Photo-physical properties of 2-(1-ethynylpyrene)-adenosine: influence of hydrogen bonding on excited state properties.

The photo-physical properties of 2-(1-ethynylpyrene)-adenosine (PyA), a fluorescent probe for RNA dynamics, were examined by solvation studies. The excited-state dynamics display the influence of the vicinity on the spectral features. Combining improved transient absorption and streak camera measurements along with a new analysis method provide a detailed molecular picture of the photophysics. After intramolecular vibrational energy redistribution (IVR), two distinct states are observed. Solvent class (protic/aprotic) and permittivity strongly affect the properties of these states and their population ratio. As a result their emission spectrum is altered, while the fluorescence quantum yield and the overall lifetime remain nearly unchanged. Consequently, the hitherto existing model of the photophysics is herein refined and extended. The findings can serve as basis for improving the information content of measurements with PyA as a label in RNA.

Nitroxide spin labeled RNA for long range distance measurements by EPR-PELDOR.

Long range distance measurement on RNA allow the determination of RNA folds. Here we report the site specific incorporation of nitroxide spin labels at U,C and A by "on column synthesis". PELDOR (Pulsed Electron Double Resonance) measurements of several RNAs in the range of 2-6 nm were successful.

The fluoride cleavable 2-(cyanoethoxy)methyl (CEM) group as reversible 3'-O-terminator for DNA sequencing-by-synthesis - synthesis, incorporation, and cleavage.

A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast determination of DNA sequences. 2'-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM) group at the 3'-O-position are potential reversible terminators for the SBS technology. Herein we describe the synthesis, the incorporation by several polymerases, and the cleavage of this 3'-O-blocking group using 3'-O-CEM-thymidinyl-5'-O-triphosphate 7 as an example.

RNA recognition by fluor-aromatic substituted.

RNA exhibits a higher structural diversity than DNA and is an important molecule in the biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops, etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing, and hydrogen bond is limited. We synthesized different fluoromodifications of RNA building blocks: 1'-deoxy-1'-phenyl-beta-D-ribofuranose (B), 1'-deoxy-1'-(4-fluorophenyl)-beta-D-ribofuranose (4 FB), 1'-deoxy-1'-(2,4-difluorophenyl)-beta-D-ribofuranose (2,4 DFB), 1'-deoxy- 1'-(2,4,5-trifluorophenyl)-beta-D-ribofuranose (2,4,5 TFB), 1'-deoxy- 1'-(2,4, 6-trifluorophenyl)-beta-D-ribofuranose, 1'-deoxy- 1'-(pentafluorophenyl)-beta-D-ribofuranose (PFB), 1'-deoxy-1'-(benzimidazol-1-yl)-beta-D-ribofuranose (BI), 1'-deoxy-1'-(4-fluoro-1H-benzimidazol-1-yl)-1-beta-ribofuranose (4 FBI), 1'-deoxy- 1'-(6-fluoro- 1H-benzimidazol-1-yl)-beta-D-ribofuranose (6FBI), 1'-deoxy- 1'-(4, 6-difluoro- 1H-benzimidazol- 1-yl)-beta-D-ribofuranose (4,6 DFBI), 1'-deoxy- 1'-(4-trifluoromnethyl- H-benzimidazol-1-yl)-beta-D-ribofuranose (4 TFM), 1'-deoxy-1'-(5-trifluoromnethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (5 TFM), and 1'-deoxy-1'-(6-trifluoromethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (6 TFM). These amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5-CUU UUCXUU CUU-3' paired with 3'-GAA AAG YAA GAA-5'). Only one position was modified, marked as X and Y, respectively. UV melting profiles of those oligonucleotides were measured.

Homo and heterodimers of ddI, d4T and AZT: influence of (5'-5') thiolcabonate-carbamate linkage on anti-HIV activity.

New homo and heterodimers of ddI, d4T and AZT with (5-5) thiolcarbonate-carbamate linkages have been prepared with the aim of testing them against wild type and NNRTI resistant HIV mutants. The prepared dimers showed a low activity in comparison to the parent drug.

Synthesis and purification of fluorinated benzimidazole and benzene nucleoside-5'-triphosphates.

The expression "universal base" is very often used to express hybridization properties and recognition patterns of nucleosides. Their behaviour in biological applications, however, is of great interest regarding, e.g.,' their incorporation by polymerases. The 4,6-difluorobenzimidazole and the 2,4-difluorobenzene nucleoside analogues have proven to be universal bases that do not discriminate between the four natural nucleobases in RNA duplexes. Therefore, we synthesized the corresponding triphosphates to evaluate their behavior in polymerase catalyzed reactions and to investigate their ability to serve as substrates for the T7 RNA polymerase.

Doped natural phosphate: a new and environmentally friendly catalyst in nucleoside synthesis.

Doped natural phosphate is used as acidic or basic catalyst in nucleoside and acyclonucleoside synthesis. Some examples are given.

Synthesis of new homo and heterodimers of 2',3'-dideoxyinosine (ddI) using ester linkages.

A series of new homo and heterodimers of ddI has been synthesized. A glutarate diester spacer was used to covalently couple ddI onto ddI, AZT or d4T.

Synthesis of modified RNA-oligonucleotides for structural investigations.

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1'-deoxy-1'-(2,4,6-trifluorophenyl)-beta-D-ribofuranose (F), 1'-deoxy-1'-(2,4,5-trifluorophenyl)-beta-D-ribofuranose (M) and 1'-deoxy-1'-(5-trifluoromethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5'-CUU UUC XUU CUU-3' paired with 3'-GAA AAG YAA GAA-5') (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.

