On the mechanism of the methyl green-pyronin stain for nucleic acids.

Abstract

The combination of pyronin, methyl green, malachite green, crystal violet, and Alcian Blue with a large number of polynucleotides and acidic polysaccharides has been investigated. The “critical electrolyte concentration” approach has been used to provide a measure of affinity between dye and substrate. The interaction of methyl green with DNA, RNA and heparin has been examined spectroscopically. Previously published results are re-examined, and with the new experiments permit consistent interpretations of the specificities of dye binding in terms of modern ideas of nucleic acid and dye structure. All dyestuffs except Alcian Blue bind more strongly to polynucleotides than would be expected if solely electrostatic bonds were present. Pyronin and planar monovalent cationic dyes interact best with polynucleotides in which purine and pyrimidine bases are freely accessible, as in single stranded molecules without extensive secondary structure, such as RNA, denatured DNA, etc. Non-planar triphenylmethane dyes, e.g. methyl green, malachite green etc. bind less strongly to such substrates, but because of their shape they fit well into the secondary structure of native DNA. Tumour RNA and DNA did not differ from “normal” RNA and DNA. By varying the electrolyte concentration, pyronin-methyl green selectivity e.g. for DNA or RNA, can be controlled, and non-nucleotide staining suppressed. The relevance of the new interpretation to the ribonuclease-pyronin technique is discussed.