Molecular machines have previously been designed that are propelled by DNAzymes1–3, protein enzymes4–6 and strand-displacement7–9. These engineered machines typically move along precisely defined one- and two-dimensional tracks. Here, we report a DNA walker that uses hybridisation to drive walking on DNA-coated microparticle surfaces. Through purely DNA:DNA hybridisation reactions, the nanoscale movements of the walker can lead to the generation of a single-stranded product and the subsequent immobilisation of fluorescent labels on the microparticle surface. This suggests that the system could be of use in analytical and diagnostic applications, similar to how strand exchange reactions in solution have been used for transducing and quantifying signals from isothermal molecular amplification assays10,11. The walking behaviour is robust and the walker can take more than 30 continuous steps. The traversal of an unprogrammed, inhomogeneous surface is also due entirely to autonomous decisions made by the walker, behaviour analogous to amorphous chemical reaction network computations12,13 that have been shown to lead to pattern formation14–17.

ConspectusWhile the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes.Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston–Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently.Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options.

Summary CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ~107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a Type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising three-nucleotide periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeqnuclease digestion and deep sequencing-a massively parallel platform that measures cleavage kinetics and captures the timeresolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a-reported as a more specific nuclease in cells-has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

There are few methods for the assembly of defined protein oligomers and higher order structures that could serve as novel biomaterials. Using fluorescent proteins as a model system, we have engineered novel oligomerization states by combining oppositely supercharged variants. A well-defined, highly symmetrical 16mer (two stacked, circular octamers) can be formed from alternating charged proteins; higher order structures then form in a hierarchical fashion from this discrete protomer. During SUpercharged PRotein Assembly (SuPrA), electrostatic attraction between oppositely charged variants drives interaction, while shape and patchy physicochemical interactions lead to spatial organization along specific interfaces, ultimately resulting in protein assemblies never before seen in nature.