Bioluminescent detection of RNA with sequence-specificity using RNA binding protein-luciferase fusion protein.
A novel bifunctional reagent has been synthesized for RNA detection by genetic fusion of a sequence-specific RNA binding protein and firefly luciferase. The RNA binding protein used in this study recognizes the oligoribonucleotide rH4 which contains four (UUAGGG)-repeat sequence, while luciferase works as a bioluminescent marker. The constructed fusion protein exhibited both sequence-specific RNA binding and bioluminescent activities. The rH4 and rECGF, which has an unrelated sequence having the same length as rH4, were immobilized on a nylon membrane. The membrane was pre-treated by 5% bovine serum albumin and soaked into a fusion protein solution. Bioluminescent detection has successfully been performed at more than 50 pmol of rH4, so the detection limit using this protein was 50 pmol. However, no appreciable bioluminescence was induced by rECGF.
Kajita
Y
Y
Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Kobatake
E
E
Ishikawa
F
F
Aizawa
M
M
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008156:Luciferases / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D009843:Oligoribonucleotides; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA; D008156:Luciferases