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Michael Petersen

Publications and source records attributed to Michael Petersen.

At least 19 recordsLinked to original sources
PubMed2006

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure.

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.

Base Sequence↗
PubMed2006

Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

5' Untranslated Regions↗
PubMed2006

Prediction of molecular alignment of nucleic acids in aligned media.

We demonstrate--using the data base of all deposited DNA and RNA structures aligned in Pf1-medium and RDC refined--that for nucleic acids in a Pf1-medium the electrostatic alignment tensor can be predicted reliably and accurately via a simple and fast calculation based on the gyration tensor spanned out by the phosphodiester atoms. The rhombicity is well predicted over its full range from 0 to 0.66, while the alignment tensor orientation is predicted correctly for rhombicities up to ca. 0.4, for larger rhombicities it appears to deviate somewhat more than expected based on structural noise and measurement error. This simple analytical approach is based on the Debye-Huckel approximation for the electrostatic interaction potential, valid at distances sufficiently far away from a poly-ionic charged surface, a condition naturally enforced when the charge of alignment medium and solute are of equal sign, as for nucleic acids in a Pf1-phage medium. For the usual salt strengths and nucleic acid sizes, the Debye-Huckel screening length is smaller than the nucleic acid size, but large enough for the collective of Debye-Huckel spheres to encompass the whole molecule. The molecular alignment is then purely electrostatic, but it's functional form is under these conditions similar to that for steric alignment. The proposed analytical expression allows for very fast calculation of the alignment tensor and hence RDCs from the conformation of the nucleic acid molecule. This information provides opportunities for improved structure determination of nucleic acids, including better assessment of dynamics in (multi-domain) nucleic acids and the possibility to incorporate alignment tensor prediction from shape directly into the structure calculation process. The procedures are incorporated into MATLAB scripts, which are available on request.

Bacteriophage Pf1↗
PubMed2006

A cyclic dinucleotide with a four-carbon 5'-C-to-5'-C connection; synthesis by RCM, NMR-examination and incorporation into secondary nucleic acid structures.

A 5'-C-allylthymidine derivative was prepared from thymidine by the application of a stereoselective allylation procedure and its 5'(S)-configuration was confirmed. From this nucleoside derivative, appropriately protected building blocks were prepared and coupled using standard phosphoramidite chemistry to afford a dinucleotide with two 5'-C-allylgroups. This molecule was used as a substrate for a ring-closing metathesis (RCM) reaction and after deprotection, a 1 : 1 mixture of E- and Z-isomers of a cyclic dinucleotide with an unsaturated 5'-C-to-5'-C connection was obtained. Alternatively, a hydrogenation of the double bond and deprotection afforded a saturated cyclic dinucleotide. An advanced NMR-examination confirmed the constitution of this molecule and indicated a restriction in its overall conformational freedom. After variation of the protecting group strategy, a phosphoramidite building block of the saturated cyclic dinucleotide with the 5'-O-position protected as a pixyl ether and the phosphate protected as a methyl phosphotriester was obtained. This building block was used in the preparation of two 14-mer oligonucleotides with a central artificial bend due to the cyclic dinucleotide moiety. These were found to destabilise duplexes, slightly destabilise bulged duplexes but, to some extent, stabilise a three-way junction in high Mg(2+)-concentrations.

Models, Molecular↗
PubMed2006

NMR solution structures of LNA (locked nucleic acid) modified quadruplexes.

We have determined the NMR solution structures of the quadruplexes formed by d(TGLGLT) and d(TL4T), where L denotes LNA (locked nucleic acid) modified G-residues. Both structures are tetrameric, parallel and right-handed and the native global fold of the corresponding DNA quadruplex is retained upon introduction of the LNA nucleotides. However, local structural alterations are observed owing to the locked LNA sugars. In particular, a distinct change in the sugar-phosphate backbone is observed at the G2pL3 and L2pL3 base steps and sequence dependent changes in the twist between tetrads are also seen. Both the LNA modified quadruplexes have raised thermostability as compared to the DNA quadruplex. The quadruplex-forming capability of d(TGLGLT) is of particular interest as it expands the design flexibility for stable parallel LNA quadruplexes and shows that LNA nucleotides can be mixed with DNA or other modified nucleic acids. As such, LNA-based quadruplexes can be decorated by a variety of chemical modifications. Such LNA quadruplex scaffolds might find applications in the developing field of nanobiotechnology.

