Hayashi Gosuke, Yanase Masafumi, Takeda Katsuya, Sakakibara Daisuke, Sakamoto Ryosuke, Wang Dan Ohtan, Okamoto Akimitsu
†Department of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
‡Institute for Integrated Cell-Material Science (iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, 606-8501, Japan.
Bioconjug Chem. 2015 Mar 18;26(3):412-7. doi: 10.1021/acs.bioconjchem.5b00090. Epub 2015 Mar 2.
Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes.
由于小寡核苷酸探针的扩散特性，在特定细胞内位置进行活细胞RNA成像在技术上受到限制。设计并合成了在寡核苷酸末端带有链霉亲和素或金纳米颗粒的大分子荧光点亮探针。这些大分子探针分别通过核内和胞质显微注射实现了对内源mRNA的细胞核和细胞质选择性监测。同时使用与不同荧光染料偶联的大分子和小分子探针，能够对细胞核和细胞质中存在的mRNA进行双色成像。此外，我们观察到，在转染大分子探针后，活的HeLa细胞边缘附近的荧光会随着时间推移，与伪足突起的动态形成和变形同步移动。
