Incorporating 2‑Thiouracil into Short Double-Stranded RNA-Binding Peptide Nucleic Acids for Enhanced Recognition of A‑U Pairs and for Targeting a MicroRNA Hairpin Precursor

Chemically modified short peptide nucleic acids (PNAs) recognize RNA duplexes under near physiological conditions by major-groove PNA·RNA–RNA triplex formation and show great promise for the development of RNA-targeting probes and therapeutics. Thymine (T) and uracil (U) are often incorporated into PNAs to recognize A-U pairs through major-groove T·A-U and U·A-U base triple formation. Incorporation of a modified nucleobase, 2-thiouracil (s²U), into triplex-forming oligonucleotides stabilizes both DNA and RNA triplexes. Thiolation of uracil causes a decrease in the dehydration energy penalty for triplex formation as well as a decrease in the pK_a of the N3 atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions, similar to the previously reported thiolation effect of pseudoisocytosine (J to L substitution). Here, we incorporated s²U into short PNAs, followed by binding studies of a series of s²U-modified PNAs. We demonstrated by nondenaturing polyacrylamide gel electrophoresis and thermal melting experiments that s²U and L incorporated into dsRNA-binding PNAs (dbPNAs) enhance the recognition of A-U and G-C pairs, respectively, in RNA duplexes in a position-independent manner, with no appreciable binding to the DNA duplex. Combining s²U and L modifications in dbPNAs facilitates enhanced recognition of dsRNAs and maintains selective binding to dsRNAs over ssRNAs. We further demonstrated through a cell-free assay the application of the s²U- and L-modified dbPNAs (8-mer, with a molecular mass of ∼2.3 kDa) in the inhibition of the pre-microRNA-198 maturation in a substrate-specific manner. Thus, s²U-modified dbPNAs may be generally useful for the enhanced and selective recognition of RNA duplexes and for the regulation of RNA functions.