Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design. We developed novel Ps-TFOs containing 2 & PRIME;-O,4 & PRIME;-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2, 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions. We conducted a close investigation of psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs). The triangular relationships between structural differences, photo-crosslinking efficiencies, and biological activities were examined. This research suggested that the photo-crosslinking of Ps-TFO did not reach a plateau under cell irradiation conditions, highlighting the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.+image