Inchworm-type PNA-PEG conjugate regulates gene expression based on single nucleotide recognition

In order to detect single nucleotide mutations and suppress gene expression, we synthesized an artificial nucleic acid, an inchworm-type PNA-PEG conjugate (i-PPc), that possessed a chemical structure in which 8 residues of peptide nucleic acid (PNA) were linked to both ends of a polyethylene glycol molecule. I-PPc_T7_FM, which forms a complementary strand with the T7 promoter region of luciferase-expressing mRNA, failed to suppress the amount of luciferase produced via gene expression. However, 10 μM of i-PPc_ATG_FM, targeting the start codon of luciferase (Luc⁺), suppressed approximately 85% of Luc⁺ production compared to that of the control in the cell-free protein synthesis system. Moreover, i-PPc_ATG_MM (i-PPc_ATG_FM with a single base mutation) only suppressed the amount of luciferase produced by approximately 15%, and such suppression of luciferase expression has not been achieved with block-type PPc or PNA oligos. The thermodynamic parameters suggested that the difference in stability of each PNA segment of the i-PPc contributed to single nucleotide recognition. These results indicate that the i-PPc could be used in antisense therapy to target single nucleotide polymorphisms (SNP).