9 research outputs found
The crystal structure of an ‘All Locked’ nucleic acid duplex
‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern
Deconvoluting hepatic processing of carbon nanotubes
Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans
PEDF and GDNF are key regulators of photoreceptor development and retinal neurogenesis in reaggregates from chick embryonic retina
Here, role(s) of pigment epithelial-derived factor (PEDF) and glial-derived neurotrophic factor (GDNF) on photoreceptor development in three-dimensional reaggregates from the retinae of the E6 chick embryo (rosetted spheroids) was investigated. Fully dispersed cells were reaggregated under serum-reduced conditions and supplemented with 50 ng/ml PEDF alone or in combination with 50 ng/ml GDNF. The spheroids were analyzed for cell growth, differentiation, and death using proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, and other immunocytochemical stainings and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) methods. PEDF strongly promoted synthesis of the messenger RNAs for blue and violet cone opsins and to a lesser extent on the red and green cone opsins. This correlated with an increase in the number of cone photoreceptors, as determined by the cone cell marker CERN906. Likewise, PEDF nearly completely inhibited rod differentiation, as detected by immunostaining with anti-rho4D2 and RT-PCR. Furthermore, PEDF accelerated proliferation of cells in the spheroids and inhibited apoptosis. As negative effects, PEDF inhibited the normal histotypic tissue formation of retinal aggregates and reduced the frequency of photoreceptor rosettes and IPL-like areas. Noticeably, supplementation of PEDF-treated cultures with GDNF reversed the effects of PEDF on spheroid morphology and on rod differentiation. This study establishes that PEDF strongly affects three-dimensional retinogenesis in vitro, most notably by inhibiting rod development and supporting proliferation and differentiation of cones, effects which are partially counteracted by GDNF
In vitro selection of Spiegelmers against affinity tags
Protein-Affinitäts-Tags sind Peptidsequenzen, welche zur Detektion und
Affinitätsaufreinigung von rekombinant hergestellten Proteinen eingesetzt
werden. Das Oktapeptid Strep-Tag-II gehört zu den meist verwendeten kurzen
Affinitäts-Tags. Eine hochaffine und hochspezifische Erkennung ist zur
Detektion und Aufreinigung von Strep-Tag-II-Fusionsproteinen unerlässlich.
Spiegelmere sind enantiomere Aptamere, welche durch die Anwendung der
Spiegeltechnik auf die in vitro Selektion von Nukleinsäuren gewonnen werden.
Die so erhaltenen L-Nukleinsäuren sind durch ihren Aufbau aus L-Monomeren
hochgradig stabil und besitzen dennoch die Eigenschaften von Aptameren: die
hochaffine und -spezifische Bindung des Zielmoleküls. Innerhalb dieser Arbeit
wurden erstmals Spiegelmere gegen das Strep-Tag-II-Affinitätspeptid
entwickelt. Diese können zur affinen und spezifischen Detektion,
Immobilisierung und Affinitätsaufreinigung von Strep-Tag-II-Fusionsproteinen
eingesetzt werden. Innerhalb von sieben Selektionsrunden der in vitro
Selektion wurden aus einer kombinatorischen DNA-Bibliothek mit etwa 1015
verschiedenen Molekülen sieben verschiedene Aptamere isoliert, welche alle
affin das D-Strep-Tag-II-Peptid binden. Für die konservierten Bereiche der
Aptamere wurde eine G-Quadruplexstruktur postuliert, was mit der ermittelten
Abhängigkeit der Bindung von Kalium- und Natriumionen kohärent ist. Das
Aptamer und das korrespondierende Spiegelmer ST-146 binden das D- bzw. L
-Strep-Tag-II-Peptid mit einer Dissoziationskonstante von 370 nM. Die
Bindungskonstante einer auf 43 Nukleotide verkürzten Version des ST-146 liegt
in derselben Größenordnung. Die für dieses Molekül erhaltenen Kristalle können
zu einer röntgenkristallografischen Strukturaufklärung des Strep-Tag-II-
Binders führen. Das Spiegelmer ST-146 wurde sowohl zur N-, als auch zur
C-terminalen Detektion eines Strep-Tag-II-Fusionsproteins eingesetzt. Die
affine und spezifische Erkennung von Strep-Tag-II-Fusionsproteinen durch das
Spiegelmer (Kd = 220 - 600 nM) ist stark vom pH-Wert abhängig, wobei ein pH-
Bereich von 5 - 7,5 ideal für die Bindung ist. Darüber hinaus konnte die
