Edward J. Merino

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2′-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100–200 nucleotides.Note: In the version of this article initially published online, the value for the pH range in the second paragraph on page 2 was incorrect; it should have read 7.5–8.2. In addition, two sentences were included that should have been removed from page 1. These errors have been corrected in all versions of the article.

Biological Contexts for DNA Charge Transport Chemistry

Many experiments have now shown that double helical DNA can serve as a conduit for efficient charge transport (CT) reactions over long distances in vitro. These results prompt the consideration of biological roles for DNA-mediated CT. DNA CT has been demonstrated to occur in biologically relevant environments such as within the mitochondria and nuclei of HeLa cells as well as in isolated nucleosomes. In mitochondria, DNA damage that results from CT is funneled to a crucial regulatory element. Thus, DNA CT provides a strategy to funnel damage to particular sites in the genome. DNA CT might also be important in long-range signaling to DNA-bound proteins. Both DNA repair proteins, containing Fe-S clusters, and the transcription factor, p53, which is regulated through thiol-disulfide switches, can be oxidized from a distance through DNA-mediated CT. These observations highlight a means through which oxidative stress may be chemically signaled in the genome over long distances through CT from guanine radicals to DNA-bound proteins. Moreover, DNA-mediated CT may also play a role in signaling among DNA-binding proteins, as has been proposed as a mechanism for how DNA repair glycosylases more efficiently detect lesions inside the cell.

A role for DNA-mediated charge transport in regulating p53: Oxidation of the DNA-bound protein from a distance

Charge transport (CT) through the DNA base pairs provides a means to promote redox reactions at a remote site and potentially to effect signaling between molecules bound to DNA. Here we describe the oxidation of a cell-cycle regulatory protein, p53, from a distance through DNA-mediated CT. A consensus p53 binding site as well as three DNA promoters regulated by p53 were synthesized containing a tethered DNA photooxidant, anthraquinone. Photoinduced oxidation of the protein occurs from a distance; introduction of an intervening CA mismatch, which inhibits DNA-mediated CT, prevents oxidation of p53. DNA-mediated oxidation is shown to promote dissociation of p53 from only some promoters, and this sequence-selectivity in oxidative dissociation correlates with the biological regulation of p53. Under severe oxidative stress, effected here through oxidation at long range, p53 dissociates from a promoter that activates DNA repair as well as the promoter for the negative regulator of p53, Mdm2, but not from a promoter activating cell-cycle arrest. Mass spectrometry results are consistent with disulfide bond formation in p53 upon DNA-mediated oxidation. Furthermore, DNA-bound p53 oxidation is shown in vivo by up-regulation of p53 and subsequent irradiation in the presence of a rhodium photooxidant to give a new p53 adduct that can be reversed with thiol treatment. This DNA-mediated oxidation of p53 parallels that seen by treating cells with hydrogen peroxide. These results indicate a unique mechanism using DNA-mediated CT chemistry by which p53 activity on different promoters may be controlled globally under conditions of oxidative stress.

Catalysis of amide synthesis by RNA phosphodiester and hydroxyl groups

The functional groups found among the RNA bases and in the phosphoribose backbone represent a limited repertoire from which to construct a ribozyme active site. This work investigates the possibility that simple RNA phosphodiester and hydroxyl functional groups could catalyze amide bond synthesis. Reaction of amine groups with activated esters would be catalyzed by a group that stabilizes the partial positive charge on the amine nucleophile in the transition state. 2′-Amine substitutions adjacent to 3′-phosphodiester or 3′-hydroxyl groups react efficiently with activated esters to form 2′-amide and peptide products. In contrast, analogs in which the 3′-phosphodiester is replaced by an uncharged phosphotriester or is constrained in a distal conformation react at least 100-fold more slowly. Similarly, a nucleoside in which the 3′-hydroxyl group is constrained trans to the 2′-amine is also unreactive. Catalysis of synthetic reactions by RNA phosphodiester and ribose hydroxyl groups is likely to be even greater in the context of a preorganized and solvent-excluding catalytic center. One such group is the 2′-hydroxyl of the ribosome-bound P-site adenosine substrate, which is close to the amine nucleophile in the peptidyl synthesis reaction. Given ubiquitous 2′-OH groups in RNA, there exists a decisive advantage for RNA over DNA in catalyzing reactions of biological significance.

A Redox-Active, Compact Molecule for Cross-Linking Amyloidogenic Peptides into Nontoxic, Off-Pathway Aggregates: In Vitro and In Vivo Efficacy and Molecular Mechanisms.

Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-β (Aβ) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand-peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimers disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aβ forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aβ with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.

