Dnabinding Cyanine Dyes

In recent years, unsymmetrical cyanine dyes have received considerable interest because of their excellent nucleic acid staining properties. In 1986, Lee et al.[3] showed that the unsymmetrical cyanine dye Thiazole Orange (TO; Fig. 2) exhibits 3000-fold enhancement in fluorescence intensity upon binding to RNA. Later, Rye et al.[4] showed that TO, and the similar dye, Oxazole Yellow (YO) with a benzoxazolium moiety instead of benzothiazolium, have a dramatic increase in fluorescence also upon binding to DNA. Both these studies indicated that the dyes bind in a nearest-neighbor exclusion stoichiometry typical of intercalators.

To obtain stains with high affinity for DNA, Rye et al.[5] developed TOTO and YOYO (Fig. 2), which have two dye moieties connected by a biscationic amine linker.

Their affinity for double-stranded DNA is about the square of that of the monomers as a result of bisinterca-lation and their high positive charge. To enhance the affinity for DNA of monomeric dyes, positively charged substituents (3-propyl trimethyl ammonium bromide) were added in TO-PRO and YO-PRO (Fig. 3).[6]

The absorption maximum of cyanine dyes depends on the length of the conjugated chain and the nature of the heterocyclic moieties. Today dyes are available whose absorptions span a broad range of the visible spectrum (Fig. 3).[6] They typically have several hundredfold enhancements in fluorescence upon binding DNA and a quantum yield in bound state of at least 0.1.

The fluorescence quantum yield of cyanine dyes increases when torsional motion around the methine bridge is restricted, which reduces the probability of nonradiative relaxation from the excited singlet state.[7,8] When the dyes bind DNA, internal rotation is likely to be strongly hindered, which causes the dramatic increase in fluorescence.

The interactions of cyanine dyes with nucleic acids have been thoroughly investigated. Early flow linear dichroism studies indicated that YO-PRO and YOYO intercalate in DNA.[9] Intercalation was also supported by circular dichroism, fluorescence anisotropy, and dye-nucleobase energy transfer measurements. By nuclear magnetic resonance (NMR), the solution structures of TOTO bound to short oligonucleotides have been determined.[10] Frequently, more than one complex was observed. A preferred binding site was bisintercalation in a central CTAG:CTAG binding site with the cationic linker located in the minor groove. The DNA complex is unwound by ~ 30° and extended ~ 2 A per TO moiety, which is typical of intercalation.

Thermodynamic studies of TO binding to nucleic acids of different base compositions showed that binding has little sequence selectivity.[11] At elevated dye:base pair ratios, a secondary binding mode was observed for both monomeric and dimeric dyes.[9,12] In a spectroscopic study, Nygren et al.[13] determined thermodynamic parameters of TO binding to DNA both as monomer and

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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