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Biomedical subjects

P E Nielsen

Publications and source records attributed to P E Nielsen.

At least 19 recordsLinked to original sources
PubMed2004

The influence of a chiral amino acid on the helical handedness of PNA in solution and in crystals.

The X-ray structure of a self-complementary PNA hexamer (H-CGTACG-L-Lys-NH(2)) has been determined to 2.35 A resolution. The introduction of an L-lysine moiety has previously been shown to induce a preferred left-handedness of the PNA double helices in aqueous solution. However, in the crystal structure an equal amount of interchanging right- and left-handed helices is observed. The lysine moieties are pointing into large solvent channels and no significant interactions between this moiety and the remaining PNA molecule are observed. In contrast, molecular mechanics calculations show a preference for the left-handed helix of this hexameric PNA in aqueous solution as expected. The calculations indicate that the difference in the free energy of solvation between the left-handed and the right-handed helix is the determining factor for the preference of the left-handed helix in aqueous solution.

Amino Acids↗
PubMed2001

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.

Circular Dichroism↗
PubMed2001

Inhibition of promyelocytic leukemia (PML)/retinoic acid receptor-alpha and PML expression in acute promyelocytic leukemia cells by anti-PML peptide nucleic acid.

The fusion protein promyelocytic leukemia (PML)/retinoic acid receptor (RAR)alpha is tightly linked to the pathogenesis of acute promyelocytic leukemia (APL); hence, it represents a tumor-associated, transformation-related molecule. In this study, three anti-PML adamantyl-conjugated peptide nucleic acid (PNA) oligomers previously described as in vitro inhibitors of PML/RARalpha translation were combined and used to block PML/RARalpha synthesis in NB4 cells. Cationic liposomes were used to achieve sufficient delivery of PNAs into the cells. Upon treatment of cells with the liposome/PNA mixture, enhanced cellular uptake of PNA (approximately 5-fold compared with control) was obtained. Concomitantly, a substantial reduction (>90%) of the expression of PML/RARalpha was observed when all of the three PNAs were used together. This resulted in a dramatic effect on the number and viability of NB4 cells in culture after 48 h of treatment. This phenomenon was preceded by induction of apoptosis that could be observed 24 h after treatment. No sign of granulocytic differentiation was observed after treatment. These effects were also noted on other leukemic cell lines that express PML but not the fusion transcript. These results show that it is possible to deliver PNA into hematopoietic cells and obtain specific gene inhibition, and they suggest that a growth inhibitory effect on acute promyelocytic leukemia cells can be obtained through the block of PML/RARalpha and PML expression.

Antineoplastic Agents↗
PubMed2001

Structural isomers of bis-PNA bound to a target in duplex DNA.

Upon binding of a decamer bis-PNA (H-Lys-TTCCTCTCTT-(eg1)(3)-TTCTCTCCTT-LysNH(2)) to a complementary target in a double-stranded DNA fragment, three distinct complexes were detected by gel mobility shift analysis. Using in situ chemical probing techniques (KMnO(4) and DMS) it was found that all three complexes represent bona fide sequence-specific PNA binding to the designated target, but the complexes were structurally different. One complex that preferentially formed at higher PNA concentrations contains two bis-PNA molecules per DNA target, whereas the other two complexes are genuine triplex invasion clamped structures. However, these two latter complexes differ by the path relative to the DNA target of the flexible ethylene-glycol linker connecting the two PNA oligomers that comprise a bis-PNA. We distinguish between one in which the linker wraps around the non-target DNA strand, thus making this strand part of the triplex invasion complex and another complex that encompass the target strand only. The implications of these results are discussed in terms of DNA targeting by synthetic ligands.

DNA↗
PubMed2001

Synthesis of pyrrolidinone PNA: a novel conformationally restricted PNA analogue.

To preorganize PNA for duplex formation, a new cyclic pyrrolidinone PNA analogue has been designed. In this analogue the aminoethylglycine backbone and the methylenecarbonyl linker are connected, introducing two chiral centers compared to PNA. The four stereoisomers of the adenine analogue were synthesized, and the hybridization properties of PNA decamers containing one analogue were measured against complementary DNA, RNA, and PNA strands. The (3S,5R) isomer was shown to have the highest affinity toward RNA, and to recognize RNA and PNA better than DNA. The (3S,5R) isomer was used to prepare a fully modified decamer which bound to rU10 with only a small decrease in Tm (delta Tm/mod = 1 degree C) relative to aminoethylglycine PNA.