The effect of universal fluorinated nucleobases on the catalytic activity of ribozymes.

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2, containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The Km value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM(-1), the corresponding kcat is 2.91 x 10(-3) min(-1). A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2'-(beta-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.

Specific inhibition of hepatitis C viral gene expression by non-polar (phenylalkyl)phosphonates.

Different phenylalkyl backbone modified antisense oligonucleotides complementary to the Hepatitis C virus (HCV) RNA nucleotides 326-342 were synthesized. The lipohilic character of modified oligonucleotides was determined from RP-HPLC retention times. The inhibitory effect of these antisense oligonucleotides on HCV gene expression was analyzed in an in vitro test system.

Building blocks for polyamide nucleic acids: facile synthesis using potassium fluoride doped natural phosphate as basic catalyst.

Potassium fluoride doped natural phosphate, inexpensive and environmentally friendly catalyst, is shown to be an efficient basic catalyst for the N1/N9 alkylation of different nucleobases as synthons for PNAs.

Measurement of 2J(H,C)- and 3J(H,C)-coupling constants by alpha/beta selective HC(C)H-TOCSY.

A new heteronuclear NMR pulse sequence for the measurement of nJ(C,H) coupling constants, the alpha/beta selective HC(C)H-TOCSY, is described. It is shown that the S3E element (Meissner et al., 1997a,b) can be used to obtain spin state selective coherence transfer in molecules, in which adjacent CH moieties are labeled with 13C. Application of the alpha/beta selective HC(C)H-TOCSY to a 10 nt RNA tetraloop 5'-CGCUUUUGCG-3', in which the four uridine residues are 13C labeled in the sugar moiety, allowed measurement of two bond and three bond J(C,H) coupling constants, which provide additional restraints to characterize the sugar ring conformation of RNA in cases of conformational averaging.

Phenylalkyl backbone modified oligodeoxynucleotides, their synthesis and the influence of the alkyl chain length.

Phenylalkyl modified phosphoramidites (alkyl chain length n = 1, 2, 3, 5; Fig. 1) were synthesised and incorporated into a DNA hexamer (5'-d(GCCp-GCG); p = place of modification). The obtained diastereomeres were separated by RP-HPLC. After hybridisation with the complementary DNA strand Tm-value and thermodynamic data were measured. The stability of duplexes depends on the linker length and the absolute configuration of the backbone modified oligodeoxynucleotides (Rp, Sp).

A new facile method for spin-labeling of oligonucleotides.

A new facile method for spin-labeling suitable for DNA and RNA oligonucleotides is presented. The nitroxide 3-ethenyl-2,2,5,5-tetramethyl-pyrrolin-1-yloxy was directly introduced during automated solid-phase synthesis by a Pd(0) cross coupling reaction. The main advantages of this procedure are the small amount of spin-label needed for the derivatisation of the oligonucleotide and the high coupling efficiency on the solid phase.

Design and properties of hepatitis C virus antisense oligonucleotides for liver specific drug targeting.

Different backbone modified antisense oligonucleotides (AS-ODNs) directed against the hepatitis C virus genome were 5'-conjugated to cholesterol, cholic acid or taurocholic acid to enhance liver specific drug targeting and hepatocellular uptake. The lipophilic character of modified AS-ODNs was determined from RP-HPLC retention times and duplex stability was correlated with Tm-values from UV melting curves.

6-oxocytidine containing oligonucleotides inhibit the HIV-1 integrase in vitro.

Integration of the proviral DNA into the genome of infected cells is a key step of HIV-1 replication. Integration is catalyzed by the viral enzyme integrase (IN). 6-oxocytidine-containing oligonucleotides were found to be efficient inhibitors of integrase in vitro. The inhibitory effect is sequence-specific and strictly requires the presence of the 6-oxocytidine base. It is due to the impairment of the integrase binding to its substrate and does not involve an auto-structure of the oligonucleotide.

An assay system to detect the selective advantage of anti-HIV ribozyme expressing CD4+ T-lymphocytes.

This study was undertaken to establish an assay system to detect the survival advantage of anti-HIV ribozyme expressing cells under the selective pressure of HIV infection. In a mixture with wild type cells the proportion of ribozyme expressing cells was increased up to 12-fold. As a mechanism of the selective advantage an inhibition of HIV induced apoptotic cell death could be demonstrated. Furthermore, a dose dependency of the antiviral ribozyme effects was observed.

Stacking and stability of RNA duplexes containing fluorobenzene and fluorobenzimidazole nucleosides.

Six different fluorobenzene or fluorobenzimidazole ribonucleosides and one abasic site were incorporated in oligoribonucleotides. Individual contributions of base stacking and solvation of the modified nucleosides could be determined. In fluorobenzene.fluorobenzimidazole-modified base pairs a duplex stabilizing force was found that points to a weak F...H hydrogen bond. The lipophilicity of the unprotected nucleosides were investigated by determination of 1-octanol water partition coefficients.

Synthesis of 3'-thioamido-modified 3'-deoxythymidine-5'-triphosphates and their use as chain terminators in Sanger-DNA sequencing.

The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxo- propyl)amino]thymidine 1 and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-((6-([(9H-(fluo-ren-9- ylmethoxy)carbonyl]-amino)-1-thioxohexyl)amino) thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5'-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.