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The interactions of the delta and lambda enantiomers of the chiral metal complex [Ru(phen)3]2+ (phen = 1,10-phenanthroline) with the oligonucleotide duplex [d(CGCGATCGCG)]2 have been studied with NMR and CD spectroscopy. From NOESY data it is shown that the interaction primarily takes place in the minor groove of the oligonucleotide which remains in a B-like conformation. The observed NOEs also provide evidence that the metal complexes preferentially bind to the central AT region. The observed AT specificity is more pronounced with the delta as compared to the lambda enantiomer, which interacts with a larger part of the oligonucleotide. Furthermore, the NOESY data show that neither of the enantiomers binds by classical intercalation. This is also supported by a comparison study of the analogue [Ru(phen)2DPPZ]2+ (DPPZ = dipyrido[3,2-a:2',3'c]phenazine) which intercalates in DNA. The NMR as well as the CD results show that the delta and lambea enantiomers of [Ru(phen)3]2+ bind in different modes to [d(CGCGATCGCG)]2. Comparison of CD spectra of the metal complex in the presence of [d(CGCGATCGCG)]2, poly(dAdT).poly-(dAdT), poly(dGdC).poly(dGdC), and calf thymus DNA suggests that these binding modes are independent of DNA sequence. The results are found to be compatible with binding of delta-[Ru(phen)3]2+ by insertion of two phenanthroline ligands into the minor groove, causing minor distortions of the DNA structure, whereas the lambda enantiomer binds in a mode that leaves the DNA structure unaffected.

Peptide nucleic acid (PNA) is a DNA mimic with promising properties for the development of antisense agents. Antisense PNAs targeted to Escherichia coli genes can specifically inhibit gene expression, and attachment of PNA to the cell-permeabilizing peptide KFFKFFKFFK dramatically improves antisense potency. The improved potency observed earlier was suggested to be due to better cell uptake; however, the uptake kinetics of standard or modified PNAs into bacteria had not been investigated. Here we monitored outer and inner membrane permeabilization by using chemical probes that normally are excluded from cells but can gain access at points where membrane integrity is disturbed. Membrane permeabilization was much more rapid in the presence of peptide-PNA conjugates relative to the free components used alone or in combination. Indeed, peptide-PNAs permeabilized E. coli nearly as quickly as antimicrobial peptides. Furthermore, as expected for outer membrane-active compounds, added MgCl 2 reduced cell-permeabilization. Concurrent monitoring of outer and inner membrane permeabilization indicated that passage across the outer membrane is rate-limiting for uptake. The enhanced cell-permeation properties of peptide-PNAs can explain their potent antisense activity, and the results indicate an unanticipated synergy between the peptide and PNA components.Antisense agents are designed to inhibit gene expression through sequence-specific nucleic acid binding, and this normally requires the formation of 10 or more base pairs. Oligonucleotides of such lengths are typically too large for efficient passive cellular uptake by diffusion across lipid bilayers (1), and cellular outer membranes can pose additional barriers. Recently we introduced antisense peptide nucleic acids (PNAs) 1 as antisense agents for bacterial applications. The early experiments indicated very encouraging sequence specificity against reporter genes; however, uptake appeared to be limited by bacterial cell barriers (2, 3). This limitation is not surprising as Escherichia coli and other Gram-negative bacteria have outer and inner bilayer membranes. In addition, the outer membrane contains a lipopolysaccharide (LPS) layer that stringently restricts the entry of foreign molecules (4). Nevertheless, there are opportunities to overcome these cellular barriers and even exploit membrane charge characteristics to enhance delivery.PNA is a DNA mimic with (standard) nucleobases attached to a pseudo-peptide backbone (see Fig. 1) (5). The PNA molecule provides improved hybridization affinity and specificity, chemical and enzymatic stability, and low toxicity. Antisense PNAs have outperformed DNA analogues in diverse systems (2, 6). The main limitation to wider applications in vivo appears to be poor cellular uptake; however, there are several recent examples showing improved delivery and antisense effects for PNAs attached to carrier peptides. For delivery into eukaryotic cells, PNAs have been attached to a peptide derived from the third helix domain of the Drosophila an...

Complementary peptide nucleic acids (PNA) form Watson-Crick base-paired helical duplexes. The preferred helicity of such a duplex is determined by a chiral amino acid attached to the C-terminus. We here show that the induced helicity, as measured by circular dichroism (CD), is drastically dependent on the nucleobase sequence proximal to the chiral center. Chemically linked PNA tetramer duplexes of all 16 combinations of the two bases proximal to a carboxy terminal lysine residue were studied by CD. We conclude that the base next to the chiral center must be either a guanine or a cytosine for efficient stabilization of one helical sense. In case of cytosine, the subsequent base should preferably be a purine. We also show that the side chain properties of the C-terminal amino acid influence the resulting sense of helicity. The propagation length of induced chirality in PNA duplexes is found to be around 10 base pairs. Theoretical calculations of the circular dichroism for B-DNA, using the quantum mechanical matrix method of Schellman, give spectra in reasonable agreement with those found experimentally for PNA duplexes. The rate of helix conversion of the duplexes shows first-order kinetics with a rate constant in the range of minutes. Shorter duplexes are found to have lower activation energy and larger negative activation entropy for helix conversion, in agreement with a conversion mechanism in which a perfect helix is switched to the opposite handedness.