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Jesper Wengel

Publications and source records attributed to Jesper Wengel.

At least 37 records · Page 2Linked to original sources
PubMed2005

Locked nucleic acid synthesis.

Methods and protocols for automated synthesis and purification of locked nucleic acid (LNA), a class of oligonucleotides obeying the Watson-Crick base-pairing rules but displaying unprecedented binding affinities toward complementary deoxyribonucleic acid (cDNA) and ribonucleic acid (RNA), is described. LNA and LNA-DNA chimeras containing phosphordiester or phosphorothioate linkages, or a mixture thereof, can be assembled by standard DNA synthesizers using 2-cyanoethyl DNA phosphoramidites and 2-cyanoethyl LNA phosphoramidites. Compared to the standard protocols used for DNA synthesis, slightly longer coupling time and oxidation time are needed for efficient oligomerization of LNA phosphoramidites. When the LNA has been assembled, it is removed from the solid support as the 5'-end-O-dimethoxytrityl (DMT) protected LNA oligomer by treatment with concentrated aqueous ammonia that also removes the phosphate and nucleobase protecting groups. The crude DMT protected LNA product can be purified using, for example, reversed-phase chromatography.

Chromatography, High Pressure Liquid↗
PubMed2004

Locked nucleic acids and intercalating nucleic acids in the design of easily denaturing nucleic acids: thermal stability studies.

Intercalating nucleic acids (INA(R)s) with insertions of (R)-1-O-(1-pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make "quenched" complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13-mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 degrees C) in comparison with the unmodified double-stranded DNA (Tm=48.7 degrees C) and the corresponding duplexes of LNA/DNA (Tm=81.6 degrees C) and INA/DNA (Tm=66.4 degrees C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.

DNA↗
PubMed2004

Synthesis and thermal denaturation studies of novel 2'-O,3'-C-linked bicyclic oligonucleotides with a methoxy or a piperazino group facing the major groove of nucleic acid duplexes.

With the aim of evaluating duplex stabilities of oligonucleotides (ONs) with major groove facing functionalities, two novel 2'-O,3'-C-linked bicyclic nucleoside phosphoramidite building blocks were synthesized by routes involving regioselective O-methylation or piperazine attachment using carbonyldiimidazole coupling chemistry. The novel monomers were incorporated into 9-mer mixed base ONs and the thermal stability toward complementary single stranded DNA and RNA was evaluated by thermal denaturation experiments. O-methylated ONs confirmed the applicability of the functionalized bicylic sugar unit for attachment of groups facing the major groove and satisfactory binding properties towards the RNA complement were observed. For the piperazino modified ONs, experiments were performed in aqueous buffers with low (40 mM) and medium (110 mM) salt concentrations, at pH 5 and pH 7. A change from a medium to a low salt concentration induced a significant relative increase in the thermal stability of modified duplexes toward both DNA and RNA complements, which suggests protonation of the piperazino group under the experimental conditions applied.

Molecular Structure↗
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

3'-C-Branched LNA-type nucleosides locked in an N-type furanose ring conformation: synthesis, incorporation into oligodeoxynucleotides, and hybridization studies.

Three protected 3'-C-branched LNA-type phosphoramidite building blocks 17, 27, and 38, containing furanose rings locked in an N-type conformation, were synthesized from a known 3-C-allyl allofuranose derivative using strategies relying on the introduction of the branching alkyl chain before condensation with the nucleobase. Synthesis of 3'-C-hydroxypropyl derivatives proved superior to synthesis of the 3'-C-hydroxyethyl derivatives, and the former was converted into the corresponding 3'-C-aminopropyl derivatives. Phosphoramidites 27 and 38 were subsequently applied on an automated DNA synthesizer leading to the introduction of three novel 3'-C-branched LNA-type monomers X, Y, and Z into oligodeoxynucleotides and studies of their effect on the hybridization properties. A duplex-stabilizing effect of introducing 3'-C-aminopropyl-LNA monomer Y, relative to 3'-C-hydroxypropyl-LNA monomer X, was observed, especially at low salt conditions. This indicates that the primary amino group of monomer Y is protonated under the hybridization conditions applied and that positioning of this positively charged group in the major groove has a significant duplex stabilizing effect. Monomer Y was by an on-column conjugation method further functionalized by a glycyl unit to give monomer Z that showed a less stabilizing effect than monomer Y.

Furans↗
PubMed2004

Conformationally controlled high-affinity targeting of RNA or DNA by novel 2'-amino-DNA/LNA mixmers and pyrenyl-functionalized 2'-amino-DNA.

9-Mer DNA sequences containing 2'-N-methyl-2'-N-(pyren-1-ylmethyl)-2'-amino-DNA monomers display significantly increased affinity towards DNA complements whereas the corresponding 2'-amino-DNA monomer has a detrimental effect on duplex stability. These effects are efficiently reversed by incorporation of four LNA nucleotides inducing a B-DNA to A-DNA conformational change.

DNA↗
PubMed2004

Sequence and pH effects of LNA-containing triple helix-forming oligonucleotides: physical chemistry, biochemistry, and modeling studies.

Triple helix-forming oligonucleotides (TFOs) have been demonstrated to be capable of interfering with gene expression and modifying genomic DNA in a sequence-specific manner. Partial incorporation of 2'-O,4'-C-methylene linked locked nucleic acid (LNA) residues in TFOs has been shown to enhance significantly triple helix formation, whereas the full-length LNA TFO failed to form a stable triplex. This work is aimed at understanding the triple helix-forming properties of LNA-containing TFOs and at optimally designing their sequences. Both DNA thermal melting, gel retardation, and restriction enzyme experiments as well as modeling studies by molecular mechanics were carried out to investigate the base composition/sequence and pH-dependence effects of LNA-containing TFOs, as well as their structural features underlying triple helix formation. Alternating LNA substitution every 2-3 nucleotides in TFOs is mandatory, whereas the use of thymine LNA residues should be favored under neutral pH conditions. A rule for designing optimal LNA-containing TFOs is proposed. In addition, alternative LNA and 2'-O-methyl residues in TFOs do not significantly improve triple helix formation.