Selenium effects on arsenic cytotoxicity and protein phosphorylation in human kidney cells using chip-based nanoLC-MS/MS

Although arsenic toxicity is well known, little is known of how it exerts its effects at the proteome level. Protein phosphorylation is an important post-translational modification in the regulation of cell signaling. Despite the importance of protein phosphorylation, the identification and characterization of phosphorylated proteins, as influenced by interaction between arsenic and selenium species have not been fully studied. The aim of this study is to identify phosphorylation in arsenic toxified cells, with and without selenium present. Here, we identify the phosphorylated proteins related to post translational modifications (PTMs) after inorganic arsenic (iAs) and selenomethionine (SeMet) were inoculated together with HEK293 human kidney cells. In this study, using TiO(2)-based nanoLC-phosphochip® coupled to ESI-MS we observed phosphorylated peptide enrichment and significant reduction in sample complexity. The identification of phosphorylated proteins in highly complex digests of cell lysate were markedly different with As toxification only, or when in the presence of SeMet. Several phosphorylation sites and proteins are identified using Spectrum Mill and Mascot protein data base search engines. Cytotoxicity studies showed that SeMet significantly reduces the cytotoxic effect of iAs in HEK293 cells, while inorganic selenium did not.

Common mitochondrial DNA mutations generated through DNA-mediated charge transport.

Mutation sites that arise in human mitochondrial DNA as a result of oxidation by a rhodium photooxidant have been identified. HeLa cells were incubated with [Rh(phi)(2)bpy]Cl(3) (phi is 9,10-phenanthrenequinone diimine), an intercalating photooxidant, to allow the complex to enter the cell and bind mitochondrial DNA. Photoexcitation of DNA-bound [Rh(phi)(2)bpy](3+) can promote the oxidation of guanine from a distance through DNA-mediated charge transport. After two rounds of photolysis and growth of cells incubated with the rhodium complex, DNA mutations in a portion of the mitochondrial genome were assessed via manual sequencing. The mutational pattern is consistent with dG to dT transversions in the repetitive guanine tracts. Significantly, the mutational pattern found overlaps oxidative damage hot spots seen previously. These mutations are found within conserved sequence block II, a critical regulatory element involved in DNA replication, and these have been identified as sites of low oxidation potential to which oxidative damage is funneled. On the basis of this mutational analysis and its correspondence to sites of long-range oxidative damage, we infer a critical role for DNA charge transport in generating these mutations and, thus, in regulating mitochondrial DNA replication under oxidative stress.

Investigation of fluorinated and bifunctionalized 3-phenylchroman-4-one (isoflavanone) aromatase inhibitors ☆

Fluorinated isoflavanones and bifunctionalized isoflavanones were synthesized through a one-step gold(I)-catalyzed annulation reaction. These compounds were evaluated for their in vitro inhibitory activities against aromatase in a fluorescence-based enzymatic assay. Selected compounds were tested for their anti-proliferative effects on human breast cancer cell line MCF-7. Compounds 6-methoxy-3-(pyridin-3-yl)chroman-4-one (3c) and 6-fluoro-3-(pyridin-3-yl)chroman-4-one (3e) were identified as the most potent aromatase inhibitors with IC₅₀ values of 2.5 μM and 0.8 μM. Therefore, these compounds have great potential for the development of pharmaceutical agents against breast cancer.

Novel ROS-activated agents utilize a tethered amine to selectively target acute myeloid leukemia.

This study explores the possible use of reactive oxygen-activated DNA modifying agents against acute myeloid leukemia (AML). A key amine on the lead agent was investigated via cytotoxicity assays and was found necessary for potency. The two best compounds were screened via the NCI-60 cell panel. These two compounds had potency between 200 and 800nM against many of the leukemia cancer cell types. Subsequent experiments explored activity against a transformed AML model that mimics the molecular signatures identified in primary AML patient samples. A lead compound had an IC50 of 760nM against this AML cell line as well as a therapeutic index of 7.7±3 between the transformed AML model cell line and non-cancerous human CD34+ blood stem/progenitor cells (UCB). The selectivity was much greater than the mainstays of AML treatment: doxorubicin and cytarabine. This manuscript demonstrates that this novel type of agent may be useful against AML.

Developing ICP-MS/MS for the detection and determination of synthetic DNA-protein crosslink models via phosphorus and sulfur detection

Various endogenous and exogenous agents drive the un-directed formation of covalent bonds between proteins and DNA. These complex molecules are of great biological relevance, as can derive in mutations, but are difficult to study because of their heterogeneous chemical properties. New analytical approaches with sufficient detection capabilities to detect and quantify these compounds can help to standardize study models based on synthesized standards. The use of atomic spectrometry can provide quantitative information on the DNA-protein cross-link reaction yield along with basic stoichiometry of the products, based on internal elemental tags, sulfur from Cys and Met amino acids, and phosphorus from the DNA. A new instrumental approach to remove isobaric and polyatomic interferences from 31P+ and 32S+ was developed recently, with state-of-the-art for interference removal that captures 31P+ in Q1; it reacts with O2 in an octopole collision-reaction cell generating 47PO+, therefore allowing detection in Q3 without 31NOH+/48Ca/47Ti interferences. Similarly, 32S+ is reacted to 48SO+, eliminating the polyatomic interferences at m/z = 32. In conjunction with the high resolving power of high-performance liquid chromatography (HPLC), this newer technology was applied by to the product purification of a DNA-protein cross link model and some preliminary structural studies.