Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding.

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue improves the 3'-exonuclease stability of 2'-5'-oligoadenylate-antisense conjugates.

Incorporation of a 4-hydroxy-N-acetylprolinol nucleotide analogue at the 3'-terminus of DNA or 2-5A-DNA sequences resulted in a significantly enhanced 3'-exonuclease resistance while the affinity for complementary RNA was only slightly decreased. Furthermore, the binding to and activation of human RNase L by thus modified 2-5A-DNA conjugates was not altered as compared to the parent unmodified 2-5A-DNAs.

Synthesis and RNAse L binding and activation of a 2-5A-(5')-DNA-(3')-PNA chimera, a novel potential antisense molecule.

Fully automated solid-phase synthesis gave access to a hybrid in which 5'-phosphorylated-2'-5'-linked oligoadenylate (2-5A) is connected to the 5'-terminus of DNA which, in turn, is linked at the 3'-end to PNA [2-5A-(5')-DNA-(3')-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.

2-5A-PNA complexes: a novel class of antisense compounds.

This paper presents the fully automated solid phase synthesis of 2-5A-PNA hybrids. These stable antisense probes cause RNase L mediated hydrolysis of target RNA sequences.

2,5-oligoadenylate-peptide nucleic acids (2-5A-PNAs) activate RNase L.

To potentiate the 2-5A (2',5'-oligoadenylate)-antisense and peptide nucleic acid (PNA) approaches to regulation of gene expression, composite molecules were generated containing both 2-5A and PNA moieties. 2-5A-PNA adducts were synthesized using solid-phase techniques. Highly cross-linked polystyrene beads were functionalized with glycine tethered through a p-hydroxymethylbenzoic acid linker and the PNA domain of the chimeric oligonucleotide analogue was added by sequential elongation of the amino terminus with the monomethoxytrityl protected N-(2-aminoethyl)-N-(adenin-1-ylacetyl)glycinate. Transition to the 2-5A domain was accomplished by coupling of the PNA chain to dimethoxytrityl protected N-(2-hydroxyethyl)-N-(adenin-1-ylacetyl)glycinate. Finally, (2-cyanoethyl)-N,N-diisopropyl-4-O-(4,4-dimethoxytrityl)butylphosphor amidite and the corresponding (2-cyanoethyl)-N,N-diisopropylphosphoramidite of 5-O-(4,4'-dimethoxytrityl)-3-O-(tert-butyldimethylsilyl)-N6-benzoyladeno sine were the synthons employed to add the 2 butanediol phosphate linkers and the four 2',5'-linked riboadenylates. The 5'-phosphate moiety was introduced with 2-[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]ethyl-(2-cyanoethyl) -N,N-diisopropylphosphoramidite. Deprotection with methanolic NH3 and tetraethylammonium fluoride afforded the desired products, 2-SA-pnaA4, 2-5A-pnaA8 and 2-5A-pnaA12. When evaluated for their ability to cause the degradation of two different RNA substrates by the 2-5A-dependent RNase L, these new 2-5A-PNA conjugates were found to be potent RNase L activators. The union of 2-5A and PNA presents fresh opportunities to explore the biological and therapeutic implications of these unique approaches to antisense.