Triplex‐Forming Peptide Nucleic Acids with Extended Backbones

Abstract

Peptide nucleic acid (PNA) forms a triple helix with doublestranded RNA (dsRNA) stabilized by a hydrogen-bonding zipper formed by PNA's backbone amides (NÀ H) interacting with RNA phosphate oxygens. This hydrogen-bonding pattern is enabled by the matching~5.7 Å spacing (typical for A-form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one À CH 2 À group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B-form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA-DNA triplexes compared to PNA-RNA. To test this hypothesis, we synthesized triplex-forming PNAs that had the pseudopeptide backbones extended by an additional À CH 2 À group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A-form-like conformations when binding double-stranded nucleic acids. It appears that the original six-atom-long PNA backbone is an almost perfect fit for binding to A-form nucleic acids.

Fluorobenzene Nucleobase Analogues for Triplex‐Forming Peptide Nucleic Acids

“…We synthesized six PNAs having modified nucleobases with various fluorine substitution patterns (PNAX, Figure 2) ranging from none (X=F0) to fully substituted pentafluorobeznene (X=F5). PNA monomers were synthesized using our previously reported methods starting from commercially available substituted phenylacetic acids (for details, see Supporting Information) [9–10,19] …”

“…PNA monomers were synthesized using our previously reported methods starting from commercially available substituted phenylacetic acids (for details, see Supporting Information). [9][10]19] We evaluated the stability and specificity of base triplets involving fluorinated benzenes using UV thermal melting of the triple helices formed between model hairpins and modified PNAs. UV thermal melting is a common technique to measure stability of nucleic acid secondary structures; however, for triple helices the measurements may be complicated by overlap between the triplex to duplex and duplex to single strands transitions.…”

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

“…Not surprisingly, backbone modification has been a major focus of follow up attempts to improve the original PNA design. Early studies showed that maintaining proper distances (number of bonds) along the backbone and be-tween the backbone and nucleobases of PNA was critical for effective nucleic acid binding as extension of either by additional methylene groups strongly decreased the binding affinity of PNA to either single-or double-stranded nucleic acids [46][47][48]. Furthermore, replacing amide linkages connecting the PNA's backbone and the nucleobase with a tertiary amine also destabilized PNA complexes with complementary DNA [49].…”

Mechanisms and applications of peptide nucleic acids selectively binding to double‐stranded RNA

“…Binding data suggest that increasing the length of the backbone or the backbone‐base linker significantly weakens the binding of PNAs to dsRNAs, ssRNAs, and dsDNAs. [ 64,71 ] NMR and fluorescence studies suggest that attaching a γ‐(R)‐hydroxymethyl group to PNAs containing bases T, M, and E results in a reduced PNA•dsRNA triplex formation and an enhanced invasion of a dsRNA to form a PNA•RNA‐PNA triplex. [ 55,72 ] Clearly, base modifications (Figures 3–7) combined with backbone/linker modifications (Figure 1) would allow the development of optimized PNA‐based scaffolds for the specific recognition of dsRNAs.…”