Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography.

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (Tm) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (Tm was 67.1 - 78.2 °C for 10-100 mM [Na+] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li+], 68.1 °C in 10 mM [NH4+], 65.6 °C in 10 mM [Cs+], and 56.6 °C in 10 mM [triethylammonium+] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1-3 °C, and denaturation power increases in the order MeOH

Oxidative Modification of the Potential G-Quadruplex Sequence in the PCNA Gene Promoter Can Turn on Transcription.

Because of its low redox potential, guanine (G) is the most frequent site of oxidation in the genome. Metabolic processes generate reactive oxygen species (ROS) that can oxidize G to yield 8-oxo-7,8-dihydroguanine (OG) as a key two-electron oxidation product. In a genome, G-rich sites including many gene promoters are sensitive to oxidative modification, and some of these regions have the propensity to form G-quadruplexes (G4s). Recently, OG formation in G-rich gene promoters was demonstrated to regulate mRNA expression via the base excision repair (BER) pathway. The proliferating cell nuclear antigen ( PCNA) gene was previously found to be activated by metabolic ROS, and the gene has a five G-track potential G4 in the coding strand of its promoter. Herein, we demonstrated the ability for four G runs of the PCNA promoter sequence to adopt a parallel-stranded G4. Next, we identified G nucleotides in the PCNA G4 sequence sensitive to oxidative modification. The G oxidation product OG and its initial BER product, an abasic site, were synthetically incorporated into the four- and five-track PCNA sequences at the sensitive sites followed by interrogation of G4 folding by five methods. We found the modifications impacted the G4 folds with positional dependency. Additionally, the fifth G track maintained the stability of the modified G4s by extrusion of the oxidatively modified G run. Finally, we synthetically inserted a portion of the promoter into a reporter plasmid with OG at select oxidation-prone positions to monitor expression in human glioblastoma cells. Our results demonstrate that OG formation in the context of the PCNA G4 can lead to increased gene expression consistent with the previous studies identifying that metabolic ROS activates transcription of the gene. This study provides another example of a G4 with the potential to serve as a regulatory agent for gene expression upon G oxidation.

Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells.

The bifunctional alkylating agents epichlorohydrin (ECH) and diepoxybutane (DEB) have been linked to increased cancer risks in industrial workers. These compounds react with DNA and proteins, leading to genotoxic effects. We used the comet assay to monitor formation of cross-links in HL-60 cells treated with ECH, DEB, and the structurally related anti-cancer drug mechlorethamine (HN2). We report a time- and dose-dependent cytotoxicity that correlated with cross-linking activity, following the order HN2 > DEB > ECH. The rate of cross-link repair also varied with drug, with ECH-induced lesions the fastest to repair. High drug doses led to the formation of saturating amounts of HN2 cross-links that were repaired inefficiently. DEB and ECH produced fewer overall cross-links, but some were also resistant to repair. These persistent cross-links may activate cell-cycle arrest to allow repair of damage, with prolonged arrest triggering apoptosis. Quantitative reverse transcription polymerase chain reaction experiments revealed that treatment of HL-60 cells with DEB and ECH results in up-regulation of several genes involved in the intrinsic (mitochondrial) apoptosis pathway, including BAX, BAK1, CASP-9, APAF-1, and BCL-2. These findings contribute to our understanding of the principles underlying the carcinogenic potentials of these xenobiotics.