A NEW FLUORESCENT HYDRAZIDE FOR SEQUENCING RIBONUCLEIC ACIDS

Abstract

This paper deals with the synthesis and properties of a fluorescent hydrazide and its hydrazones of nucleoside dialdehydes and of 5'-methylene nucleoside dialdehydes. Hydrazination of 1-dimethylaminonaphthalene-5-sulphonyl-glycyl ethyl ester (DNS-GlyOC 2 H 5 ) gives the fluorescent carbonyl reagent, 1-dimethylaminonaphthalene-5-sulphonyl-glycyl hydrazide (DNS-Gly-NHNH 2 ). The reaction of DNS-Gly-NHNH 2 with the nucleoside dialdehydes or the 5'-methylene nucleoside dialdehydes produced by the oxidation of the corresponding nucleosides or 5'-nucleotides with sodium periodate forms the respective fluorescent hydrazones. The coupling ratio of nucleoside dialdehydo to DNS-Gly-NHNH 2 is 1:1. The nucleoside hydrazones can be clearly resolved by polyamide plate chromatography with a sensitivity of 1 × 10 -10 mole. The ultraviolet absorption spectra, the molar extinction coefficients, the fluorescent spectra as well as the quantum yields of the hydrazide and its nucleoside hydrazones in water, ethanol and dioxane are measured. The fluorescent intensities of the hydrazone increase with the decrease of solvent polarity. Their quantum yields are about 0.4 in dioxane. A micromethod is described for sequencing oligoribonucleotides by stepwise β-elimination followed by fluorescent labeling of their 3'-terminal nucleosides. The sensitivity of this method indicates that tetraribonucleotide with A 260nm as little as 0.16 is sufficient for sequence determination.