Synthesis and RNA-Binding Properties of Extended Nucleobases for Triplex-Forming Peptide Nucleic Acids

Abstract

Triple-helix formation, using Hoogsteen hydrogen bonding of triplex-forming oligonucleotides, represents an attractive method for sequence-specific recognition of double-stranded nucleic acids. However, practical applications using triple-helix-forming oligonucleotides and their analogues are limited to long homopurine sequences. The key problem for recognition of pyrimidines is that they present only one hydrogen-bond acceptor or donor group in the major groove. Herein, we report our first attempt to overcome this problem by using peptide nucleic acids (PNAs) modified with extended nucleobases that form three hydrogen bonds along the entire Hoogsteen edge of the Watson–Crick base pair. New nucleobase triples (five) were designed, and their hydrogen bonding feasibility was confirmed by ab initio calculations. PNA monomers carrying the modified nucleobases were synthesized and incorporated in short model PNA sequences. Isothermal titration calorimetry showed that these nucleobases had a modest binding affinity for their double-stranded RNA (dsRNA) targets. Finally, molecular modeling of the modified triples in PNA–dsRNA helix suggested that the modest binding affinity was caused by subtle structural deviations from ideal hydrogen-bonding arrangements or disrupted π-stacking of the extended nucleobase scaffolds.

Extended Peptide Nucleic Acid Nucleobases Based on Isoorotic Acid for the Recognition of A–U Base Pairs in Double‐Stranded RNA

“…Presumably, this twist resulted from repulsions of the carbonyl with the O4 on the Io pyrimidine ring and repulsions of the benzamide NH 2 with the ortho ‐hydrogen (C‐6) on the benzamide ring. Nonetheless, compared to previously modelled extended nucleobases, [13] this scaffold represented the most planar base triple observed to date.…”

“…We reported an extended triazole‐containing nucleobase that showed moderate binding with a single modification, but the simple triazolyl‐substituted pyrimidine showed improved binding, similar to T control (Figure 2 c). [13] Finally, Hudson and Wojciechowski reported that the PNA 2 :RNA triplex between Ac‐TTTTTTK‐NH 2 and poly(riboadenylic acid) was stabilized, as observed by UV melting, when a single isoorotamide modification was introduced into the middle of the poly T PNA oligomer (Figure 2 d). [14] Though this phenomenon was proposed to be due to both increased base‐stacking interactions and the cationic ammonium moiety, it was not explored further.…”

“…Expanding on our strategy of using extended PNA nucleobases for RNA recognition, [13] we envisioned a scaffold containing benzamide attached through an amide linkage to an isoorotic acid derived PNA ( Io , Figure 2 e). This design relied on presumed advantages including (1) attaching the pyrimidine base to an electron‐withdrawing group to increase the H‐bond donating ability of the uracil, [12a] (2) imparting enough rigidity across the extended π‐system to afford π‐stacking opportunities and entropic advantages for binding and, (3) planning a modular synthesis allowing modification of the extended nucleobase to achieve optimum planarity and binding.…”

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

“…This study was the first demonstration of the biological effect of binding of M-and P-modified PNAs to dsRNA in live cells. Recent work from our labs [120] systematically surveyed simple nitrogen heterocycles and found that the 3-pyridazinyl nucleobase formed significantly more stable triplets with C-G than other heterocycles, including P. Several groups have explored extended PNA nucleobases for recognition of C-G base pairs [121,122]. Chen and co-workers followed up on original work by Seidman [123] and showed that Q (Figure 7) in PNAs targeting dsRNA, recognized C-G base pairs with good selectivity.…”

Recent Advancement and Innovations in CRISPR/Cas and CRISPR Related Technologies: A review

“…[96] However, TFO based technologies have shown practical issues in terms of limitations of use to only homo-purine sequences. They were not able to recognize pyrimidines as they possess only one-hydrogen acceptor in major DNA groove [97]. Further innovations to overcome this issue resulted in development of Peptide Nucleic Acids (PNAs) supplanted by nucleobases which could overcome this problem and were able to address this issue [97].…”