Pseudocomplementary PNAs containing$\text{diaminopurine}· \text{thiouracil}$base pairs have been prepared and are shown to bind with high specificity and efficiency to complementary targets in ...double-stranded DNA by a mechanism termed "double duplex invasion" in which the duplex is unwound and both DNA strands are targeted simultaneously, each by one of the two pseudocomplementary peptide nucleic acids (PNAs). On the basis of our results we predict that (for decameric targets) more than 80% of all sequences can be targeted by straightforward Watson-Crick base pairing by using this approach in its present form. Targeting of pseudocomplementary PNAs to the promoter of the T7 phage RNA polymerase effectively inhibits transcription initiation. These results have important implications in the development of gene therapeutic agents as well as for genetic diagnostic and molecular biology applications.
A novel conformationally constrained pyridazinone E
ag-base PNA-monomer
2 capable of binding thymine in a triplex motif was designed and synthesised. A bis-PNA with the E
ag-base incorporated in the ...Hoogsteen strand was hybridised with a complementary DNA. Thermal stability studies revealed an increase in
T
m (4.3
°C per mod.) compared to a no-base unit, but showed no improvement over a previously described unconstrained analogue (E,
1). Surprisingly, no significant difference was found in the thermodynamic parameters (ΔH°, ΔS° and ΔG°) for PNA–DNA triplex formation involving
2 or the unconstrained analogue
1.
A novel conformationally constrained pyridazinone E
ag-base PNA-monomer capable of binding thymine in a triplex motif was designed, synthesised and evaluated in terms of thermal stability and thermodynamic parameters (ΔH°, ΔS° and ΔG°).
Pseudo-complementary oligonucleotide analogues and mimics provide novel opportunities for targeting duplex structures in RNA and DNA. Previously, a pseudo-complementary A-T base pair has been ...introduced. Towards sequence unrestricted targeting, a pseudo-complementary G-C base pair consisting of the unnatural nucleobases N6-methoxy-2,6-diaminopurine (previously described in a DNA context), and N4-benzoylcytosine is now presented for design of pseudo-complementary PNA oligomers (pcPNAs).
The interaction of the uranyl(VI) ion (UO22+) with DNA and its light-induced cleavage of DNA has been studied using flow-linear dichroism and P-32-endlabeled oligonucleotides. It was found that ...binding of uranyl ion to DNA is a prerequisite for photocleavage; from run-off experiments the binding constant was estimated to be of the order of 10(10) M-1 at pH 4. The angular orientation of the O=U=O2+ chromophore is consistent with binding by bridging phosphate groups on opposite strands of the minor groove of DNA; at higher DNA concentration aggregation indicates intermolecular bridging as well. The uranyl-mediated photocleavage of DNA is not influenced by the presence of O2, is more efficient at low pH (
Ceric ammonium nitrate (CAN) in methanol-water gave a new N-dearylation of a series of substituted 1-(p-methoxyphenyl) pyrazoles and a 2-(p-methoxyphenyl)tetrazole producing p-benzoquinone and the ...parent azole in a mole for mole ratio. Application of this reaction to 1-(p-methoxyphenyl) pentazole at -40 degrees C produced p-benzoquinone. 15N NMR spectra suggest that pentazole, HN5, was also produced and held in solution as N5- with Zn2+ ion. The 15N signal from N5- was -10.0 +/- 2.0 ppm in agreement with calculated values.
A series of N-(2-aminoethyl)-α-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the α-amino acid position have been synthesized. These include PNA monomers ...where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T m) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0−6 °C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH2) for easy conjugation to a 18Fradiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.
An improved phosphoramidite method is described to prepare oligonucleotides modified with the acyclic, achiral monomers 1. Examination of dimers, prepared on solid support or in solution, showed that ...phosphortriester dimers containing the allylic unit 1 were unstable towards bases, whereas phosphordiester dimers were stable. Phosphordiester dimers were obtained by replacing cyanoethyl phosphoramidites 2 with phosphoramidites 3, which gave phosphordiesters directly upon oxidation. The phosphordiester dimers were found to be stable towards capping and oxidation, but were somewhat labile towards acids. By reducing the contact time to acids during detritylation it was possible to prepare oligonucleotides containing 4 or 8 modified A, G or T units. The modified oligonucleotides hybridized to complementary DNA and RNA, although with reduced affinity (DeltaT(m) per modification -1 to -5 degrees C).
The synthesis of 10 new T-PNA monomers derived from L-amino acids is presented. The monomers were incorporated into decameric PNA oligomers, and the hybridisation with RNA, DNA and PNA complements ...studied by thermal stability measurements.
The synthesis of 10 new T-PNA monomers derived from L-amino acids is presented. The monomers were incorporated into decameric PNA oligomers, and the hybridisation with RNA, DNA and PNA complements studied by thermal stability measurements.
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