Programming DNA Tube Circumferences

Synthesizing molecular tubes with monodisperse, programmable circumferences is an important goal shared by nanotechnology, materials science, and supermolecular chemistry. We program molecular tube circumferences by specifying the complementarity relationships between modular domains in a 42-base single-stranded DNA motif. Single-step annealing results in the self-assembly of long tubes displaying monodisperse circumferences of 4, 5, 6, 7, 8, 10, or 20 DNA helices

Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo

Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability

Multiplexed In Situ Hybridization Using Hybridization Chain Reaction

Choi et al. describe programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) that enable simultaneous mapping of multiple target mRNAs within whole-mount zebrafish embryos. In contrast to traditional in situ hybridization approaches, HCR in situ amplification is performed for all target mRNAs simultaneously, so the experimental timeline is independent of the number of target mRNAs. Using HCR, probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled hairpins self-assemble into tethered fluorescent amplification polymers in situ (Fig. 1), enabling straightforward multiplexing for up to five target mRNAs at a time, deep sample penetration, high signal-to-background, and subcellular signal localization. The new DNA HCR technology improves on the original RNA HCR technology to dramatically increase signal gain, reduce reagent cost, and increase reagent durability. DNA HCR kits containing probe sets, amplifiers, and buffers can be purchased from the nonprofit academic resource (www.molecularinstruments.org). Advice and technical support can be obtained by emailing [email protected]

Topological constraints in nucleic acid hybridization kinetics

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of ‘helix-driven wrapping’. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary ‘kissing hairpins’ demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex

Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

In situ hybridization based on the mechanism of the hybridization chain reaction (HCR) has addressed multi-decade challenges that impeded imaging of mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of a higher signal-to-background ratio with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables three multiplexed quantitative analysis modes: (1) qHCR imaging – analog mRNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos; (2) qHCR flow cytometry – analog mRNA relative quantitation for high-throughput expression profiling of mammalian and bacterial cells; and (3) dHCR imaging – digital mRNA absolute quantitation via single-molecule imaging in thick autofluorescent samples

Integrating DNA strand-displacement circuitry with DNA tile self-assembly

DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 µm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures

Programming biomolecular self-assembly pathways

In nature, self-assembling and disassembling complexes of proteins and nucleic acids bound to a variety of ligands perform intricate and diverse dynamic functions. In contrast, attempts to rationally encode structure and function into synthetic amino acid and nucleic acid sequences have largely focused on engineering molecules that self-assemble into prescribed target structures, rather than on engineering transient system dynamics. To design systems that perform dynamic functions without human intervention, it is necessary to encode within the biopolymer sequences the reaction pathways by which self-assembly occurs. Nucleic acids show promise as a design medium for engineering dynamic functions, including catalytic hybridization, triggered self-assembly and molecular computation. Here, we program diverse molecular self-assembly and disassembly pathways using a 'reaction graph' abstraction to specify complementarity relationships between modular domains in a versatile DNA hairpin motif. Molecular programs are executed for a variety of dynamic functions: catalytic formation of branched junctions, autocatalytic duplex formation by a cross-catalytic circuit, nucleated dendritic growth of a binary molecular 'tree', and autonomous locomotion of a bipedal walker

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization