Doubly catalytic sensing of HIV-1-related CCR5 sequence in prokaryotic cell-free translation system using riboregulator-controlled luciferase activity.
A molecular-beacon-type riboregulator (mRNA) was applied to multiply catalytic gene sensing. It consists of a reporter gene for firefly protein luciferase and, upstream thereof, a regulator hairpin domain composed of an RBS/anti-RBS stem (RBS = ribosome binding site) and a loop which is complementary to the target. The hairpin and, hence, the RBS are rendered open upon binding of a target oligonucleotide of the human CC chemokine receptor 5 sequence in a prokaryotic cell-free translation system (10 muL) to ignite ribosomal catalytic translation, or transcription/translation when using a DNA form of the probe, to produce luciferase, which is assayed by a catalytic chemiluminescence reaction. The sensing, using an unmodified RNA or even dsDNA as a probe with a chemiluminescence output, is thus doubly catalytic or amplifiable with a sensitivity at </=50 fmol in respect to the target with 4.5 fmol (1 ng/muL) of probe and a single nucleotide resolution.
Sando
Shinsuke
S
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Narita
Atsushi
A
Abe
Kenji
K
Aoyama
Yasuhiro
Y
D001483:Base Sequence; D002384:Catalysis; D002474:Cell-Free System; D005786:Gene Expression Regulation / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000235:genetics*; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics*
D015347:RNA Probes; D012333:RNA, Messenger; D019713:Receptors, CCR5; D008156:Luciferases