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Biomedical subjects

Christian J Leumann

Publications and source records attributed to Christian J Leumann.

36 records · Page 2Linked to original sources
PubMed2003

Synthesis and incorporation into PNA of fluorinated olefinic PNA (F-OPA) monomers.

[structure: see text] A fluorinated OPA monomer containing the base thymine ((Z)-t-F-OPA) was synthesized in 12 steps, featuring a highly selective allylic over homoallylic Mitsunobu substitution for the introduction of the nucleobase. F-OPA modified PNA decamers were prepared by the MMTr/acyl protection strategy. The thermal stability of duplexes of PNA decamers containing (Z)-t-F-OPA units with antiparallel complementary DNA was measured. We found a strong dependence of stability from the sequential position of the (Z)-t-F-OPA units, ranging from DeltaT(m) of +2.4 to -8.1 degrees C/modification relative to unmodified PNA.

Alkenes↗
PubMed2003

Recent improvements in antigene technology.

DNA triple-helix-based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother-like treatment in comparison to the flourishing field of oligonucleotide-based control of translation (antisense technology). This was mostly due to lack of affinity of triplex-forming oligonucleotides to their DNA target, to sequence restriction constraints imposed by the triple helical recognition motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine-purine inversion sites as well as sugar modifications, for example, the 2'-aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid sugar unit, which greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence-specific genomic DNA manipulation is starting to become a useful tool in biotechnology.

Base Pairing↗
PubMed2003

A short, efficient synthesis of 2'-deoxypseudoisocytidine based on Heck-chemistry.

A novel synthesis of 2'-deoxypseudoisocytidine as well as of its phosphoramidite building block for oligonucleotide synthesis is presented. The synthesis is based on Heck-coupling between N-protected pseudoisocytosine and a silyl protected furanoid glycal. With this procedure the corresponding phosphoramidite building block is obtained in 5 steps and an overall yield of 28%.

Deoxycytidine↗
PubMed2002

Bicyclo[3.2.1]amide-DNA: a chiral, nonchiroselective base-pairing system.

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analogue, are reported. This analogue consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to which the nucleo-bases were attached through an extended, acyclic amide linker. Homobasic adenine-containing bca decamers form duplexes with complementary oligonucleotides containing bca, DNA, RNA, and, surprisingly, also L-RNA backbones. UV and CD spectroscopic investigations revealed the duplexes with D- or L-complements to be of similar stability and enantiomorphic in structure. Bca oligonucleotides that contain all four bases form strictly antiparallel, left-handed complementary duplexes with themselves and with complementary DNA, but not with RNA. Base-mismatch discrimination is comparable to that of DNA, while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior to those of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA-containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association.

Amides↗
PubMed2002

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands.

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic, and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of nonequilibrium UV-melting and annealing curves in the range of pH 5.1-6.7 in a 10 mM citrate/phosphate buffer containing 0.1 M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both the association and dissociation process in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability, as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.

Base Sequence↗
PubMed2002

DNA binding properties of oligodeoxynucleotides containing pyrrolidino C-nucleosides.

We have incorporated pyrrolidino-C-nucleosides (pyrrolidino-pseudonucleosides) containing the base uracil and N-1-methyl uracil into oligodeoxynucleotides and compared their thermal duplex and triplex stabilities with unmodified or pseudouridine-containing oligodeoxynucleotides. We find relative destabilizations of triplex formation by ca. -13 to -1 degrees C per modification (relative to thymidine) in a strongly sequence dependent mode. Duplex formation is less destabilizing and more homogeneous with -4 to -2 degrees C per modification.

Base Sequence↗
PubMed2002

Antisense properties of tricyclo-DNA.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Alternative Splicing↗
PubMed2002

Watson-Crick base-pairing properties of tricyclo-DNA.

Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues. It differs structurally from DNA by an additional ethylene bridge between the centers C(3') and C(5') of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. The synthesis of the hitherto unknown tricyclodeoxynucleosides containing the bases cytosine and guanine and of the corresponding phosphoramidite building blocks is described, as well as a structural description of a representative of an alpha- and a beta-tricyclodeoxynucleoside by X-ray analysis. Tricyclodeoxynucleoside building blocks of all four bases were used for the synthesis of fully modified mixed-base oligonucleotides. Their Watson-Crick pairing properties with complementary DNA, RNA, and with itself were investigated by UV melting curves, CD spectroscopy, and molecular modeling. Tricyclo-DNA was found to be a very stable Watson-Crick base-pairing system. A UV melting curve analysis of the decamers tcd(pcgtgacagtt) and tcd(paactgtcacg) showed increased thermal stabilities of up to DeltaT(m)/mod. = +1.2 degrees C with complementary DNA and +2.4 degrees C with complementary RNA. With itself, tricyclo-DNA showed an increase in stability of +3.1 degrees C/base pair relative to DNA. Investigations into the thermodynamic properties of these decamers revealed an entropic stabilization and an enthalpic destabilization for the tricyclo-DNA/DNA duplexes. CD spectroscopic structural investigations indicated that tricyclo-DNA containing duplexes preferrably exist in an A-conformation, a fact which is in agreement with results from molecular modeling.

Base Pair Mismatch↗
PubMed2002

DNA analogues: from supramolecular principles to biological properties.

Mainly driven by the needs of antisense research, a large number of oligonucleotide analogues have been prepared and evaluated over the last 15 years. Besides minor structural modifications of the building blocks of DNA and RNA itself, a considerable effort has been devoted to the de novo design of nucleoside analogues with improved binding properties. A particularly successful concept turned out to be that of conformational restriction. This review focuses on recent advances in this area and tries to summarize scope and limitations of this design principle.

DNA↗
PubMed2002

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides.

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions.

DNA↗
PubMedDate not supplied

Pyrrolidino-DNA.

We synthesized pyrrolidino-C-nucleosides, incorporated them into oligodeoxynucleotides and investigated their pairing properties. The thermal duplex and triplex stabilities were measured. While triplex formation is destabilized in the case of pyrrolidino-pseudo-U and -T, pyrrolidino-pseudo-iso-C leads to an increase of the Tm value for third strand dissociation. Duplexes are destabilized with all pyrrolidino-C-nucleosides.

Cytosine↗
PubMedDate not supplied

Fluorinated peptide nucleic acid.

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.

Base Sequence↗
PubMedDate not supplied

Transition metal ligands as novel DNA-base substitutes.

The base modified nucleoside dBP, carrying a non-hydrogen-bonding non-shape complementary base was incorporated into oligonucleotides (Brotschi, C.; Häberli, A.; Leumann C.J. Angew. Chem. Int. Ed. 2001, 40, 3012-3014). This base was designed to coordinate transition metal ions into well defined positions within a DNA double helix. Melting experiments revealed that the stability of a dBP:dBP base couple in a DNA duplex is similar to a dG:dC base pair even in the absence of transition metal ions. In the presence of transition metal ions, melting experiments revealed a decrease in duplex stability which is on a similar order for all metal ions (Mn2+, Cu2+, Zn2+, Ni2+) tested.

Base Pairing↗
PubMedDate not supplied

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.

DNA↗
PubMedDate not supplied

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.

Adenine↗
PubMedDate not supplied

Olefinic peptide nucleic acid (OPA).

The olefinic peptide nucleic acid analogues (OPA) monomers containing the bases thymine and adenine were synthesised in 11 steps. Fully modified oligomers containing these units were prepared and their pairing properties assessed by means of UV-melting experiments.

Adenine↗