Due to their restricted conformational flexibility, cyclic peptides are of great interest in connection with structure-activity relationships, especially the elucidation of bioactive conformations. For linear peptides that do not contain turn structure-inducing amino acid residues, the cyclization reaction may be an inherently improbable or slow process, and side reactions, such as cyclodimerization and epimerization at the C-terminal residue, may dominate. A number of 1-hydroxy-7-azabenzotriazole-based onium salts were examined for cyclization of thymopentin-derived pentapeptides and the results compared with data from more conventional coupling reagents. The azabenzotriazol-derived coupling reagents stood out as being the most effective by far. The cyclizations proceed extremely rapidly, and in contrast to other coupling reagents, C-terminal epimerization was generally less than 10%. C-terminal D-amino acid residues favor the formation of monocyclic pentapeptide rings. A similar effect was observed for cyclization of linear N-methylamino acid-containing peptides, suggesting that reversible amide bond alkylation such as Hmb-modification should be useful in promoting the cyclization of pepitdes devoid of turn-inducing amino acid residues.

In order to evaluate the ability of the cell-penetrating a-helical amphipathic model peptide KLALKLALKALK AALKLA-NH 2 (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5¢-GGAGCAGGAAAG-3¢ directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAPconjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three-and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10-and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.Keywords: cell-penetrating peptides; cellular uptake; PNApeptide conjugates.The wider application of peptide nucleic acids (PNAs) [1] as antisense agents appears to be limited mainly by poor cellular uptake [2,3]. Improved delivery into mammalian cells and enhanced antisense activity have been achieved after covalent coupling of PNAs to cell-penetrating peptides (CPPs), which are able to enter cells in a nonendocytic but as yet unknown mode [3][4][5][6][7][8]. The structural requirements for the delivery activity of peptides have been unclear until now. In order to contribute to an elucidation of structuredelivery activity relationships we have previously investigated the cellular uptake and biological activity of CPP-phosphorothioate oligonucleotide conjugates using the cell-penetrating amphipathic model peptide MAP (KLALKLALKALKAALKLA-NH 2 ) [9,10] as the lead compound [11]. The value of the results of this study was limited, however, by a high cell toxicity of the phosphorothioate oligonucleotide-peptide conjugates. Therefore, in the present study we evaluated the suitability of PNA to serve as the cargo molecule in MAP-based structuredelivery activity investigations. To this end we investigated cellular uptake and biological activity of a 12-mer peptide nucleic acid (5¢-GGAGCAGGAAAG-Lys-3¢; compound I; Table 1) complementary to bases 12-23 of the translated region of the nociceptin/orphanin FQ receptor, proven previously to be sensitive to antisense attacks [12,13], and of its conjugate with MAP (compound II; Table 1). For assessing the cellular uptake, we developed a protocol based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) providing absolute quantities of internalized PNA which was used supplementally with confocal laser scanning microscop...

Nucleic Acids Res. 21:5332-5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs (rDNAs) which are not frequently found in clone libraries generated by standard PCR from complex microbial consortia. Three different PNA molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) overlapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved either by competitive binding between the PNA molecules and the forward or reverse primers (competitive clamping) or by hindering polymerase readthrough (elongation arrest). Gene libraries generated from mixed rDNA templates by using PCR clamping are enriched for clones that do not contain sequences homologous to the appropriate PNA oligomer. This effect of PCR clamping was exploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping with PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different sequences present in an artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences which represented recently described phylogenetic groups (NKB19, TM6, cluster related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might be a useful supplement to standard PCR amplification in rDNA-based studies of microbial diversity and could be used to selectively recover members of undescribed phylogenetic clusters from complex microbial communities.

No abstract

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.