No abstract

Bimodal PNAs are new PNA constructs designed to bind two different cDNA sequences synchronously to form double duplexes. They are synthesized on solid phase using sequential coupling and click reaction to introduce a second base in each monomer at Cα via alkyltriazole linker. The ternary bimodal PNA:DNA complexes show stability higher than that of individual duplexes. Bimodal PNAs are appropriate to create higher-order fused nucleic acid assemblies.

Cα-bimodal peptide nucleic acids (bm-Cα-PNA) are PNAs with two faces and are designed homologues of PNAs in which each aminoethylglycine (aeg) repeating unit in the standard PNA backbone hosts a second nucleobase at Cα through a spacer chain with a triazole linker. Such bm-Cα-PNA with mixed sequences can form double duplexes by simultaneous binding to two complementary DNAs, one to the base sequence on t-amide side and the other to the bases on the Cα side chain. The synthesis of bm-Cα-PNA with homothymine (T 7 ) on the t-amide face and homocytosine (C 5 ) on the Cα side chain through the triazole linker was achieved by solid phase synthesis with the global click reaction. In the presence of complementary DNAs dA 8 and dG 6 at neutral pH, bm-Cα-PNA 1 forms a higher order pentameric double duplex of a triplex composed of two bm-Cα-PNA-C 5 :dG 5 duplexes built on a core (bm-Cα-PNA-T 7 ) 2 :dA 8 triplex. Circular dichroism studies showed that assembly can be achieved by either triplex first and duplex later or vice versa. Isothermal titration calorimetry data indicated that the assembly is driven by favorable enthalpy. These results validate concurrent multiple complex formation by bimodal PNAs with additional nucleobases at Cα or Cγ on the aeg-PNA backbone and open up ways to design programmed supramolecular assemblies.

Peptide nucleic acids (PNAs) are DNA analogs that bind with high affinity to DNA and RNA in a sequence-specific manner but have poor cell permeability, limiting use as therapeutic agents. The work described here is motivated by recent reports of efficient gene silencing specifically in hepatocytes by small interfering RNAs conjugated to triantennary N-acetyl galactosamine (GalNAc), the ligand recognized by the asialoglycoprotein receptor (ASGPR). PNAs conjugated to either triantennary GalNAc at the N-terminus (the branched architecture) or monomeric GalNAc moieties anchored at C γ of three consecutive PNA monomers of N-(2-aminoethyl)glycine (aeg) scaffolds (the sequential architecture) were synthesized on the solid phase. These formed duplexes with complementary DNA and RNA as shown by UV and circular dichroism spectroscopy. The fluorescently labeled analogs of GalNAc-conjugated PNAs were internalized by HepG2 cells that express the ASGPR but were not taken up by HEK-293 cells that lack this receptor. The sequential conjugate was internalized about 13-fold more efficiently than the branched conjugate into HepG2 cells, as demonstrated by confocal microscopy. The results presented here highlight the potential significance of the architecture of GalNAc conjugation for efficient uptake by target liver cells and indicate that GalNAc-conjugated PNAs have possible therapeutic applications.

Peptide nucleic acids (PNAs) are linear equivalents of DNA with a neutral acyclic polyamide backbone that has nucleobases attached via tert-amide link on repeating units of aminoethylglycine. They bind complementary DNA or RNA with sequence specificity to form hybrids that are more stable than the corresponding DNA/RNA self-duplexes. A new type of PNA termed bimodal PNA [Cγ­(S/R)-bm-PNA] is designed to have a second nucleobase attached via amide spacer to a side chain at Cγ on the repeating aeg units of PNA oligomer. Cγ-bimodal PNA oligomers that have two nucleobases per aeg unit are demonstrated to concurrently bind two different complementary DNAs, to form duplexes from both tert-amide side and Cγ side. In such PNA:DNA ternary complexes, the two duplexes share a common PNA backbone. The ternary DNA 1:Cγ­(S/R)-bm-PNA:DNA 2 complexes exhibit better thermal stability than the isolated duplexes, and the Cγ­(S)-bm-PNA duplexes are more stable than Cγ­(R)-bm-PNA duplexes. Bimodal PNAs are first examples of PNA analogues that can form DNA2:PNA:DNA1 double duplexes via recognition through natural bases. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-level assemblies.