Fluorescence lifetime for on-the-fly multiplex detection of DNA restriction fragments in capillary electrophoresis.

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.

Concerted intercalation and minor groove recognition of DNA by a homodimeric thiazole orange dye.

The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5'-CTAG-3', a 5'-CTTAG-3', and a 5'-CTATAG-3' sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.

Fluorescence spectral characteristics of novel asymmetric monomethine cyanine dyes in nucleic acid solutions.

Six new asymmetric monomethine cyanine dyes have been synthesized and their fluorescence characteristics in the presence of nucleic acids studied. The new dyes have no fluorescence of their own in water solutions upon excitation at 480 nm but they become strongly fluorescent in the presence of nucleic acids. The fluorescence maxima of the investigated dyes are found at 525-545 nm when bound to dsDNA and around 600 nm upon binding to RNA and ssDNA. Fluorescence quenching studies with increasing concentrations of NaCl indicate that the cyanine dyes have a mixed (intercalating and groove binding) type of interaction with dsDNA.