CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In addition, efficient “primed” spacer acqui sition and viral degradation (interference) both require the Cascade complex along with the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Dynamic assembly of macromolecules in biological systems is one of the fundamental processes that facilitates life. Although such assembly most commonly uses noncovalent interactions, a set of dynamic reactions involving reversible covalent bonding is actively being exploited for the design of functional materials, bottom-up assembly, and molecular machines. This Minireview highlights recent implementations and advancements in the area of tunable orthogonal reversible covalent (TORC) bonds for these purposes, and provides an outlook for their expansion, including the development of synthetically encoded polynucleotide mimics.

Sequence-defined polymers show promise for biomimetics, self-assembly, catalysis, and information storage, wherein the primary structure begets complex chemical processes. Here we report the solution-phase and the high-yielding solid-phase syntheses of discrete oligourethanes and methods for their self-immolative sequencing, resulting in rapid and robust characterization of this class of oligomers and polymers, without the use of MS/MS. Crucial to the sequencing is the inherent reactivity of the terminal alcohol to "unzip" the oligomers, in a controlled and iterative fashion, releasing each monomer as a 2-oxazolidinone. By monitoring the self-immolation reaction via LC/MS, an applied algorithm rapidly produces the sequence of the oligourethane. Not only does this process provide characterization of structurally complex molecules, it works as a reader of molecular information.Sequence-defined polymers, e.g., β-peptides, γ-peptides, peptoids, polyureas, and polycarbamates, 1 have garnered significant interest over recent decades, to the point that their size and structural complexity are nearing those of biopolymers. 2 This complexity renders sequence elucidation difficult, at times -impossible. When successful, analysis has relied upon an assortment of 1 and 2D NMR spectroscopy together with sophisticated mass spectrometry techniques. 3 Molecular sequencing techniques such as Edman degradation for peptides and Sanger sequencing for DNA are among the most significant chemical achievements of the 20th century. Modern proteomic studies rely on comparisons to databases for protein identification, 4 wherein many of the protein sequences were elucidated via Edman degradations. Notably, very few techniques analogous to Edman or Sanger sequencing exist for synthetic macromolecules, likely due to the fact that only recently has synthetic methodology been capable of creating monodisperse macromolecules as structurally complex as biopolymers. 1 Peptoids are one exception, with Zuckerman realizing their stepwise chemical degradation on resin. 5 As more examples of sequence-defined

A new autoinductive cascade employing benzoyl fluoride as a latent source of fluoride is reported for signal amplification and optical detection of fluoride. The autoinduction leads to a maximum 4-fold signal enhancement for each fluoride generated, as well as a self-propagating cycle that generates three fluorophores for each single fluoride released. A two-step integrated protocol creates a more rapid autoinductive cascade than previously reported, as well as a highly sensitive diagnostic assay for the ultratrace quantitation of a phosphoryl fluoride nerve agent surrogate.

SUMMARY Molecular encoding in sequence-defined polymers shows promise as a new paradigm for data storage. Here, we report what is, to our knowledge, the first use of self-immolative oligourethanes for storing and reading encoded information. As a proof of principle, we describe how a text passage from Jane Austen’s Mansfield Park was encoded in sequence-defined oligourethanes and reconstructed via self-immolative sequencing. We develop Mol.E-coder, a software tool that uses a Huffman encoding scheme to convert the character table to hexadecimal. The oligourethanes are then generated by a high-throughput parallel synthesis. Sequencing of the oligourethanes by self-immolation is done concurrently in a parallel fashion, and the liquid chromatography-mass spectrometry (LC-MS) information decoded by our Mol.E-decoder software. The passage is capable of being reproduced wholly intact by a third-party, without any purifications or the use of tandem MS (MS/MS), despite multiple rounds of compression, encoding, and synthesis.