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Birte Vester

Publications and source records attributed to Birte Vester.

At least 19 recordsLinked to original sources
PubMed2006

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets.

BACKGROUND: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. RESULTS: We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and alpha-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two alpha-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1). CONCLUSION: The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

Base Sequence↗
PubMed2006

The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics.

A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.

Anti-Bacterial Agents↗
PubMed2006

Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center.

Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the peptidyl transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the peptidyl transferase cavity.

Anti-Bacterial Agents↗
PubMed2005

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start.

The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0 degrees C) than the wild-type double-stranded DNA (dsDNA, 26.0 degrees C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0 degrees C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2'-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0 degrees C) than duplexes with the complementary ssDNAs (31.5 and 19.5 degrees C). The excimer band observed in the ds2'-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2'-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2'-OMe-RNA.

DNA↗
PubMed2005

The archaeon Haloarcula marismortui has few modifications in the central parts of its 23S ribosomal RNA.

Post-transcriptional modifications were mapped in domains II, IV and V of 23S RNA from the archaeon Haloarcula marismortui. The RNA was investigated by two primer extension techniques using reverse transcriptase and three mass spectrometry techniques. One primer extension technique utilized decreasing concentrations of deoxynucleotide triphosphates to detect 2'-O-ribose methylations and other polymerase blocking modifications. In the other, the rRNA was chemically modified, followed by mild alkaline hydrolysis to map pseudo-uridine groups (Psis). RNA fragments for mass spectrometry were isolated from 23S rRNA by site-directed RNase H or mung bean nuclease digestion followed by gel purification. Modified RNase digestion fragments were identified with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the modifications were further studied by tandem MS. Psis suggested by the primer extension technique were verified by specific cyanoethylation and mass spectrometric detection. A total of only five post-transcriptionally methylated nucleotides and three Psis were detected in the three 23S rRNA domains. One of the methylated nucleotides has not been reported while a dispute about the number of Psis is solved. The limited number of modified nucleotides suggests that H. marismortui does not have special needs for extensive rRNA modifications. We have performed detailed investigations on the three-dimensional location and molecular interactions of the modified nucleotides by computer analysis. Our results show that all the modified positions are at regions with RNA-RNA contacts and all except one are at the surface of the subunit and in functionally important regions.

Haloarcula marismortui↗
PubMed2005

Design, synthesis and ribosome binding of chloramphenicol nucleotide and intercalator conjugates.

Molecular modelling based on X-ray structures of the antibiotic drug chloramphenicol bound in a bacterial ribosome has been used for design of chloramphenicol derivatives. Conjugates of the chloramphenicol amine through appropriate linkers to either a pyrene moiety or to a mono- or dinucleotide moiety were designed to improve binding to ribosomes by providing specific interactions in the peptidyl transferase site or to the P-loop in the ribosome. Specific binding of the conjugates were investigated by footprinting analysis using chemical modifications of accessible nucleotides in ribosomal RNA. The pyrene chloramphenicol conjugate shows enhanced binding to the chloramphenicol binding site compared to the native chloramphenicol, whereas the four nucleotide conjugates could not be shown to bind to the chloramphenicol binding site or to the P-loop.

Binding Sites↗
PubMed2005

A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503.

The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid-encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI-TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.

Anti-Bacterial Agents↗
PubMed2004

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA.

Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.

DNA, Complementary↗
PubMed2004

Hexofuranosyl thymidines inserted into oligodeoxynucleotides via their two exocyclic hydroxy groups. Oligo synthesis and RNase H activity.

Hexofuranosyl nucleosides are considered as conformationally restricted acyclic nucleosides using a furanose ring to link the diol backbone to the nucleobase. The phosphoramidite of 1-(2,3-dideoxy-beta-D-erythro-hexofuranosyl)thymine was synthesized from thymidine with formation of a new stereocentre at C-5' and the nucleoside was used in oligodeoxynucleotide (ODN) synthesis. Binding of mixed sequence ODNs towards complementary DNA and RNA showed decreased affinity compared to the wild-type oligos. Insertion in the middle of poly alphaT sequence led to stabilization of ODN/dA(14) duplexes at low ionic strength, but a decrease was observed in medium and high salt buffers compared to d(alphaT)(14)/dA(14). Both beta and alpha hexofuranosyl thymidines allowed cleavage of complementary mixed-sequence RNA by RNase H to the 3'-site of the modification in ODNs whereas a limited inhibition was detected from the 5'-site.

Base Sequence↗
PubMed2004

Mutations in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated with reduced susceptibility to tiamulin in Brachyspira spp. isolates.

The pleuromutilin antibiotic tiamulin binds to the ribosomal peptidyl transferase centre. Three groups of Brachyspira spp. isolates with reduced tiamulin susceptibility were analysed to define resistance mechanisms to the drug. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA (Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 (Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.

Amino Acid Substitution↗
PubMed2003

Xylo-Configured oligonucleotides (XNA, xylo nucleic acid): synthesis of conformationally restricted derivatives and hybridization towards DNA and RNA complements.

