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Peptide nucleic acid (PNA) forms a triple helix with double‐stranded RNA (dsRNA) stabilized by a hydrogen‐bonding zipper formed by PNA’s backbone amides (N‐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 ‐CH2‐ group may bring the distance between PNA amide N‐H closer to 7 Å, favourable for hydrogen‐bonding to the B‐form dsDNA phosphate oxygens. Extension of 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 ‐CH2‐ 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 PNA may have an inherent preference for A‐form‐like conformations when binding double‐stranded nucleic acids. It appears that the original six atoms long PNA backbone is an almost perfect fit for binding to A‐form nucleic acids.
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Improved Triplex‐Forming Isoorotamide PNA Nucleobases for A−U Recognition of RNA Duplexes**
Abstract Four new isoorotamide (Io)‐containing PNA nucleobases have been designed for A−U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A−U in a PNA:RNA2triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A‐binding nucleobases. Molecular dynamics simulations provided further insights into binding ofIobases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomerIo8additionally gave the highest affinity observed for an A‐binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.
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- PAR ID:
- 10523938
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Chemistry – A European Journal
- Volume:
- 29
- Issue:
- 64
- ISSN:
- 0947-6539
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/chem.202302390
