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Abstract Triplex-forming peptide nucleic acids (PNAs) require chemical modifications for efficient sequence-specific recognition of DNA and RNA at physiological pH. Our research groups have developed 2-aminopyridine (M) as an effective mimic of protonated cytosine in C+•G-C triplets. M-modified PNAs have a high binding affinity and sequence specificity as well as promising biological properties for improving PNA applications. This communication reports the optimization of synthetic procedures that give PNA M monomer in seven steps, with minimal need for column chromatography and in good yields and high purity. The optimized route uses inexpensive reagents and easily performed reactions, which will be useful for the broad community of nucleic acid chemists. Thought has also been given to the potential for future development of industrial syntheses of M monomers. 

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. 

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. 

Peptide nucleic acid (PNA) clamps modified with 2-aminopyridine (M) nucleobase invaded double-stranded DNA under physiological salt conditions. In contrast, PNAs carrying common nucleobases could not fully invade DNA under these conditions. M-modified PNAs may overcome the problematic requirement for low salt concentration, a long-standing DNA invasion problem. 

Over the last three decades, triplex-forming PNAs have emerged as ligands for the recognition of double-stranded RNA. Strong and sequence selective binding using synthetic nucleobases offers opportunity for modulation of biological function of endogenous RNA transcripts. 

In triplex-forming peptide nucleic acid, a novel 2-guanidyl pyridine nucleobase (V) enables recognition of up to two cytosine interruptions in polypurine tracts of dsRNA by engaging the entire Hoogsteen face of C–G base pair. Ab initio and molecular dynamics simulations provided insights into H-bonding interactions that stabilized V·C–G triplets. Our results provided insights for future design of improved nucleobases, which is an important step towards the ultimate goal of recognition of any sequence of dsRNA.