Xylo-Configured oligonucleotides (XNA) containing a novel conformationally restricted 2'-deoxy-2'-fluoro-beta-D-xylofuranosyl nucleotide monomer, a novel conformationally locked 2'-amino-2'-deoxy-2'-N,4'-C-methylene-beta-D-xylofuranosyl nucleotide monomer, and a known 2'-deoxy-beta-D-xylofuranosyl nucleotide monomer (XNA monomers) have been synthesized and their hybridization towards DNA and RNA complements studied. Thermal denaturation studies of nine-mer mixed-base sequences composed of a mixture of XNA monomers and DNA monomers revealed preferential hybridization towards RNA complements relative to DNA complements. For 14-mer homo-thymine XNAs containing thirteen XNA monomers, stable complexes towards single-stranded DNA and RNA were formed at pH 7. Gel-shift experiments revealed these complexes to involve at least two XNA strands per DNA or RNA target strand.

DNA↗
PubMed2003

Methylphosphonate LNA: a locked nucleic acid with a methylphosphonate linkage.

Synthesis of an oligonucleotide containing one methylphosphonate locked nucleic acid (LNA) thymine monomer using the phosphoramidite approach is described. The binding affinity of this 9-mer methylphosphonate LNA towards complementary DNA and RNA oligonucleotides was increased compared to the reference DNA, but decreased compared to the reference LNA. In the 9-mer sequence context studied, introduction of a single methylphosphonate LNA monomer, contrary to a single LNA monomer, efficiently inhibits 3'-exonucleolytic degradation.

DNA↗
PubMed2003

The avilamycin resistance determinants AviRa and AviRb methylate 23S rRNA at the guanosine 2535 base and the uridine 2479 ribose.

Avilamycin is an orthosomycin antibiotic that has shown considerable potential for clinical use, although it is presently used as a growth promoter in animal feed. Avilamycin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit. The ribosomes of the producer strain, Streptomyces viridochromogenes Tü57, are protected from the drug by the action of three resistance factors located in the avilamycin biosynthetic gene cluster. Two of the resistance factors, aviRa and aviRb, encode rRNA methyltransferases that specifically target 23S rRNA. Recombinant AviRa and AviRb proteins retain their activity after purification, and both specifically methylate in vitro transcripts of 23S rRNA domain V. Reverse transcriptase primer extension indicated that AviRa is an N-methyltransferase that targets G2535 within helix 91 of the rRNA, whereas AviRb modified the 2'-O-ribose position of nucleotide U2479 within helix 89. MALDI mass spectrometry confirmed the exact positions of each of these modifications, and additionally established that a single methyl group is added at each nucleotide. Neither of these two nucleotides have previously been described as a target for enzymatic methylation. Molecular models of the 50S subunit crystal structure show that the N-1 of the G2535 base and the 2'-hydroxyl of U2479 are separated by approximately 10 A, a distance that can be spanned by avilamycin. In addition to defining new resistance mechanisms, these data refine our understanding of the probable ribosome contacts made by orthosomycins and of how these antibiotics inhibit protein synthesis.

Animals↗
PubMed2003

Resistance to the peptidyl transferase inhibitor tiamulin caused by mutation of ribosomal protein l3.

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

Anti-Bacterial Agents↗
PubMed2002

LNAzymes: incorporation of LNA-type monomers into DNAzymes markedly increases RNA cleavage.

Incorporation of two alpha-L-LNA/LNA nucleotides into each of the two binding arms of a "10-23" DNAzyme has been accomplished and the RNA cleavage with these novel LNAzymes studied. In comparison with the unmodified DNAzyme, the LNAzymes show significantly improved cleavage of the phosphodiester backbone at the target nucleotide in a small RNA substrate (58n RNA) under single-turnover conditions. The LNAzymes show efficient multiple turnover. With the LNAzymes, efficient cleavage was accomplished also of a naturally occurring ribosomal RNA at a target site within a highly structured region. The reference DNAzyme was ineffective at cleaving the ribosomal RNA target.

DNA, Catalytic↗
PubMed2002

Interaction of avilamycin with ribosomes and resistance caused by mutations in 23S rRNA.

The antibiotic growth promoter avilamycin inhibits protein synthesis by binding to bacterial ribosomes. Here the binding site is further characterized on Escherichia coli ribosomes. The drug interacts with domain V of 23S rRNA, giving a chemical footprint at nucleotides A2482 and A2534. Selection of avilamycin-resistant Halobacterium halobium cells revealed mutations in helix 89 of 23S rRNA. Furthermore, mutations in helices 89 and 91, which have previously been shown to confer resistance to evernimicin, give cross-resistance to avilamycin. These data place the binding site of avilamycin on 23S rRNA close to the elbow of A-site tRNA. It is inferred that avilamycin interacts with the ribosomes at the ribosomal A-site interfering with initiation factor IF2 and tRNA binding in a manner similar to evernimicin.

Anti-Bacterial Agents↗
PubMedDate not supplied

LNA and alpha-L-LNA: towards therapeutic applications.

LNA and alpha-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we report dose-dependent inhibition of HIV-1 Tat-dependent trans activation by a 12-mer chimeric alpha-L-LNA/DNA oligomer. This oligomer exhibits a dose-dependency similar to that of the corresponding 12-mer chimeric LNA/2'-O-Me-RNA oligomer. In addition, we show that incorporation of alpha-L-LNA or LNA monomers into each of the two binding arms of a "10-23" DNAzyme markedly increases cleavage of the target RNA.

Base Sequence↗