Base Sequence↗
PubMed2004

DNA-selective hybridization and dual strand invasion of short double-stranded DNA using pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA.

Incorporation of a novel pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA monomer into oligodeoxynucleotides leads to increased thermal stability of duplexes with DNA complements but reduced thermal stability of duplexes with RNA complements. This DNA-selective hybridization is explored for recognition of double-stranded DNA by a novel dual strand invasion approach.

Base Sequence↗
PubMed2004

Transcription factor decoy oligonucleotides modified with locked nucleic acids: an in vitro study to reconcile biostability with binding affinity.

Double-stranded oligonucleotides (ODNs) containing the consensus binding sequence of a transcription factor provide a rationally designed tool to manipulate gene expression at the transcriptional level by the decoy approach. However, modifications introduced into oligonucleotides to increase stability quite often do not guarantee that transcription factor affinity and/or specificity of recognition are retained. We have previously evaluated the use of locked nucleic acids (LNA) in the design of decoy molecules for the transcription factor kappaB. Oligo nucleotides containing LNA substitutions displayed high resistance to exo- and endonucleolytic degradation, with LNA-DNA mix-mers being more stable than LNA-DNA-LNA gap-mers. However, insertion of internal LNA bases resulted in a loss of affinity for the transcription factor. This latter effect apparently depended on positioning of the internal LNA substitutions. Indeed, here we demonstrate that intra- and inter-strand positioning of internal LNAs has to be carefully considered to maintain affinity and achieve high stability, respectively. Unfortunately, our data also indicate that LNA positioning is not the only parameter affecting transcription factor binding, the interference in part being dependent on the intrinsic conformational properties of this nucleotide analog. To circumvent this problem, the successful use of an alpha-L-ribo- configured LNA is demonstrated, indicating LNA-DNA-alpha-L-LNA molecules as promising new decoy agents.

Base Sequence↗
PubMed2004

Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology.

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.

Animals↗
PubMed2004

LNA-antisense rivals siRNA for gene silencing.

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications.

Animals↗
PubMed2003

Nucleic acid nanotechnology-towards Angstrom-scale engineering.

Nucleic acids and analogues are suitable building blocks for reliable self-assembly of nanometer-sized two- or three-dimensional materials. In order to mimic or approach nature with respect to size and function, Angstrom-scale chemical engineering is emerging as pivotal for future developments. Efforts within nucleic acid nanotechnology will be focussed on generating rigid and stable low nanometer-sized structures carrying functionalities with predictable spatial positioning allowing, by encoded self-assembly, functional nucleic acid architectures to be built towards applications within the biological and material sciences.

Chelating Agents↗
PubMed2003

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world.

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.

Base Sequence↗
PubMed2003

Oligodeoxynucleotides containing alpha-L-ribo configured LNA-type C-aryl nucleotides.

Synthesis of 2[prime or minute]-O,4[prime or minute]-C-methylene-[small alpha]-l-ribofuranosyl derivatives containing phenyl and 1-pyrenyl aglycons, i.e., novel [small alpha]-l-ribo configured LNA-type C-aryl nucleosides, has been accomplished. Key synthetic steps included stereoselective Grignard reactions on tetrahydrofuran aldehyde, configurational inversion of the resulting alcohol into alcohol, and concomitant Mitsonobu cyclization furnishing the desired bicyclic furanosyl skeleton with a locked conformation. The phosphoramidite derivatives and were used for automated synthesis of 9-mer DNA and [small alpha]-L-LNA oligonucleotides containing the [small alpha]-L-LNA-type C-aryl monomers ([small alpha]L)Ph(L) and ([small alpha]L)Py(L) containing a phenyl and pyrenyl aglycon, respectively. Thermal denaturation studies showed universal base pairing behavior for the pyrenyl monomer ([small alpha]L)Py(L) when incorporated into a DNA or an [small alpha]-L-LNA oligonucleotide.

Deoxyribonucleosides↗
PubMed2003

2'-Spiro ribo- and arabinonucleosides: synthesis, molecular modelling and incorporation into oligodeoxynucleotides.

We have synthesized four conformationally restricted bicyclic 2'-spiro nucleosides via 2'-C-allyl nucleosides as key intermediates. The ribo-configured 2'-spironucleosides 9b and 14b were obtained by a convergent strategy starting from 2-ketofuranose 1 whereas the arabino-configured 2'-spironucleosides 21 and 27 were obtained by a linear strategy with a 2'-ketouridine derivative as starting material. The furanose ring of 9b/14b adopts N-type conformations whereas the furanose ring of 21/27 exists as an N<==>S equilibrium. These compounds showed no anti-HIV-1 activity or cytotoxicity. Incorporation of the four 2'-spironucleosides (as monomers X4 and X5) into oligodeoxynucleotides was accomplished using the phosphoramidite approach on an automated DNA synthesizer. Irrespective of monomeric configuration, hybridization studies revealed that these 2'-spironucleotide monomers (X4 and X5) induce decreased duplex thermostabilities compared with the corresponding DNA:DNA and DNA:RNA duplexes. Molecular modelling indicated that steric constraints are a possible reason for the lowered binding affinities of the modified oligodeoxynucleotides towards complementary single-stranded DNA and single-stranded RNA complements.

Anti-HIV Agents↗
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↗