a fluorescent RNA:small-molecule adduct. We demonstrate that our method is sensitive, detecting activity from low nanomolar concentrations of several families of Cas nucleases, and can be conducted in a high-throughput microplate fashion with a simple fluorescent-based readout. We provide a mathematical framework for quantifying the activities of these nucleases and demonstrate two applications of our method, namely the development of a logic circuit and the characterization of an anti-CRISPR protein. We anticipate our method will be valuable to those studying Cas nucleases and will allow the application of Cas nuclease beyond the field of life sciences.

Autonomous dynamic control of DNA nanostructure self-assembly.

Biological cells routinely reconfigure their shape using dynamic signalling and regulatory networks that direct self-assembly processes in time and space, through molecular components that sense, process and transmit information from the environment. A similar strategy could be used to enable life-like behaviours in synthetic materials. Nucleic acid nanotechnology offers a promising route towards this goal through a variety of sensors, logic and dynamic components and self-assembling structures. Here, by harnessing both dynamic and structural DNA nanotechnology, we demonstrate dynamic control of the self-assembly of DNA nanotubes-a well-known class of programmable DNA nanostructures. Nanotube assembly and disassembly is controlled with minimal synthetic gene systems, including an autonomous molecular oscillator. We use a coarse-grained computational model to capture nanotube length distribution dynamics in response to inputs from nucleic acid circuits. We hope that these results may find use for the development of responsive nucleic acid materials, with potential applications in biomaterials science, nanofabrication and drug delivery.

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

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.

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

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.

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.

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.

Correction: Programmable RNA microstructures for coordinated delivery of siRNAs.

Correction for 'Programmable RNA microstructures for coordinated delivery of siRNAs' by Jaimie Marie Stewart et al., Nanoscale, 2016, 8, 17542-17550.

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

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.

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.

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.

Covalent Linkage of One-Dimensional DNA Arrays Bonded by Paranemic Cohesion.

The construction of DNA nanostructures from branched DNA motifs, or tiles, typically relies on the use of sticky-ended cohesion, owing to the specificity and programmability of DNA sequences. The stability of such constructs when unligated is restricted to a specific range of temperatures, owing to the disruption of base pairing at elevated temperatures. Paranemic (PX) cohesion was developed as an alternative to sticky ends for the cohesion of large topologically closed species that could be purified reliably on denaturing gels. However, PX cohesion is also of limited stability. In this work, we added sticky-ended interactions to PX-cohesive complexes to create interlocked complexes by functionalizing the sticky ends with psoralen, which can form cross-links between the two strands of a double helix. We were able to reinforce the stability of the constructs by creating covalent linkages between the 3'-ends and 5'-ends of the sticky ends; the sticky ends were added to double crossover domains via 3'-3' and 5'-5' linkages. Catenated arrays were obtained either by enzymatic ligation or by UV cross-linking. We have constructed finite-length one-dimensional arrays linked by interlocking loops and have positioned streptavidin-gold particles on these constructs.

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.