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 noncanonical interactions and uridine participation in 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 red-shifted base absorption (365 nm), colored base (yellow), and higher base acidity (pKa = 7.85) reduced by an 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 (∼1.8-fold) 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.