Fluorescent determination of micro-quantities of RNA using Hoechst 33258 and binase.

Detection of small amounts of RNA in various biological samples is an important applied task. Using fluorescence spectroscopy, the hydrolysis by binase of rRNA and tRNA, stained with Hoechst 33258, in aqueous solutions was investigated. The binding constant of Hoechst with rRNA is 106 M-1. Specific hydrolysis of rRNA and tRNA by binase during 1-2 min at room temperature leads to a multiple decrease in fluorescence of the dye. This rapid hydrolysis goes to large polynucleotide fragments, but not to short oligonucleotides. The binding constant of binase with rRNA is about of 2.5 × 106 M-1, which is several dozen times higher than with oligonucleotides. The susceptibility to binase attack depends on the secondary structure of RNA, determined by non-canonical ribonucleotides. The developed highly sensitive fluorescent method can be used for the rapid selective detection of trace amounts of rRNA or tRNA, as well as for studying the physicochemical properties of these RNAs. Using the proposed method, one can confidently detect RNA from 10-7 M.

Determination of Micro-Quantities of DNA Using DNAse and Fluorescence of Hoechst 33258 and Light-Scattering.

The DNA hydrolysis by deoxyribonuclease (DNAse I) in aqueous solution was studied, using fluorescence spectroscopy and high-sensitive light-scattering detection. Specific hydrolysis of high-polymer DNA or fragmented DNA by the enzyme led to a strong decrease in the fluorescence of the Hoechst dye. The hydrolysis of mitochondrial DNA was accompanied by a decrease in the fluorescence of the dye only in 1.6 times. Hydrolysis within minutes and even hours led to appearance of large polynucleotide fragments, but not to short oligonucleotides, that was confirmed using polarized fluorescence and highly sensitive measurement of light-scattering. At the moment of the time of formation of a complex between DNA and DNAse I, a strong light-scattering occurred, which then dropped sharply during hydrolysis of high-molecular DNA, and slowly decreased during hydrolysis of fragmented DNA. The proposed methods can be applied for selective detection of trace amounts of various types of DNA, as well as for studying their physic-chemical properties.