A two-unnatural-base-pair system toward the expansion of the genetic code.
Toward the site-specific incorporation of amino acid analogues into proteins, a two-unnatural-base-pair system was developed for coupled transcription-translation systems with the expanded genetic code. A previously designed unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was used for the site-specific incorporation of yTP into RNA opposite s in templates by T7 RNA polymerase. For the site-specific incorporation of sTP into RNA, a newly developed unnatural base, imidazolin-2-one (denoted by z), is superior to y as a template base for pairing with s in T7 transcription. The combination of the s-y and s-z pairs provides a powerful tool to prepare both y-containing mRNA and s-containing tRNA for efficient coupled transcription-translation systems, in which the genetic code is expanded by the codon-anticodon interactions mediated by the s-y pair. In addition, the nucleoside of s is strongly fluorescent, and thus the s-z pair enables the site-specific fluorescent labeling of RNA molecules. These unnatural-base-pair studies provide valuable information for understanding the mechanisms of replication and transcription.
Hirao
Ichiro
I
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. ihirao@postman.riken.jp
Harada
Yoko
Y
Kimoto
Michiko
M
Mitsui
Tsuneo
T
Fujiwara
Tsuyoshi
T
Yokoyama
Shigeyuki
S
D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005815:Genetic Code; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011687:Purines / Q000378:metabolism*; D011728:Pyridones / Q000378:metabolism*; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins
C435686:2-amino-6-(2-thienyl)purine; D011687:Purines; D011728:Pyridones; D012333:RNA, Messenger; D014764:Viral Proteins; D012313:RNA; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases