The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions

The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate U DzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. U DzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.

Potential desiccation cracks on Mars: A synthesis from modeling, analogue-field studies, and global observations

Potential desiccation polygons (PDPs) are polygonal surface patterns that are a common feature in Noachian-to-Hesperian-aged phyllosilicate- and chloride-bearing terrains and have been observed with size scales that range from cm-wide (by current rovers) to 10s of meters-wide. The global distribution of PDPs shows that they share certain traits in terms of morphology and geologic setting that can aid identification and distinction from fracturing patterns caused by other processes. They are mostly associated with sedimentary deposits that display spectral evidence for the presence of Fe/Mg smectites, Al-rich smectites or less commonly kaolinites, carbonates, and sulfates. In addition, PDPs may indicate paleolacustrine environments, which are of high interest for planetary exploration, and their presence implies that the fractured units are rich in smectite minerals that may have been deposited in a standing body of water. A collective synthesis with new data, particularly from the HiRISE camera suggests that desiccation cracks may be more common on the surface of Mars than previously thought. A review of terrestrial research on desiccation processes with emphasis on the theoretical background, field studies, and modeling constraints is presented here as well and shown to be consistent with and relevant to certain polygonal patterns on Mars.

Crystalline, Mixed-Valence Manganese Analogue of Prussian Blue: Magnetic, Spectroscopic, X-ray and Neutron Diffraction Studies

The compound of stoichiometry Mn(II)3[Mn(III)(CN)6]2·zH2O (z = 12−16) (1) forms air-stable, transparent red crystals. Low-temperature single crystal optical spectroscopy and single crystal X-ray diffraction provide compelling evidence for N-bonded high-spin manganese(II), and C-bonded low-spin manganese(III) ions arranged in a disordered, face-centered cubic lattice analogous to that of Prussian Blue. X-ray and neutron diffraction show structured diffuse scattering indicative of partially correlated (rather than random) substitutions of [Mn(III)(CN)6] ions by (H2O)6 clusters. Magnetic susceptibility measurements and elastic neutron scattering experiments indicate a ferrimagnetic structure below the critical temperature Tc = 35.5 K.

Synthetic studies towards a novel, chemical stable, abasic site analogue of DNA

We synthesized the phosphinate 7 via photoaddition of methanol to the alpha, beta unsaturated deoxyribono lactone as the key step, followed by an Arbusov reaction for the introduction of phosphorous. Precursor 7 serves for the synthesis and incorporation into DNA of a novel chemically stable abasic site analogue that might act as an inhibitor for DNA glycosylases

Synthesis of a cyclopentane amide DNA analogue and its base pairing properties

cpa-DNA monomers containing the bases adenine and thymine have been synthesized starting from the known compound 1 in 12 steps. Partially and fully modified cpa-thymidine and cpa-adenosine containing oligodeoxynucleotides were synthesized by standard oligonucleotide chemistry. Fully modified homo-cpa-A sequences lead to duplex destabilization by -1.4 degrees C/mod. relative to DNA. As its congener bca-DNA, cpa-DNA prefers left-handed duplex formation where possible

Synthesis and incorporation into a-DNA of a novel conformationally constrained alpha-nucleoside analogue

We describe the synthesis and incorporation into alpha-DNA of a novel conformationally constrained alpha-nucleoside analogue. The carbohydrate part of this analogue was prepared in 4 steps from the known bicyclic precursor 1 via a stereospecific, intramolecular, Et 3B mediated radical addition to a keto-function as the key step. The thus obtained intermediate 4 was transformed stereoselectively into the corresponding alpha-nucleoside analogues 7 and 8 containing the bases adenine and thymine, and were further elaborated into the phosphoramidite building blocks 11 and 12 . Both building blocks were incorporated into alpha-oligodeoxynucleotides and their pairing behavior to parallel complementary DNA studied by UV-melting experiments. Single substitutions of alpha-deoxyribnucleoside units by the new analogues in the center of duplexes were found to be thermally destabilizing by only -0.8 to -3.1›C.

