13 research outputs found
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 (6â4) 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 (6â4) 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 (6â4) 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 3â˛-folate, 3â˛-cholesterol,
and, as a new entity, 3â˛-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 HPLCâMS
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>)âConfigured 2â˛-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 2â˛-(<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 2â˛-(<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>N<sup>2</sup></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><sup>2</sup>)-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><sup>2</sup>)-AAF/dG) calculated for multiple transfections with independent preparations of the expression constructs (nâ=â3, +/â 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 MgCl<sub>2</sub> 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 MgCl<sub>2</sub>,
zincation using TMP<sub>2</sub>Zn¡2LiCl¡2MgCl<sub>2</sub> occurs at the position C(6). This metalation method provides easy
access to functionalized uracils and uridines. Using TMP<sub>2</sub>Zn¡2LiCl¡2MgCl<sub>2</sub> also allows to functionalize
cytidine derivatives at the position C(6)
Modulation of the effect of dG(<i>N<sup>2</sup></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><sup>2</sup>)-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