| Budget Amount *help |
¥16,640,000 (Direct Cost: ¥12,800,000、Indirect Cost: ¥3,840,000)
Fiscal Year 2016: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2015: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2014: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000)
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| Outline of Final Research Achievements |
Photochemical activation of caged oligonucleotides enable the regulation of genes of interest within a single-cell level of spatial resolution. In the course of our study of the development of caged oligonucleotides, we designed and synthesized new precursor molecules of photo-releasable nucleotides having an affinity tag for purification and detection. A precursor molecule, Bio-Bhc-diazo comprises three components: biotin for an affinity tag, photoactive Bhc group, and phosphate reactive diazomethyl moiety. We developed a method to modify arbitrary sequence regions of long-chain double-stranded DNA and plasmid DNA with photolabile protecting groups by chemo-enzymatic reactions. We demonstrated that a caged compound of RNA can be synthesized using a novel nucleotide caging reagent having a biotin tag. We demonstrated that this technique can be applied to the gRNA of CRISPR / Cas9 system to synthesize caged gRNA protecting target DNA recognition site or Cas9 binding site.
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