Direct physical measurements on substituted agarose gels: evidence for intercalation of gel-bound ethidium into transfer RNA.
The cation of the salt ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) has been covalently linked to an agarose matrix through an intermediate 3,3'-diaminodipropylaminosuccinyl spacer arm. Partition binding and visible absorption spectral measurements on the gel were used to monitor the binding of transfer RNA to the covalently bound ethidium group. Direct fluorescence measurements of the formation of the gel-bound complex indicate that this binding involves the intercalation of the ethidium groups into the tRNA molecule. Dissociation of the ethidium-tRNA complex was monitored as a function of sodium chloride concentration by both direct solution spectral measurement of the released tRNA and by fluorescence quenching measurements of the dissociation of the intercalation complex. The derivatized gel has been shown to be capable of the fractionation of tRNA species by elution with a positive salt gradient under column flow conditions.
Thomas
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Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014, USA.
Schechter
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D002846:Chromatography, Affinity / Q000379:methods; D002847:Chromatography, Agarose / Q000379:methods; D004996:Ethidium; D005456:Fluorescent Dyes; D005782:Gels; D007364:Intercalating Agents; D012329:RNA, Bacterial / Q000302:isolation & purification; D012343:RNA, Transfer / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D005782:Gels; D007364:Intercalating Agents; D012329:RNA, Bacterial; D012343:RNA, Transfer; D004996:Ethidium