An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA.
Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, gamma-amidotriphosphates, as substrates of 3' to 5' exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.
Hirao
Ichiro
I
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. ihirao@riken.jp
Kimoto
Michiko
M
Mitsui
Tsuneo
T
Fujiwara
Tsuyoshi
T
Kawai
Rie
R
Sato
Akira
A
Harada
Yoko
Y
Yokoyama
Shigeyuki
S
D020029:Base Pairing / Q000235:genetics*; D001665:Binding Sites; D019921:Biotinylation; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D009690:Nucleic Acid Conformation; D011725:Pyridines / Q000378:metabolism*; D011758:Pyrroles / Q000378:metabolism*; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000378:metabolism
D011725:Pyridines; D011758:Pyrroles; D014764:Viral Proteins; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases