A 6'-Fluoro-Substituent in Bicyclo-DNA Increases Affinity to Complementary RNA Presumably by CF-HC Pseudohydrogen Bonds

The synthesis of a novel bicyclic thymidine analogue carrying a β-fluoro substituent at C6' (6'F-bcT) has been achieved. Key steps of the synthesis were an electrophilic fluorination/stereospecific hydrogenation sequence of a bicyclo sugar intermediate, followed by an N-iodo-succinimide-induced stereoselective nucleosidation. A corresponding phosphoramidite building block was then prepared and used for oligonucleotide synthesis. Tm measurements of oligonucleotides with single and double incorporations showed a remarkable stabilization of duplex formation particularly with RNA as complement without compromising pairing selectivity. Increases in Tm were in the range of +1-2 °C compared to thymidine and +1-3 °C compared to a standard bc-T residue. Structural investigations of the 6'F-bcT nucleoside by X-ray crystallography showed an in-line arrangement of the fluorine substituent with H6 of thymine, however, with a distance that is relatively long for a nonclassical CF-HC hydrogen bond. In contrast, structural investigations in solution by (1)H and (13)C NMR clearly showed scalar coupling of fluorine with H6 and C6 of the nucleobase, indicating the existence of at least weak electrostatic interactions. On the basis of these results, we put forward the hypothesis that these weak CF-HC6 electrostatic interactions increase duplex stability by orienting and partially freezing torsion angle χ of the 6'F-bcT nucleoside.

Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bcen-T and iso-tricyclo-T nucleosides

We present the synthesis of the two novel nucleosides iso-tc-T and bcen-T, belonging to the bicyclo-/tricyclo-DNA molecular platform. In both modifications the torsion around C6’–C7’ within the carbocyclic ring is planarized by either the presence of a C6’–C7’ double bond or a cyclopropane ring. Structural analysis of these two nucleosides by X-ray analysis reveals a clear preference of torsion angle γ for the gauche orientation with the furanose ring in a near perfect 2’-endo conformation. Both modifications were incorporated into oligodeoxynucleotides and their thermal melting behavior with DNA and RNA as complements was assessed. We found that the iso-tc-T modification was significantly more destabilizing in duplex formation compared to the bcen-T modification. In addition, duplexes with complementary RNA were less stable as compared to duplexes with DNA as complement. A structure/affinity analysis, including the already known bc-T and tc-T modifications, does not lead to a clear correlation of the orientation of torsion angle γ with DNA or RNA affinity. There is, however, some correlation between furanose conformation (N- or S-type) and affinity in the sense that a preference for a 3’-endo like conformation is associated with a preference for RNA as complement. As a general rule it appears that Tm data of single modifications with nucleosides of the bicyclo-/tricyclo-DNA platform within deoxyoligonucleotides are not predictive for the stability of fully modified oligonucleotides.

Synthesis and Properties of 6′-Fluoro-tricyclo-DNA

The synthesis of the two fluorinated tricyclic nucleosides 6?-F-tc-T and 6?-F-tc-5MeC, as well as the corresponding building blocks for oligonucleotide assembly, was accomplished. An X-ray analysis of N4-benzoylated 6?-F-tc-5MeC reavealed a 2?-exo (north) conformation of the furanose ring, characterizing it as an RNA mimic. In contrast to observations in the bicyclo-DNA series, no short contact between the fluorine atom and the H6 of the base, reminiscent of a nonclassical F···H hydrogen bond, could be observed. Tm measurements of modified oligodeoxynucleotides with complementary RNA showed slightly sequence-dependent duplex stabilization profiles with maximum ?Tm/mod values of +4.5 °C for 6?-F-tc-5MeC and +1 °C for 6?-F-tc-T. In comparison with parent tc-modified oligonucleotides, no relevant changes in Tm were detected, attributing the fluorine substituent a neutral role in RNA affinity. A structural analysis of duplexes with DNA and RNA by CD-spectroscopy revealed a shift from B- to A-type conformation induced by the 6?-F-tc-nucleosides. This is not a specific ?fluorine effect?, as the same is also observed for the parent tc-modifications. The two fluorinated tc-nucleosides were also incorporated into a pure tricyclo-DNA backbone and showed no discrimination in Tm with complementary RNA, demonstrating that 6?-F substitution is also compatible within fully modified tc-oligonucleotides.

The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis.

During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.

Synthesis of cyclopentane amide DNA (cpa-DNA) and its pairing properties

We recently reported on the synthesis and pairing properties of the DNA analogue bicyclo[3.2.1]amide DNA (bca-DNA). In this analogue the nucleobases are attached via a linear, 4-bond amide-linker to a structurally preorganized sugar-phosphate backbone unit. To define the importance of the degree of structural rigidity of the bca-backbone unit on the pairing properties, we designed the structurally simpler cyclopentane amide DNA (cpa-DNA), in which the bicyclo[3.2.1]-scaffold was reduced to a cyclopentane unit while the base-linker was left unchanged. Here we present a synthetic route to the enantiomerically pure cpa-DNA monomers and the corresponding phosphoramidites containing the bases A and T, starting from a known, achiral precursor in 9 and 12 steps, respectively. Fully modified oligodeoxynucleotides were synthesized by standard solid-phase oligonucleotide chemistry, and their base-pairing properties with complementary oligonucleotides of the DNA-, RNA-, bca-DNA-, and cpa-DNA-backbones were assessed by UV melting curves and CD-spectroscopic methods. We found that cpa-oligoadenylates form duplexes with complementary DNA that are less stable by -2.7 degrees C/mod. compared to DNA. The corresponding cpa-oligothymidylates do not participate in complementary base-pairing with any of the investigated backbone systems except with its own (homo-duplex). As its congener bca-DNA, cpa-DNA seems to prefer left-handed helical duplex structures with DNA or with itself as indicated by the CD spectra

Synthesis and Thermodynamic and Biophysical Properties of Tricyclo-DNA

The DNA analogue tricyclo-DNA, built from conformationally rigid nucleoside analogues that were linked via tertiary phosphodiester functions, can efficiently be synthesized from the corresponding phosphoramidites by conventional solid-phase cyanoethyl phosphoramidite chemistry. 5'-End phosphorylated tricyclo-DNA sequences are chemically stable in aqueous, pH-neutral media at temperatures from 0 to 90 C. Tricyclo-DNA sequences resist enzymatic hydrolysis by the 3'-exonuclease snake venom phosphodiesterase. Homobasic adenine- and thymine-containing tricyclo-DNA octa- and nonamers are extraordinarily stable A-T base-pairing systems, not only in their own series but also with complementary DNA and RNA. Base mismatch formation is strongly destabilized. As in bicyclo-DNA, the tricyclo-DNA purine sequences preferentially accept a complementary strand on the Hoogsteen face of the base. A thermodynamic analysis reveals entropic benefits in the case of hetero-backbone duplex formation (tricyclo-DNA/DNA duplexes) and both an enthalpic and entropic benefit for duplex formation in the pure tricyclo-DNA series compared to natural DNA. Stability of tricyclo-DNA duplex formation depends more strongly on monovalent salt concentration compared to natural DNA. Homopyrimidine DNA sequences containing tricyclothymidine residues form triplexes with complementary double-stranded DNA. Triple-helix stability depends on the sequence composition and can be higher when compared to that of natural DNA. The use of one tricyclothymidine residue in the center of the self-complementary dodecamer duplex (d(CGCGAAT t CGCG), t = tricyclothymidine) strongly stabilizes its monomolecular hairpin loop structure relative to that of the corresponding pure DNA dodecamer ( T m = +20 C), indicating (tetra)loop-stabilizing properties of this rigid nucleoside analogue.

Distamycin-NA: A DNA analog with an aromatic heterocyclic polyamide backbone. Part 2. Solid-phase synthesis of distamycin-NAs containing the nucleobase uracil: Unexpected solvent participation in the coupling step

10.1002/hlca.19980810512.abs The synthesis of the Fmoc-protected amino acid 2 is presented. First attempts of amide-bond formation to the homodimer 4 in solution showed only poor coupling yields indicative for the low reactivity of the amino and carboxy groups in the building blocks 1 and 2, respectively (Scheme 1). Best coupling yields were found using dicyclohexylcarbodiimide (DCC) without any additive. The oligomerization of building block 2 adopting the Fmoc ((9H-fluoren-9-ylmethoxy)carbonyl) solid-phase synthesis yielded a mixture of N-terminal-modified distamycin-NA derivatives. By combined HPLC and MALDI-TOF-MS analysis, the N-terminal functional groups could be identified as acetamide and N,N-dimethylformamidine functions, arising from coupling of the N-terminus of the growing chain with residual AcOH or DCC-activated solvent DMF. An improved preparation of building block 2 and coupling protocol led to the prevention of the N-terminal acetylation. However, ‘amidination’ could not be circumvented. A thus isolated tetramer of 2, containing a lysine unit at the C-terminus and a N,N-dimethylformamidine-modified N-terminus, not unexpectedly, showed no complementary base pairing to DNA and RNA, as determined by standard UV-melting-curve analysis.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.

The effect of RNA base lesions on mRNA translation

The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell. Here we report on the effect of the commonly known RNA base-lesions 8-oxo-rG, 8-oxo-rA, ε-rC, ε-rA, 5-HO-rC, 5-HO-rU and the RNA abasic site (rAS) on ribosomal translation. To this end we have developed an in vitro translation assay based on the mRNA display methodology. A short synthetic mRNA construct containing the base lesion in a predefined position of the open reading frame was 32P-labeled at the 5′-end and equipped with a puromycin unit at the 3′-end. Upon in vitro translation in rabbit reticulocyte lysates, the encoded peptide chain is transferred to the puromycin unit and the products analyzed by gel electrophoresis. Alternatively, the unlabeled mRNA construct was used and incubated with 35S-methionine to prove peptide elongation of the message. We find that all base-lesions interfere substantially with ribosomal translation. We identified two classes, the first containing modifications at the base coding edge (ε-rC, ε-rA and rAS) which completely abolish peptide synthesis at the site of modification, and the second consisting of 8-oxo-rG, 8-oxo-rA, 5-HO-rC and 5-HO-rU that significantly retard full-length peptide synthesis, leading to some abortive peptides at the site of modification.

Syncytiotrophoblast vesicles show altered micro-RNA and haemoglobin content after ex-vivo perfusion of placentas with haemoglobin to mimic preeclampsia.

BACKGROUND Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content. METHODS The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR. RESULTS All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs. CONCLUSION This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

An intergenic non-coding RNA targets and regulates the ribosome in H. volcanii

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the non-protein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation and seems to inhibit translation in vitro. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.

Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves

Senescence-associated coordination in amounts of enzymes localized in different cellular compartments were determined in attached leaves of young wheat (Triticum aestivum L. cv. Arina) plants. Senescence was initiated at the time of full leaf elongation based on declines in total RNA and soluble protein. Removal of N from the growth medium just at the time of full leaf elongation enhanced the rate of senescence. Sustained declines in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), and a marked decrease in the rbcS transcripts, just after full leaf elongation indicated that Rubisco synthesis/degradation was very sensitive to the onset of senescence. Rubisco activase amount also declined during senescence but the proportion of rca transcript relative to the total poly A RNA pool increased 3-fold during senescence. Thus, continued synthesis of activase may be required to maintain functional Rubisco throughout senescence. N stress led to declines in the amount of proteins located in the chloroplast, the peroxisome and the cytosol. Transcripts of the Clp protease subunits also declined in response to N stress, indicating that Clp is not a senescence-specific protease. In contrast to the other proteins, mitochondrial NADH-glutamate dehydrogenase (EC 1.4.1.2) was relatively stable during senescence and was not affected by N stress. During natural senescence with adequate plant nitrate supply the amount of nitrite reductase (EC 1.7.7.1) increased, and those of glutamine synthetase (EC 1.4.7.1) and glutamate synthase (EC 6.3.1.2) were stable. These results indicated that N assimilatory capacity can continue or even increase during senescence if the substrate supply is maintained. Differential stabilities of proteins, even within the same cellular compartment, indicate that proteolytic activity during senescence must be highly regulated.