Magnetic Resonance Spectroscopy↗
PubMed2001

Comparison of electron capture dissociation and collisionally activated dissociation of polycations of peptide nucleic acids.

Electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry coupled with electrospray ionization enhances the sequence elucidation of peptide nucleic acids compared with conventional low-energy collisionally activated dissociation (CAD). Examples are shown where ECD produced complete or extensive sequence coverage in PNAs six to ten nucleobases long. However, facile base losses from the reduced species and low abundances of backbone ECD fragments presented a significant problem. This was rationalized through the lower degree of charge solvation on the backbone compared to polypeptides. Combination of both CAD and ECD data is advantageous, as these techniques produce cleavages at different sites.

Cations↗
PubMed2001

Investigating the kinetics of DNA-DNA and PNA-DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy.

Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin-streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.

Base Sequence↗
PubMed2001

Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology.

During the past ten years, the DNA mimic peptide nucleic acid has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and characterisation of nucleic acids. Most of these methods have taken advantage of the very favourable DNA and RNA hybridisation properties of peptide nucleic acids combined with the unique properties and opportunities offered by peptide chemistry. Within the past year, significant progress in in situ hybridisation technology has been achieved, which has resulted, in particular, in reliable and sensitive methods for detection of bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction clamping method have been expanded, and novel ways of exploiting complexes of peptide nucleic acids with double-stranded DNA, such as double duplex invasion complexes and PD loops, have been developed.

Bacterial Infections↗
PubMed2001

Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid).

Mixed pyrimidine-purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA(2)-DNA triplex with Watson-Crick complementary adenine-cytosine oligonucleotides and to bind complementary adenine-cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine-purine target in double stranded DNA and illustrate a novel binding mode of PNA.

Base Composition↗
PubMed2001

Bactericidal antisense effects of peptide-PNA conjugates.

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide-PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 microM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide-PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.

Amino Acid Sequence↗
PubMed2001

Perinatal outcome following fetal single umbilical artery diagnosis.

OBJECTIVE: We report the frequency of associated congenital abnormalities in fetuses with a single umbilical artery as well as the sensitivity, specificity, positive predictive value and negative predictive value of ultrasound for detecting these abnormalities. We also report the pregnancy outcome of fetuses complicated by single umbilical artery, both isolated and with other congenital anomalies. METHODS: All pregnancies complicated by fetal single umbilical artery from 1995 to 1999 were identified. A retrospective chart review was performed on both the prenatal records and the ultrasound records of these pregnancies, determining the nature and incidence of other congenital abnormalities. Delivery data were collected to include gestational age at delivery, Apgar score, birth weight, mode of delivery, fetal gender and any complications. RESULTS: Ninety-two pregnancies were identified with a fetal single umbilical artery, of which outcome data were available for 65. Forty-eight (74%) cases were identified as isolated single umbilical artery. Seventeen (26%) cases had other congenital abnormalities. High-resolution ultrasound had 100% sensitivity and specificity for identifying single umbilical artery and an 85% sensitivity and 98% specificity for detecting other congenital abnormalities. Compared to isolated single umbilical artery, pregnancies complicated by single umbilical artery with other abnormalities had a statistically significantly increased rate of fetal aneuploidy, lower birth weight, preterm delivery and Cesarean delivery. CONCLUSION: Pregnancies complicated by fetal single umbilical artery, especially when associated with other congenital abnormalities, are at increased risk for adverse pregnancy outcome.

Adult↗
PubMed2001

Peptide nucleic acids as antibacterial agents via the antisense principle.

Peptide nucleic acid (PNA) is a peptide-like DNA mimic that was introduced almost ten years ago. It was immediately predicted that PNA would have a bright future in gene therapeutic drug development, but progress in this direction has been rather modest thus far. This is predominantly due to inefficient uptake of PNA by most living cells. However, within the past couple of years a variety of methods have been devised to address this problem and the stage should now be set for more rapid progress. Several studies have demonstrated antisense effects ex vivo in cells in culture and two reports on direct injection of PNA into the brain of rats are also interesting. Only a few studies have addressed the possible exploitation of the antisense principle for development of antibacterial drugs. However, the first in vitro results using antiribosomal RNA PNAs and antisense PNAs targeted to the beta-lactamase gene on Escherichia coli cultures were quite promising. Most recently, these preliminary studies have been extended to demonstrate in vivo efficacy of antibacterial PNAs in an E. coli peritonitis/sepsis mouse model. Therefore, PNA drug development again is rapidly picking up pace.

Amino Acid Sequence↗