10 research outputs found
2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling
Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms
AbstractThe flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms
Surprising Base Pairing and Structural Properties of 2′-Trifluoromethylthio-Modified Ribonucleic Acids
The chemical synthesis of ribonucleic
acids (RNA) with novel chemical
modifications is largely driven by the motivation to identify eligible
functional probes for the various applications in life sciences. To
this end, we have a strong focus on the development of novel fluorinated
RNA derivatives that are powerful in NMR spectroscopic analysis of
RNA folding and RNA ligand interactions. Here, we report on the synthesis
of 2′-SCF<sub>3</sub> pyrimidine nucleoside containing oligoribonucleotides
and the comprehensive investigation of their structure and base pairing
properties. While this modification has a modest impact on thermodynamic
stability when it resides in single-stranded regions, it was found
to be destabilizing to a surprisingly high extent when located in
double helical regions. Our NMR spectroscopic investigations on short
single-stranded RNA revealed a strong preference for C2′-endo
conformation of the 2′-SCF<sub>3</sub> ribose unit. Together
with a recent computational study (L. Li, J. W. Szostak, <i>J.
Am. Chem. Soc</i>. <b>2014</b>, <i>136</i>, 2858–2865)
that estimated the extent of destabilization caused by a single C2′-endo
nucleotide within a native RNA duplex to amount to 6 kcal mol<sup>−1</sup> because of disruption of the planar base pair structure,
these findings support the notion that the intrinsic preference for
C2′-endo conformation of 2′-SCF<sub>3</sub> nucleosides
is most likely responsible for the pronounced destabilization of double
helices. Importantly, we were able to crystallize 2′-SCF<sub>3</sub> modified RNAs and solved their X-ray structures at atomic
resolution. Interestingly, the 2′-SCF<sub>3</sub> containing
nucleosides that were engaged in distinct mismatch arrangements, but
also in a standard Watson–Crick base pair, adopted the same
C3′-endo ribose conformations as observed in the structure
of the unmodified RNA. Likely, strong crystal packing interactions
account for this observation. In all structures, the fluorine atoms
made surprisingly close contacts to the oxygen atoms of the corresponding
pyrimidine nucleobase (O2), and the 2′-SCF<sub>3</sub> moieties
participated in defined water-bridged hydrogen-bonding networks in
the minor groove. All these features allow a rationalization of the
structural determinants of the 2′-SCF<sub>3</sub> nucleoside
modification and correlate them to base pairing properties
2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling
Chemical modification can significantly enrich the structural
and
functional repertoire of ribonucleic acids and endow them with new
outstanding properties. Here, we report the syntheses of novel 2′-azido
cytidine and 2′-azido guanosine building blocks and demonstrate
their efficient site-specific incorporation into RNA by mastering
the synthetic challenge of using phosphoramidite chemistry in the
presence of azido groups. Our study includes the detailed characterization
of 2′-azido nucleoside containing RNA using UV-melting profile
analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic
analysis of 2′-azido uridine and 2′-azido adenosine
modified RNAs reveals crucial structural details of this modification
within an A-form double helical environment. The 2′-azido group
supports the C3′-<i>endo</i> ribose conformation
and shows distinct water-bridged hydrogen bonding patterns in the
minor groove. Additionally, siRNA induced silencing of the brain acid
soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated
that 2′-azido modifications are well tolerated in the guide
strand, even directly at the cleavage site. Furthermore, the 2′-azido
modifications are compatible with 2′-fluoro and/or 2′-<i>O</i>-methyl modifications to achieve siRNAs of rich modification
patterns and tunable properties, such as increased nuclease resistance
or additional chemical reactivity. The latter was demonstrated by
the utilization of the 2′-azido groups for bioorthogonal Click
reactions that allows efficient fluorescent labeling of the RNA. In
summary, the present comprehensive investigation on site-specifically
modified 2′-azido RNA including all four nucleosides provides
a basic rationale behind the physico- and biochemical properties of
this flexible and thus far neglected type of RNA modification
Thermodynamics of HIV‑1 Reverse Transcriptase in Action Elucidates the Mechanism of Action of Non-Nucleoside Inhibitors
HIV-1 reverse transcriptase (RT)
is a heterodimeric enzyme that
converts the genomic viral RNA into proviral DNA. Despite intensive
biochemical and structural studies, direct thermodynamic data regarding
RT interactions with its substrates are still lacking. Here we addressed
the mechanism of action of RT and of non-nucleoside RT inhibitors
(NNRTIs) by isothermal titration calorimetry (ITC). Using a new incremental-ITC
approach, a step-by-step thermodynamic dissection of the RT polymerization
activity showed that most of the driving force for DNA synthesis is
provided by initial dNTP binding. Surprisingly, thermodynamic and
kinetic data led to a reinterpretation of the mechanism of inhibition
of NNRTIs. Binding of NNRTIs to preformed RT/DNA complexes is hindered
by a kinetic barrier and NNRTIs mostly interact with free RT. Once
formed, RT/NNRTI complexes bind DNA either in a seemingly polymerase-competent
orientation or form high-affinity dead-end complexes, both RT/NNRTI/DNA
complexes being unable to bind the incoming nucleotide substrate