O4-Modified thymidine analogues with photolabile o-nitrobenzyl group derivatives for modulating polymerase primer extension reaction

• A series of sterically bulky o-nitrobenzyl-modified thymidine derivatives were synthesized and successfully incorporated into oligonucleotides. • All derivatives destabilized duplex formation, as evidenced by a consistent decrease in melting temperature. • Polymerase-mediated primer extension was completely blocked regardless of substituent size. • Aromatic substituents, including the NPOM group, induced primer degradation via exonuclease activity. • Bulky non-aromatic photocaging groups provide a promising platform for controlled modulation of polymerase function.