Real-time monitoring of RNA synthesis in a phospholipid-coated microdroplet as a live-cell model.
We constructed a phospholipid-coated water-in-oil microdroplet (20-60 microm in diameter) that encapsulated plasmid DNA containing a human beta-actin cDNA sequence, fluorescent oligonucleotide probes, and other components of the transcription reaction. Transcription inside individual microdroplets was investigated in real time by using fluorescence microscopy. The progress of the transcription reaction was successively monitored by fluorescence resonance energy transfer (FRET), which was derived from the specific hybridization of fluorescent oligonucleotide probes to the beta-actin mRNAs synthesized. In microdroplets composed of phosphatidylethanolamine, DNAs were located in the aqueous phase or on the membrane surface depending on the Mg(II) concentration. FRET images showed that transcription occurred on DNA in both states. Moreover, individual DNA molecules undergoing transcription were visualized as discrete FRET signals in cases in which small numbers of DNAs were present in the microdroplet.
Tsuji
Akihiko
A
Spatio-Temporal Order, ICORP, Japan Science and Technology Agency, Kyoto 606-8502, Japan. tsuji@chem.scphys.kyoto-u.ac.jp
Yoshikawa
Kenichi
K
D000199:Actins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008823:Micelles; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis*; D014158:Transcription, Genetic
D000199:Actins; D008823:Micelles; D015345:Oligonucleotide Probes; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C483858:phosphatidylethanolamine; D012313:RNA