Abstract:The Nucleic Acid Package (NUPACK) is a growing software suite for the analysis and design of nucleic acid systems. The NUPACK web server (http://www.nupack.org) currently enables:• Analysis: thermodynamic analysis of dilute solutions of interacting nucleic acid strands.• Design: sequence design for complexes of nucleic acid strands intended to adopt a target secondary structure at equilibrium.• Utilities: evaluation, display, and annotation of equilibrium properties of a complex of nucleic acid strands.NUPACK algorithms are formulated in terms of nucleic acid secondary structure. In most cases, pseudoknots are excluded from the structural ensemble.

We introduce the concept of hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment. In the simplest version of this process, two stable species of DNA hairpins coexist in solution until the introduction of initiator strands triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers. The average molecular weight of the HCR products varies inversely with initiator concentration. Amplification of more diverse recognition events can be achieved by coupling HCR to aptamer triggers. This functionality allows DNA to act as an amplifying transducer for biosensing applications. Biosensors require both a recognition component for detection and a transduction component for readout. In gene chips, recognition is performed by single-stranded DNAs that screen for complementary nucleic acid fragments, and transduction is typically performed by optical or electrochemical means (1, 2). Nucleic acid aptamers obtained by in vitro selection methods (3, 4) generalize this recognition capability to a wide range of target analytes (5, 6) and are amenable to optical transduction approaches (7,8). Here, we demonstrate that DNA can also play the transduction role via an amplification approach termed hybridization chain reaction (HCR). This class of mechanisms suggests the possibility of constructing biosensors solely from unmodified single-stranded DNA.Single-stranded DNA is a versatile construction material (9) that can be programmed (10 -15) to self-assemble into complex structures (10, 16 -23) driven by the free energy of base pair formation. Synthetic DNA machines can be powered by strand displacement interactions initiated by the sequential introduction of auxiliary DNA fuel strands (24 -31). Typically, various DNA strands begin to associate as soon as they are mixed together. Catalytic fuel delivery provides a conceptual approach to powering autonomous DNA machines by storing potential energy in loops that are difficult to access kinetically except in the presence of a catalyst strand (32). In the system described here, monomer DNA building blocks are mixed together but do not hybridize on an experimental time scale. Exposure of an initiator strand triggers a chain reaction of hybridization events similar to living chain polymerization but without covalent bond formation. This system introduces the concept of triggered self-assembly of DNA nanostructures. MethodsSystem Specifications. DNA sequences were designed by using a combination of criteria (15): sequence symmetry minimization (10), the probability of adopting the target secondary structure at equilibrium (12), the average number of incorrect nucleotides at equilibrium relative to the target structure (15), and hybridization kinetics (33). The sequences for the basic HCR system of Fig. 1 and the aptamer HCR system of Fig. 2 are shown in Table 1. The aptamer system required new sequence designs to ensure compatibility with the fixed sequence of the aptamer. For ...

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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 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. 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.