Dewar Lesion Formation in Single- and Double-Stranded DNA is Quenched by Neighboring Bases

UV-induced Dewar lesion formation is investigated in single- and double-stranded oligonucleotides with ultrafast vibrational spectroscopy. The quantum yield for the conversion of the (6–4) lesion to the Dewar isomer in DNA strands is reduced by a factor of 4 in comparison to model dinucleotides. Time resolved spectroscopy reveals a fast process in the excited state with spectral characteristics of bases which are adjacent to the excited (6–4) lesion. These kinetic components have large amplitudes and indicate that an additional quenching channel acts in the stranded DNA systems and reduces the Dewar formation yield. Presumably relaxation evolves via a charge transfer to the neighboring guanine and the paired cytosine participates in a double-stranded oligomer. Changes in the decay of the relaxed excited electronic state of the (6–4) chromophore point to modifications in the excited state energy landscape which may lead to an additional reduction of the Dewar formation yield

Click-Modified Anandamide siRNA Enables Delivery and Gene Silencing in Neuronal and Immune Cells

Click chemistry of alkyne-modified RNA with different receptor ligand azides was used to prepare 3′-folate, 3′-cholesterol, and, as a new entity, 3′-anandamide-modified RNA in high yields and excellent purity. The anandamide-modified RNA shows surprisingly high transfection properties and enables the delivery of siRNA even into difficult-to-transfect RBL-2H3 cells which model neuronal uptake. Furthermore, the system was employed in human immune cells (BJAB), demonstrating silencing effects similar to those of a cationic, benchmark transfection reagent. In addition, the anandamide conjugates were found to be nontoxic. The reported chemistry and the described properties of the anandamide siRNA extend the possibilities of using siRNA-based gene silencing in neuronal and immune cells

Discovery and Mutagenicity of a Guanidinoformimine Lesion as a new Intermediate of the Oxidative Deoxyguanosine Degradation Pathway

Oxidative degradation of DNA is a major mutagenic process. Reactive oxygen species (ROS) produced in the course of oxidative phosphorylation or by exogenous factors are known to attack preferentially deoxyguanosine. The latter decomposes to give mutagenic lesions, which under physiological conditions are efficiently repaired by specialized maintenance systems in the cell. Although many intermediates of the degradation pathway are today well-known, we report in this study the discovery of a new intermediate with an interesting guanidinoformimine structure. The structure elucidation of the new lesion was possible by using HPLC–MS techniques and organic synthesis. Finally we report the mutagenic potential of the new lesion in comparison to the known lesions imidazolone and oxazolone using primer extension and pyrosequencing experiments

UV-Induced Charge Transfer States in DNA Promote Sequence Selective Self-Repair

Absorption of UV-radiation in nucleotides initiates a number of photophysical and photochemical processes, which may finally cause DNA damage. One major decay channel of photoexcited DNA leads to reactive charge transfer states. This study shows that these states trigger self-repair of DNA photolesions. The experiments were performed by UV spectroscopy and HPLC on different single and double stranded oligonucleotides containing a cyclobutane pyrimidine dimer (CPD) lesion. In a first experiment we show that photoexcitation of adenine adjacent to a CPD has no influence on this lesion. However, excitation of a guanine (G) adenine (A) sequence leads to reformation of the intact thymine (T) bases. The involvement of two bases for the repair points to a long-living charge transfer state between G and A to be responsible for the repair. The negatively charged A radical anion donates an electron to the CPD, inducing ring splitting and repair. In contrast, a TA sequence, having an inverted charge distribution (T radical anion, A radical cation), is not able to repair the CPD lesion. The investigations show that the presence of an adjacent radical ion is not sufficient for repair. More likely it is the driving power represented by the oxidation potential of the radical ion, which controls the repair. Thus, repair capacities are strongly sequence-dependent, creating DNA regions with different tendencies of self-repair. This self-healing activity represents the simplest sequence-dependent DNA repair system

Synthesis of (<i>R</i>)‑Configured 2′-Fluorinated mC, hmC, fC, and caC Phosphoramidites and Oligonucleotides

Investigation of the function of the new epigenetic bases requires the development of stabilized analogues that are stable during base excision repair (BER). Here we report the synthesis of 2′-(<i>R</i>)-fluorinated versions of the phosphoramidites of 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). For oligonucleotides containing 2′-(<i>R</i>)-F-fdC, we show that these compounds cannot be cleaved by the main BER enzyme thymine-DNA glycosylase (TDG)

Exclusive requirement of TC-NER for the removal of transcription-blocking dG(<i>N²</i>)-AAF.

<p>Host cell reactivation in human cell lines of the specified nucleotide excision repair complementation groups (CS-A, CS-B, XP-C and XP-A) and in the repair proficient cell line (MRC-5). The expression constructs contained a unique dG(<i>N</i>²)-AAF or dG in the specified DNA strand. (A) Overlaid fluorescence distribution plots obtained in a representative experiment. All cell lines were transfected in parallel with the same constructs. (B) Mean relative expression (dG(<i>N</i>²)-AAF/dG) calculated for multiple transfections with independent preparations of the expression constructs (n = 3, +/− s.d.).</p

Effects of single adducts on the EGFP gene expression in the GM16095 (CS-B) cell line.

<p>The protein coding sequence (arrow) and the transcription start (broken arrow) are represented out of scale, along with the adduct position (star) with respect to the transcription unit. Overlaid fluorescence distribution plots were obtained by flow cytometric analyses of cells transfected with vectors carrying the specified adducts (amber line) and with the control constructs obtained by incorporation of the unmodified deoxyribo-oligonucleotide (blue line).</p

Lewis Acid Triggered Regioselective Magnesiation and Zincation of Uracils, Uridines, and Cytidines

The Lewis acid MgCl₂ allows control of the metalation regioselectivity of uracils and uridines. In the absence of the Lewis acid, metalation of uracil and uridine derivatives with TMPMgCl·LiCl occurs at the position C(5). In the presence of MgCl₂, zincation using TMP₂Zn·2LiCl·2MgCl₂ occurs at the position C(6). This metalation method provides easy access to functionalized uracils and uridines. Using TMP₂Zn·2LiCl·2MgCl₂ also allows to functionalize cytidine derivatives at the position C(6)

Modulation of the effect of dG(<i>N²</i>)-AAF by CSB.

<p>Host cell reactivation of the EGFP expression in pairs of isogenic cell lines with different CSB expression statuses. Cells were transfected with constructs containing a unique dG(<i>N</i>²)-AAF (amber colors) or dG (blue colors) in the transcribed strand of the EGFP gene. Cells were selected according to the expression of DsRed as a transfection marker (the gated region shown by square brackets near the dot density plots) to generate the overlaid fluorescence distribution plots (on the right). (A) The effect of complementation of the CSB deficiency by expression of the CSB cDNA. (B) The effect of CSB knockdown by expression of the specific shRNA (CSBsh). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094405#pone.0094405.s003" target="_blank">Figures S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094405#pone.0094405.s004" target="_blank">S4</a>.</p

Effects of single adducts on the EGFP gene expression in the GM16094 (CS-A) cell line.

<p>Overlaid fluorescence distribution plots of cells transfected with vectors carrying the specified adducts (amber line) and with the control constructs obtained by incorporation of the unmodified deoxyribo-oligonucleotide (blue line).</p