Nucleobase and Linker Modification for Triple‐Helical Recognition of Pyrimidines in RNA Using Peptide Nucleic Acids

Abstract

Triple-helical recognition of any sequence of double-stranded RNA requires high affinity Hoogsteen hydrogen binding to pyrimidine interruptions of polypurine tracts. Because pyrimidines have only one hydrogen bond donor/acceptor on Hoogsteen face, their triple-helical recognition is a formidable problem. The present study explored various five-membered heterocycles and linkers that connect nucleobases to backbone of peptide nucleic acid (PNA) to optimize formation of X * C-G and Y * U-A triplets. Molecular modeling and biophysical (UV melting and isothermal titration calorimetry) results revealed a complex interplay between the heterocyclic nucleobase and linker to PNA backbone. While the five-membered heterocycles did not improve pyrimidine recognition, increasing the linker length by four atoms provided promising gains in binding affinity and selectivity. The results suggest that further optimization of heterocyclic bases with extended linkers to PNA backbone may be a promising approach to triple-helical recognition of RNA.

Triplex-forming peptide nucleic acids as emerging ligands to modulate structure and function of complex RNAs

“…While the K a for PNA6-HRP5 and PNA7-HRP6 was 1 and 3 × 10 6 M −1 , respectively, stability of PNA8-HRP7 and PNA9-HRP8 was too low to be measured by ITC or UV melting under our usual conditions. 51 Taken together, these results show that recognition of pyrimidines using simple heterocycles forming one hydrogen bond is a challenge and remains an unsolved problem for triplex-forming PNAs.…”

“…The extended linker that connected E to PNA backbone was expected to circumvent steric hindrance from the 5-Me of T and allow E to form a single hydrogen bond with the C4 carbonyl of T. 50 We found that E also recognized U-A base pairs in RNA with good affinity (Fig. 4, K a = 11 × 10 6 M −1 and T m = 54 °C) albeit the sequence specificity was lower than for M and T. 46,51 Molecular modeling suggested that in PNA-dsRNA triplexes E formed one hydrogen bond with U (Fig. 3) as originally proposed for the E•T-A triplet in DNA.…”

“…50 Interestingly, using the longer linker in P 14 did not significantly change the binding properties of P 9 . 51 Molecular dynamics simulations suggested that the longer linker pushed P 14 out of optimal stacking alignment, which likely off-set any gains from optimized hydrogen bond alignment. 51 Most importantly, placing P 9 and E in PNA6-PNA9 designed to recognize several pyrimidine interruptions in HRP5-HRP8 (Fig.…”

“…51 Molecular dynamics simulations suggested that the longer linker pushed P 14 out of optimal stacking alignment, which likely off-set any gains from optimized hydrogen bond alignment. 51 Most importantly, placing P 9 and E in PNA6-PNA9 designed to recognize several pyrimidine interruptions in HRP5-HRP8 (Fig. 4) resulted in notable losses of binding affinity.…”