Base Pairing↗
PubMed2005

An alpha-D-configured bicyclic nucleoside restricted in an E-type conformation: synthesis and parallel RNA recognition.

An alpha-D-arabino configured bicyclic nucleoside strongly restricted in an E-type conformation by a 2'-3'-fused oxetane ring is synthesized. Several synthetic strategies toward the target compound are described, and the successful preparation from a D-xylose derivative is based on a ruthenium-mediated cleavage of a double bond, an S(N)2-inversion at the 2-position to give an arabino-configuration, nucleobase coupling, and finally ring closure to give the oxetane ring. The E-type conformation is confirmed by molecular modeling and NMR. The nucleoside is incorporated into short alpha-DNA sequences. In a mixed pyrimidine context, these recognize complementary parallel RNA-sequences with mainly increased affinity and complementary parallel DNA-sequences with decreased affinity. The present bicyclic analogue represents the first conformationally restricted alpha-DNA-analogue to improve nucleic acid recognition in mixmers with alpha-DNA monomers.

Molecular Structure↗
PubMed2005

Mid-term findings on echocardiography and computed tomography after RVOT-reconstruction: comparison of decellularized (SynerGraft) and conventional allografts.

OBJECTIVE: The immune response against human-leucocyte-antigens on donor-cells may be an important factor contributing to the degeneration of allograft-valves. We have previously reported that the use of the decellularized allograft SynerGraft (CryoLife) reduces the immunologic response of the allograft-recipient. In this study we compare the echocardiographic and computed tomography angiographic (CTA) findings of SynerGrafts with conventional cryopreserved allografts. METHODS: 22 patients who received a pulmonary SynerGraft (SG-group) (21 during a Ross-procedure) underwent CTA and resting echocardiography (median: 10 months postoperatively). 47 randomly chosen patients who underwent a Ross-procedure served as controls (C-group) (median: 32 months postoperatively). RESULTS: Neither the pressure gradients (mean: SG=9+/-4 vs C=10+/-4mmHg; P=0.64) across the allograft, nor the effective orifice area (EOAI) (SG=0.93+/-0.80 vs C=0.93+/-0.42cm(2)/m(2); P=0.96) differed between the groups. The EOAI showed a significant correlation with the smallest allograft-conduit-area measured on CTA (r=0.81; P<0.001) which was most frequently (n=34) found in the proximal postvalvular tubular part of the conduit. Calcifications (n=11) or a fibroproliferative reaction (n=15) were rarely observed. Overall, there were no radiologic differences between the groups. On CTA, the smallest diameter of the allograft-conduits was significantly smaller than the diameter given on the cryopreservation protocol (SG=16+/-3 and C=17+/-3mm vs 25mm in both groups; P<0.001 each) whereas the diameter of the distal part of the allograft was not (SG=24+/-2, P=0.066, and C=25+/-3mm, P=0.82). CONCLUSIONS: Despite a significant shorter follow-up in the SynerGraft-group, no functional or radiologic differences were observed as compared to control-patients. The smallest diameter is located almost exclusively at the proximal level of allograft-conduits.

Adult↗
PubMed2005

QF-PCR examination of parental and meiotic origin of trisomy 21 in Central and Eastern Europe.

Study of parental/meiotic origin of free trisomy 21 in nuclear families from Russia (70 cases), Ukraine (32 cases), and 22 from Germany revealed maternal nondisjunction in 77.3% (Germany), 93.8% (Ukraine), and 91.4% (Russia), paternal origin in 13.6%, 6.2%, and 8.6%, respectively. Maternal meiosis I errors were found in 84.4% (Ukraine), 77.1% (Russia), paternal origin in 3.1% (Ukraine), 2.9% (Russia). Maternal meiosis II errors occurred in 9.4% and 14.3% and paternal in 3.1% and 5.7% in Ukraine and Russia, respectively. No significant differences were found in maternal/paternal origin among Ukraine, Russia, Germany, and published data from other European regions.

