Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.DOI: http://dx.doi.org/10.7554/eLife.02501.001

Pseudoisocytosine (J), a neutral analogue of protonated cytosine, is currently the gold standard modified nucleobase in peptide nucleic acids (PNAs) for the formation of J•G-C triplets that are stable at physiological pH. This study shows that triple-helical recognition of RNA and DNA is significantly improved by using 2-aminopyridine (M) instead of J. The positively charged M forms 3-fold stronger M+•G-C triplets than J with uncompromised sequence selectivity. Replacement of six Js with Ms in a PNA 9-mer increased its binding affinity by ∼2 orders of magnitude. Mmodified PNAs prefer binding double-stranded RNA over DNA and disfavor off-target binding to single-stranded nucleic acids. Taken together, the results show that M is a promising modified nucleobase that significantly improves triplex-forming PNAs and may provide breakthrough developments for therapeutic and biotechnology applications.

Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA’s chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA’s binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA’s binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.

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.

Conjugation with cationic lysine residues improves the biophysical and biological properties of peptide nucleic acids (PNAs). A single lysine is routinely used to improve the solubility and prevent aggregation of the neutral and hydrophobic amide backbone of PNA. Literature precedents include the attachment of lysine at either the Nor the C-terminus. Moreover, conjugation with short lysine peptides (four to eight residues) improves the cellular uptake of PNA akin to more complex cell-penetrating peptides. Herein, we report a systematic study of the effect of lysine location (N-vs Cterminus) and chirality (Dvs L-) on triple-helical binding of PNA to double-stranded RNA and DNA (dsRNA and dsDNA). The results confirmed our earlier findings that conjugation with lysine significantly increased the stability of PNA-dsRNA and PNA-dsDNA triplexes and that PNA affinity for dsRNA was about an order of magnitude higher than for the same sequence of dsDNA. In contrast, conjugation of PNA with noncharged amino acids decreased the affinity of PNA. Surprisingly, neither the location nor the chirality of lysine had significant impact on PNA affinity for either dsRNA or dsDNA. The results are consistent with the lack of chiral preorganization of single-stranded PNAs, even after conjugation with four D-or L-amino acids. Instead, the positive charge of lysine appears to be the main driving force behind the increased affinity.