Elucidation of the gas-phase structure of a sugar-modified DNA analogue

Antisense oligonucleotides are medical agents for the treatment of genetic diseases that are designed to interact specifically with mRNA. This interaction either induces enzymatic degradation of the targeted RNA or modifies processing pathways, e.g. by inducing alternative splicing of the pre-mRNA. The latter mechanism applies to the treatment of Duchenne muscular dystrophy with a sugar-modified DNA analogue called tricyclo-DNA (tcDNA). In tcDNA the ribose sugar-moiety is extended to a three-membered ring system, which augments the binding affinity and the selectivity of the antisense oligonucleotide for its target. The advent of chemically modified nucleic acids for antisense therapy presents a challenge to diagnostic tools, which must be able to cope with a variety of structural analogues. Mass spectrometry meets this demand for non-enzyme based sequencing methods ideally, because the technique is largely unaffected by structural modifications of the analyte. Sequence coverage of a fully modified tcDNA 15mer can be obtained in a single tandem mass spectrometric experiment. Beyond sequencing experiments, tandem mass spectrometry was applied to elucidate the gas-phase structure and stability of tcDNA:DNA and tcDNA:RNA hybrid duplexes. Most remarkable is the formation of truncated duplexes upon collision-induced dissociation of these structures. Our data suggest that the cleavage site within the duplex is directed by the modified sugar-moiety. Moreover, the formation of truncated duplexes manifests the exceptional stability of the hybrid duplexes in the gas-phase. This stability arises from the modified sugar-moiety, which locks the tcDNA single strand into a conformation that is similar to RNA in A-form duplexes. The conformational particularity of tcDNA in the gas-phase was confirmed by ion mobility-mass spectrometry experiments on tcDNA, DNA, and RNA.

Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

3CAPS – a structural AP-site analogue as a tool to investigate DNA base excision repair

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3′-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

VIS-NIR reflectance of water ice/regolith analogue mixtures and implications for the detectability of ice mixed within planetary regoliths

Permanently shadowed regions at the poles of the Moon and Mercury have been pointed out as candidates for hosting water ice at their surface. We have measured in the laboratory the visible and near infrared spectral range (VIS-NIR) bidirectional reflectance of intimate mixtures of water ice and the JSC-1AF lunar simulant for different ice concentrations, particle sizes, and measurement geometries. The nonlinearity between the measured reflectance and the amount of ice in the mixture can be reproduced to some extent by the mixing formulas of standard reflectance models, in particular, those of Hapke and Hiroi, which are tested here. Estimating ice concentrations from reflectance data without knowledge of the mixing coefficientsstrongly dependent on the size/shape of the grainscan result in large errors. According to our results, it is possible that considerable amounts of water ice might be intimately mixed in the regolith of the Moon and Mercury without producing noticeable photometric signatures.

Field investigation of dried lakes in western United States as an analogue to desiccation fractures on Mars

Potential Desiccation Polygons (PDPs), tens to hundreds of meters in size, have been observed in numerous regions on Mars, particularly in ancient (>3Gyr old) terrains of inferred paleolacustrine/playa geologic setting, and in association with hydrous minerals such as smectites. Therefore, a better understanding of the conditions in which large desiccation polygons form could yield unique insight into the ancient climate on Mars. Many dried lakebeds/playas in western United States display large (>50m wide) desiccation polygons, which we consider to be analogues for PDPs on Mars. Therefore, we have carried out fieldwork in seven of these dried lakes in San Bernardino and the Death Valley National Park regions complemented with laboratory and spectral analysis of collected samples. Our study shows that the investigated lacustrine/playa sediments have (a) a soil matrix containing 40-75% clays and fine silt (by volume) where the clay minerals are dominated by illite/muscovite followed by smectite, (b) carbonaceous mineralogy with variable amounts of chloride and sulfate salts, and significantly, (c) roughly similar spectral signatures in the visible-near-infrared (VIS-NIR) range. We conclude that the development of large desiccation fractures is consistent with water table retreat. In addition, the comparison of the mineralogical to the spectral observations further suggests that remote sensing VIS-NIR spectroscopy has its limitations for detailed characterization of lacustrine/playa deposits. Finally, our results imply that the widespread distribution of PDPs on Mars indicates global or regional climatic transitions from wet conditions to more arid ones making them important candidate sites for future in situ missions.