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.

The reactivity of an RNA ribose hydroxyl is shown to be exquisitely sensitive to local nucleotide flexibility because a conformationally constrained adjacent 3'-phosphodiester inhibits formation of the deprotonated, nucleophilic oxyanion form of the 2'-hydroxyl group. Reaction with an appropriate electrophile, N-methylisatoic anhydride, to form a 2'-O-adduct thus can be used to monitor local structure at every nucleotide in an RNA. We develop a quantitative approach involving Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to map the structure of and to distinguish fine differences in structure for tRNAAsp transcripts at single nucleotide resolution. Modest extensions of the SHAPE approach will allow RNA structure to be monitored comprehensively and at single nucleotide resolution for RNAs of arbitrary sequence and structural complexity and under diverse solution environments.

Current models assume that RNA folding is strongly hierarchical such that the base-paired secondary structure is more stable than and forms independently of the tertiary structure. This model has been difficult to test due to the experimental inability to interrogate the local environment at every nucleotide as a comprehensive function of the RNA folding state. Reaction of an RNA 2'-hydroxyl group with N-methylisatoic anhydride to form a nucleotide 2'-ester is governed by the extent to which the nucleotide forms base pairing or tertiary interactions. Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is shown to be an RNA analogue of the protein hydrogen exchange experiment. Single nucleotide resolution SHAPE analysis emphasizes a complexity for the unfolding of tRNA(Asp) transcripts that is not anticipated by current models for RNA folding. We quantify six well-defined transitions for tRNA(Asp) transcripts between 35 and >75 degrees C, including asymmetric unfolding of the two strands in a single helix, multistep loss of tertiary interactions, and a multihelix conformational shift. The three lowest temperature transitions each involve coupled interactions between the secondary and tertiary structure. Thus, even for this simple RNA, multiple nonhierarchical and complex interactions dominate the equilibrium transitions most accessible from the native state.

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 Alzheimer’s 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.

SUMMARYMany 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 critical 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 DNAbound proteins. Moreover, DNA-mediated CT may also play a role in signaling among DNAbinding proteins, as has been proposed as a mechanism for how DNA repair glycosylases more efficiently detect lesions inside the cell.

Metallo prodrugs that take advantage of the inherent acidity surrounding cancer cells have yet to be developed. We report a new class of pH-activated metallo prodrugs (pHAMPs) that are activated by light- and pH-triggered ligand dissociation. These ruthenium complexes take advantage of a key characteristic of cancer cells and hypoxic solid tumors (acidity) that can be exploited to lessen the side effects of chemotherapy. Five ruthenium complexes of the type [(N,N)Ru(PL)] were synthesized, fully characterized, and tested for cytotoxicity in cell culture (1: N,N = 2,2'-bipyridine (bipy) and PL, the photolabile ligand, = 6,6'-dihydroxybipyridine (6,6'-dhbp); 2: N,N = 1,10-phenanthroline (phen) and PL = 6,6'-dhbp; 3: N,N = 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline (dop) and PL = 6,6'-dhbp; 4: N,N = bipy and PL = 4,4'-dimethyl-6,6'-dihydroxybipyridine (dmdhbp); 5: N,N = 1,10-phenanthroline (phen) and PL = 4,4'-dihydroxybipyridine (4,4'-dhbp). The thermodynamic acidity of these complexes was measured in terms of two pK values for conversion from the acidic form (X) to the basic form (X) by removal of two protons. Single-crystal X-ray diffraction data is discussed for 2, 2, 3, 4, and 5. All complexes except 5 showed measurable photodissociation with blue light (λ = 450 nm). For complexes 1-4 and their deprotonated analogues (1-4), the protonated form (at pH 5) consistently gave faster rates of photodissociation and larger quantum yields for the photoproduct, [(N,N)Ru(HO)]. This shows that low pH can lead to greater rates of photodissociation. Cytotoxicity studies with 1-5 showed that complex 3 is the most cytotoxic complex of this series with IC values as low as 4 μM (with blue light) versus two breast cancer cell lines. Complex 3 is also selectively cytotoxic, with sevenfold higher toxicity toward cancerous versus normal breast cells. Phototoxicity indices with 3 were as high as 120, which shows that dark toxicity is avoided. The key difference between complex 3 and the other complexes tested appears to be higher uptake of the complex as measured by inductively coupled plasma mass spectrometry, and a more hydrophobic complex as compared to 1, which may enhance uptake. These complexes demonstrate proof of concept for dual activation by both low pH and blue light, thus establishing that a pHAMP approach can be used for selective targeting of cancer cells.

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.DNA electron transfer ͉ oxidative stress ͉ redox regulation

Ruthenium drugs are potent anti-cancer agents, but inducing drug selectivity and enhancing their modest activity remain challenging. Slow Ru ligand loss limits the formation of free sites and subsequent binding to DNA base pairs. Herein, we designed a ligand that rapidly dissociates upon irradiation at low pH. Activation at low pH can lead to cancer selectivity, since many cancer cells have higher metabolism (and thus lower pH) than non-cancerous cells. We have used the pH sensitive ligand, 6,6′-dihydroxy-2,2′-bipyridine (66′bpy(OH)2), to generate [Ru(bpy)2(66′(bpy(OH)2)]2+, which contains two acidic hydroxyl groups with pKa1 = 5.26 and pKa2 = 7.27. Irradiation when protonated leads to photo-dissociation of the 66′bpy(OH)2 ligand. An in-depth study of the structural and electronic properties of the complex was carried out using X-Ray crystallography, electrochemistry, UV/visible spectroscopy, and computational techniques. Notably, Ru-N bond lengths in the 66′bpy(OH)2 complex are longer (by ~0.3 Å) than in polypyridyl complexes that lack 6 and 6′ substitution. Thus, the longer bond length predisposes the complex for photo-dissociation and leads to the anti-cancer activity. When the complex is deprotonated, the 66′bpy(O−)2 ligand molecular orbitals mix heavily with the ruthenium orbitals, making new mixed metal-ligand orbitals that lead to a higher bond order. We investigated the anti-cancer activities of [Ru(bpy)2(66′(bpy(OH)2)]2+, [Ru(bpy)2(44′(bpy(OH)2)]2+, and [Ru(bpy)3]2+ (44′(bpy(OH)2 = 4,4′-dihydroxy-2,2′-bipyridine) in HeLa cells, which have a relatively low pH. It is found that [Ru(bpy)2(66′(bpy(OH)2)]2+ is more cytotoxic than the other ruthenium complexes studied. Thus, we have identified a pH sensitive ruthenium scaffold that can be exploited for photo-induced anti-cancer activity.