Comparison of 2-Aminopyridine and 4-Thiopseudisocytosine PNA Nucleobases for Hoogsteen Recognition of Guanosine in RNA

Abstract

Peptide nucleic acid (PNA) is emerging as a promising ligand for triple-helical recognition of folded biologically relevant RNA. Chemical modifications are actively being developed to achieve high affinity and sequence specificity under physiological conditions. In this study, we compared two modified PNA nucleobases, 2-aminopyridine (M) and 4-thiopseudisocytosine (L), as alternatives to protonated cytosine (unfavorable under physiological conditions), to form more stable triplets than C+·G-C. Both nucleobases formed M+·G-C and L·G-C triplets of similar stability; however, the L-modified PNAs showed somewhat reduced sequence specificity. In conclusion, M and L represent two alternative solutions to the problem of cytosine protonation in triple-helical recognition of RNA. In M, the pK a is increased to favor partial protonation, which improves solubility and cellular uptake of M-modified PNAs. In L, the sulfur substitution enhances favorable hydrophobic interactions, which may have advantages in avoiding off-target effects that may be caused by cationic modifications. However, our results showed that substituting Ms with Ls did not restore the sequence specificity of a PNA containing cationic groups.