Synthesis and RNA Transcription of Colored 4-Selenouridine Triphosphate with Atom Mutagenesis

4-Selenouridine (4SeU) is one of the naturally-occurring modifications of tRNAs, possibly maintaining the three-dimensional structural stability of tRNA and playing a critical role in stress responses. By using the 4-Se-atomic probe, previously we have revealed more insights into the RNA non-canonical interactions and uridine participation in the diversified RNA functions and structures. To further explore the Se-atomic probe, herein we report the first synthesis of 4-selenouridine triphosphate (4SeUTP) and the enzymatic incorporation of 4SeUTP into RNAs. Interestingly, we found that 4SeUTP possessed unique properties, including the red-shifted base absorption (365 nm), colored base (yellow) and higher base acidity (pKa = 7.85) reduced by Se-atom. Further, 4SeUTP was recognized by T7 RNA polymerase as well as UTP counterpart, and its transcription of RNAs (such as 4SeU-hammerhead ribozyme RNA) was up to the native level. Furthermore, the transcribed 4SeU-hammerhead ribozyme had higher cleaving activity (approximately 1.8 folds) than the corresponding native ribozyme. In conclusion, 4SeUTP can be a fine substrate of RNA polymerase, and the 4SeU-RNAs can be enzymatically synthesized with better catalytic activity, opening a new avenue for investigating the biochemical functions and structures of RNAs, especially catalytic RNAs.