Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex.

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 type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and 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.

Electrostatic and Covalent Assemblies of Anionic Hydrogel-Coated Gold Nanoshells for Detection of Dry Eye Biomarkers in Human Tears.

Although dry eye is highly prevalent, many challenges exist in diagnosing the symptom and related diseases. For this reason, anionic hydrogel-coated gold nanoshells (AuNSs) were used in the development of a label-free biosensor for detection of high isoelectric point tear biomarkers associated with dry eye. A custom, aldehyde-functionalized oligo(ethylene glycol)acrylate (Al-OEGA) was included in the hydrogel coating to enhance protein recognition through the formation of dynamic covalent (DC) imine bonds with solvent-accessible lysine residues present on the surface of select tear proteins. Our results demonstrated that hydrogel-coated AuNSs, composed of monomers that form ionic and DC bonds with select tear proteins, greatly enhance protein recognition due to changes in the maximum localized surface plasmon resonance wavelength exhibited by AuNSs in noncompetitive and competitive environments. Validation of the developed biosensor in commercially available pooled human tears revealed the potential for clinical translation to establish a method for dry eye diagnosis.

Sequencing of Sequence-Defined Oligourethanes via Controlled Self-Immolation.

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.

Capture and Release of Protein-Nanoparticle Conjugates by Reversible Covalent Molecular Linkers.

A hybrid approach to covalently detachable molecules for nanoparticle capture and release from several custom-functionalized surfaces is described. This new surface chemistry capability provides a means for reversible binding of functionalized nanoparticles without relying on costly nucleic acid-based complexation. A new surface linker motif was devised wherein custom molecules were synthesized with components for surface anchoring, cleavage, and target capture through biotin-streptavidin binding. All capture-and-release chemistry is performed using physiological conditions (aqueous, pH 7). Covalent cleavage of linker molecules was achieved through incorporation of a tunable orthogonal reversible covalent (TORC) hydrazone functional group which underwent exchange with a competitive hydrazide aided by an aniline catalyst. The influence of the linker architecture on hydrazone exchange and nanoparticle release was probed by altering the distance between hydrazone and biotin groups using different length PEG spacers. Cleavable linkers were used to functionalize microwells, magnetic separation beads, and gold-coated glass surfaces. Upon functionalization, all surface types bound streptavidin and conjugated nanoparticles regardless of the linker structure. Conversely, the extent of hydrazone exchange as well as release of nanoparticles were influenced both by the hydrazone surface density and the linker molecular structure.

Tunable Orthogonal Reversible Covalent (TORC) Bonds: Dynamic Chemical Control over Molecular Assembly.

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.

New Autoinductive Cascade for the Optical Sensing of Fluoride: Application in the Detection of Phosphoryl Fluoride Nerve Agents.

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.

Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9.

Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X-ray crystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm. Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand-induced rigidification of Ubc9 as a mechanism of inhibition.

Molecular Encryption and Steganography Using Mixtures of Simultaneously Sequenced, Sequence-Defined Oligourethanes.

Molecular encoding in abiotic sequence-defined polymers (SDPs) has recently emerged as a versatile platform for information and data storage. However, the storage capacity of these sequence-defined polymers remains underwhelming compared to that of the information storing biopolymer DNA. In an effort to increase their information storage capacity, herein we describe the synthesis and simultaneous sequencing of eight sequence-defined 10-mer oligourethanes. Importantly, we demonstrate the use of different isotope labels, such as halogen tags, as a tool to deconvolute the complex sequence information found within a heterogeneous mixture of at least 96 unique molecules, with as little as four micromoles of total material. In doing so, relatively high-capacity data storage was achieved: 256 bits in this example, the most information stored in a single sample of abiotic SDPs without the use of long strands. Within the sequence information, a 256-bit cipher key was stored and retrieved. The key was used to encrypt and decrypt a plain text document containing The Wonderful Wizard of Oz. To validate this platform as a medium of molecular steganography and cryptography, the cipher key was hidden in the ink of a personal letter, mailed to a third party, extracted, sequenced, and deciphered successfully in the first try, thereby revealing the encrypted document.

Efficient molecular encoding in multifunctional self-immolative urethanes.

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.

Nanogel receptors for high isoelectric point protein detection: influence of electrostatic and covalent polymer-protein interactions.

An aldehyde acrylate-based functional monomer was incorporated into poly(N-isopropylacrylamide-co-methacrylic acid) nanogels for use as protein receptors. The aldehyde component forms dynamic imines with surface exposed lysine residues, while carboxylic acid/carboxylate moieties form electrostatic interactions with high isoelectric point proteins. Together, these interactions effect protein adsorption and recognition.

Sortase-mediated fluorescent labeling of CRISPR complexes.

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.