Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage.

The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12a ) to test mechanistic conservation.

CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity.

Cas12a (also known as "Cpf1") is a class 2 type V-A CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises a single protein and a CRISPR-encoded small RNA (crRNA) and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands.

Experimental Validation of the ALLNOX Program for Studying Protein-Nucleic Acid Complexes.

Measurement of distances between spectroscopic labels (e.g., spin labels, fluorophores) attached to specific sites of biomolecules is an important method for studying biomolecular complexes.

Isolation and characterization of a high-spin mixed-valent iron dinitrogen complex.

We report a rare example of a mixed-valence iron compound with an FeNNFe core, which gives insight into the structural, spectroscopic, and magnetic influences of single-electron reductions and oxidations. In the new compound, the odd electron is localized as judged from Mössbauer spectra at 80 K and infrared spectra at room temperature, and the backbonding into the N2 unit is intermediate between diiron(i) and diiron(0) congeners. Magnetic susceptibility and relaxation studies on the series of FeNNFe compounds show significant magnetic anisotropy, but through-barrier pathways enable fairly rapid magnetic relaxation.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5'-deoxyadenosyl radical (5'-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme.

A structurally-characterized peroxomanganese(iv) porphyrin from reversible O binding within a metal-organic framework.

The role of peroxometal species as reactive intermediates in myriad biological processes has motivated the synthesis and study of analogous molecular model complexes. Peroxomanganese(iv) porphyrin complexes are of particular interest, owing to their potential ability to form from reversible O binding, yet have been exceedingly difficult to isolate and characterize in molecular form. Alternatively, immobilization of metalloporphyrin sites within a metal-organic framework (MOF) can enable the study of interactions between low-coordinate metal centers and gaseous substrates, without interference from bimolecular reactions and axial ligation by solvent molecules.

Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis.

Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain.

Naringenin Ameliorates Doxorubicin Toxicity and Hypoxic Condition in Dalton's Lymphoma Ascites Tumor Mouse Model: Evidence from Electron Paramagnetic Resonance Imaging.

Doxorubicin (DOX) is a well-known cytotoxic agent used extensively as a chemotherapeutic drug to eradicate a wide variety of human cancers. Reactive oxygen species (ROS)-mediated oxidative stress during DOX treatment can induce cardiac, renal, and hepatic toxicities, which can constrain its use as a potential cytotoxic agent. The present work investigates the antioxidant potential of naringenin (NAR) against DOXinduced toxicities of a Dalton's lymphoma ascites (DLA) tumor-bearing mouse model.