Down Syndrome↗
PubMed2004

Solution structure of a dsDNA:LNA triplex.

We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5'-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19 degrees C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1.

Base Sequence↗
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

NMR structure of an alpha-L-LNA:RNA hybrid: structural implications for RNase H recognition.

Alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) is a nucleotide analogue that raises the thermostability of nucleic acid duplexes by up to approximately 4 degrees C per inclusion. We have determined the NMR structure of a nonamer alpha-L-LNA:RNA hybrid with three alpha-L-LNA modifications. The geometry of this hybrid is intermediate between A- and B-type, all nucleobases partake in Watson-Crick base pairing and base stacking, and the global structure is very similar to that of the corresponding unmodified hybrid. The sugar-phosphate backbone is rearranged in the vicinity of the modified nucleotides. As a consequence, the phosphate groups following the modified nucleotides are rotated into the minor groove. It is interesting that the alpha-L-LNA:RNA hybrid, which has an elevation in melting temperature of 17 degrees C relative to the corresponding DNA:RNA hybrid, retains the global structure of this hybrid. To our knowledge, this is the first example of such a substantial increase in melting temperature of a nucleic acid analogue that does not act as an N-type (RNA) mimic. alpha-L-LNA:RNA hybrids are recognised by RNase H with subsequent cleavage of the RNA strand, albeit with slow rates. We attempt to rationalise this impaired enzyme activity from the rearrangement of the sugar-phosphate backbone of the alpha-L-LNA:RNA hybrid.

Base Sequence↗
PubMed2003

NMR solution structure of dsDNA containing a bicyclic D-arabino-configured nucleotide fixed in an O4'-endo sugar conformation.

[3.2.0]bcANA is a D-arabino-configured bicyclic nucleotide with a 2'-O,3'-C-methylene bridge. We here present the high-resolution NMR structure of a [3.2.0]bcANA modified dsDNA nonamer with one modified nucleotide incorporated. NOE restraints were obtained by analysis of NOESY cross peak intensities using a full relaxation matrix approach, and subsequently these restraints were incorporated into a simulated annealing scheme for the structure determination. In addition, the furanose ring puckers of the deoxyribose moieties were determined by analysis of COSY cross peaks. The modified duplex adopts a B-like geometry with Watson-Crick base pairing in all base pairs and all glycosidic angles in the anti range. The stacking arrangement of the nucleobases appears to be unperturbed relative to the normal B-like arrangement. The 2'-O,3'-C-methylene bridge of the modified nucleotide is located at the brim of the major groove where it fits well into the B-type duplex framework. The sugar pucker of the [3.2.0]bcANA nucleotide is O4'-endo and this sugar conformation causes a change in the delta backbone angle relative to the C2'-endo deoxyribose sugar pucker. This change is absorbed locally by slight changes in the epsilon and zeta angles of the modified nucleotide. Overall, the [3.2.0]bcANA modifications fits very well into a B-like duplex framework and only small and local perturbations are observed relative to the unmodified dsDNA of identical base sequence.

Arabinose↗
PubMed2003

LNA: a versatile tool for therapeutics and genomics.

Locked nucleic acid (LNA) is a nucleic acid analogue that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics. For example, Tat-dependent transcription and telomerase activity have been efficiently suppressed by LNA oligomers, and efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes ('LNAzyme'). Furthermore, convincing examples of the application of LNA to nucleic acid diagnostics have been reported, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.

Biomimetic Materials↗
PubMed2002

alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic study.

We have used NMR and CD spectroscopy to study and characterise two alpha-L-LNA:DNA duplexes, a nonamer that incorporates three alpha-L-LNA nucleotides and a decamer that incorporates four alpha-L-LNA nucleotides, in which alpha-L-LNA is alpha-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson-Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H-1H NOE measurements. In general, the deoxyriboses in both of the alpha-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified alpha-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H-1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the alpha-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson-Crick base pairing.

DNA↗