Erkennung von Strep-Tag-II-Fusionsproteinen aus einem Gemisch von Proteinen
gezeigt werden. Analog zu ST-146 wurde ein Spiegelmer gegen einen neuen
Affinitäts-Tag, den in silico generierten Spiegel-Tag entwickelt. Die
Aminosäuresequenz dieses neuen Affinitäts-Tags ist GSHKRNQTIEGR. Das
Spiegelmer SP-56 bindet den Spiegel-Tag affin mit einer Dissoziationskonstante
von 550 nM. Auch für die Interaktion des SP-56 mit dem Spiegel-Tag wurde eine
starke Abhängigkeit vom pH-Wert ermittelt. Locked Nucleic Acids (LNAs) sind
Nukleinsäureanalogons, welche häufig zur Stabilisierung von siRNAs und
Aptameren eingesetzt werden. Im zweiten Teil der Arbeit wurde erstmals eine
komplett aus LNA-Monomeren aufgebaute Doppelhelix kristallisiert und
röntgenkristallografisch vermessen. Die Struktur der ersten LNA/LNA-
Doppelhelix vervollständigt das Wissen um die strukturellen und molekularen
Eigenschaften dieser Nukleinsäureklasse.Protein affinity tags are peptide sequences, which are used for the detection
and affinity purification of recombinant proteins. The octapeptide Strep-Tag-
II is among the most commonly used short affinity tags. A sensitive and high
specific recognition of Strep-Tag-II fusion proteins is essential for the
detection and purification of these proteins. Spiegelmers are enantiomeric
aptamers, which are generated by applying the mirror-image technology to the
in vitro selection of nucleic acids. These L-nucleic acids are highly stable
by means of being composed of L-monomers that are not susceptible to
degradation by nucleases. However, they exhibit the qualities of aptamers:
binding to the target with high affinity and high specificity. In this work
for the first time, Spiegelmers against the affinity tag Strep-tag-II were
developed. They can be used for affinity and high specificity detection,
immobilization and affinity purification of Strep-tag-II fusion proteins.
Seven different aptamers were isolated by Systematic Evolution of Ligands by
Exponential Enrichment (SELEX) from a random DNA library of approximately 1015
different types of molecules. Each of them binds to the target molecule D
-Strep-tag-II peptide. A G-quadruplex structure is postulated for the
conserved sequences of the aptamers. This is coherent with the observed
dependence of the binding-affinity on potassium and sodium ions. The aptamer
and the corresponding Spiegelmer ST-146 bind with a binding constant of 370 nM
to the D- and L-Strep-tag-II peptide, respectively. A binding constant in the
same magnitude was obtained for the binding of a truncated 43 nucleotides
version of ST-146. The crystals obtained for this molecule might lead to the
X-ray crystallographic structure determination of the Strep-tag-II binder. The
Spiegelmer ST-146 was employed for the detection of fusion proteins with N-
and C-terminal Strep-tag-II, respectively. The specific recognition of Strep-
tag-II fusion proteins by ST-146 (Kd = 220 - 600 nM) strongly depends on pH
whereas values from pH 5 to 7.5 are ideal. Furthermore, ST-146 was shown to
detect Strep-Tag-II fusion proteins from a protein mixture. Similar to ST-146,
a Spiegelmer against a new affinity tag, the in silico derived ‘Spiegel-Tag’,
was developed. The amino acid sequence of the new tag is GSHKRNQTIEGR.
Spiegelmer SP-56 binds the ‘Spiegel-Tag’ with a binding constant of 550 nM. A
strong dependence on pH was detected for the interaction of SP-56 with the
Spiegel-Tag as well. Locked Nucleic Acids (LNAs) are modified nucleic acids,
which are commonly used for protecting siRNAs and aptamers from degradation.
In the second part of this work, for the first time, a double helix consisting
exclusively of LNA monomers was crystallized and analyzed by X-ray
crystallography. The structure of the first LNA/LNA duplex completes the
knowledge of the structural and molecular properties of this class of nucleic
acids
Crystallization and X-ray diffraction analysis of an ‘all-locked’ nucleic acid duplex derived from a tRNASer microhelix
A completely ‘all-locked’ nucleic acid duplex was designed from an E. coli tRNASer microhelix. The helix consists exclusively of LNA building blocks and was crystallized. The crystals diffracted to 1.9 Å resolution
Deconvoluting hepatic processing of carbon nanotubes
Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans