Marking the polarity of RNA molecules for electron microscopy by covalent attachment of psoralen-DNA restriction fragments.
We have developed a general procedure for covalently attaching complementary restriction fragments to RNA molecules and visualizing the fragment in the electron microscope after denaturation. This is accomplished by creating psoralen monoadducts on the restriction fragment by irradiation at 390 nm before hybridization to the RNA and subsequently converting the monoadducts to RNA . DNA crosslinks by irradiation at 360 nm. Using this procedure, we have determined the location of a crosslink placed in Escherichia coli 165 rRNA by a previous irradiation. A consistent assignment is obtained by marking the crosslinked RNA with restriction fragments complementary to the 5' end or the 3' end of the 165 rRNA molecule.
D004247:DNA; D004262:DNA Restriction Enzymes; D011564:Furocoumarins; D008854:Microscopy, Electron; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012335:RNA, Ribosomal; D014307:Trioxsalen / Q000031:analogs & derivatives
D011564:Furocoumarins; D012335:RNA, Ribosomal; D004247:DNA; C018822:aminomethyltrioxsalen; D004262:DNA Restriction Enzymes; D014307:Trioxsalen