Binding Agents and Their Use as Local Environmental Probes
4.1. Small Molecule–Nucleic Acid Interactions
4.1.2. Fluorescent DNA Intercalators
Rapid, highly-sensitive, and even sequence-specific detection of nucleic acids is crucial for medical diagnosis and for understanding biomolecular mechanisms and became a major issue for researchers when the desire to sequence complete genomes emerged. Interest in non-radioactive detec-tion of nucleic acids has led to the search for stains that are fluorescent and stable under gel electrophoretic conditions [384, 385]. A real break-through in this area, which enabled an important step into the genomic era, was made in the early 1990s with the discovery of the oxazole yellow (YO) and thiazole orange (TO) dye families [386, 387]. YO and TO are asymmetric cyanine dyes with a monomethine bridge connecting a
benzo-1,3-oxazole and a benzo-1,2-thiazole moiety, respectively, to a quinoline group (Figure 4.1). Their homodimeric relatives YOYO and TOTO consist of two such chromophores connected with a biscationic linker similar to those of the ethidium homodimer, which is capable of bisinter-calating into dsDNA [384]. These dyes have very high extinction coeffi-cients (XH10 cm5 !1&M!1), are virtually non-fluorescent when free in solu-tion, and form highly fluorescent and stable intercalation complexes with dsDNA. These attractive characteristics enabled for the first time DNA to be detected at sensitivity comparable with that of radioactive probes but without the danger inherent in radioactivity [388].
Since the initial report, derivatives with improved characteristics and covering a large range of emission wavelengths in the visible region of the light spectrum have blossomed [389]. Three features enable a fine tuning of the absorption and fluorescence of these dyes: (1) the nature of the heteroatom in the benzoxazole or benzothiazole ring, (2) the size of the other aromatic cycle (pyridine or quinoline), and (3) the length of the methine bridge connecting the two aromatic parts of the molecule. Other substituents and counterions will affect the solubility and the affinity of the dye to DNA, but the electronic transition only to a minor extent. Figure 4.2 shows the fluorescence spectrum of four dyes of this family. If the oxygen in the benzoxazole ring of the YO derivative is substituted for a sulphur atom yielding a TO derivative, the fluorescence is shifted to higher wavelengths. Replacement of the quinoline ring of YO or TO by a
N+
Figure 4.1. Molecular structure of dyes of the oxazole yellow and thiazole orange families.
pyridine ring, yielding BO and PO derivatives respectively (Figure 4.1), causes the fluorescence to move to lower wavelengths. The effect of in-creasing the methine bridge length from a monomethine (YOYO-1, TOTO-1) to a trimethine (YOYO-3, TOTO-3) also leads to a shift of the fluorescence to the red [389]. Most of the monomeric dyes are also avail-able as homodimers [389].
Although some sequence specificity which might lead to inhomogene-ous staining has been demonstrated for both TOTO and, to a lesser ex-tent, YOYO [390-392], these cyanines and some derivatives have found application as general DNA stains in numerous DNA detection and quantitation assays [393], such as the polymerase chain reaction [394, 395], DNA staining and fragment sizing [396-400], DNA damage detec-tion [401, 402], flow cytometry [398, 403, 404], evaluadetec-tion of biological activity [405, 406], DNA imaging [407-410], or DNA photocleavage [411-413]. YO and TO have also been covalently linked to oligonucleotides and inserted into peptide nucleic acids constructs, which become fluores-cent upon hybridisation of the light-up probe to a specific complementary strand [414-420].
Figure 4.2. Intensity-normalised fluorescence spectra of derivatives of BO, PO, YO, and TO (from left to right). The molecular structures of these derivatives are identical to that of their analogues described in Figure 4.1 except for the substituent on the pyridine, respectively quinoline ring.
The question of the way how these dyes interact with DNA has been addressed through a variety of spectroscopic studies and, like many other cyanines, both dimeric (YOYO, TOTO) and monomeric (YO, TO) dyes have been shown to display two binding modes to DNA [421-423]. The first one is bisintercalation between the base pairs (Figure 4.3), which is favoured at high DNA base pair/dye ratio, that is, when the dye concen-tration is low compared with that of the DNA base pairs. The occurrence of this binding mode is supported by circular dichroism (CD) and fluo-rescence anisotropy measurements, and linear dichroism spectra of ori-ented DNA reveal that the bound dyes have a perpendicular orientation relatively to the helical axis [421]. NMR studies of TOTO-1 and YOYO-1 bound to DNA in solution indicate that the chromophore lies across the base pairs with the benzothiazole, respectively the benzoxazole ring located between the pyrimidines and the quinoline moiety located be-tween the purines. Additionally, the two chromophores of TOTO-1 do not lie collinearly on top of each other but make a twist angle of about 83°
[392, 424]. Absorption and CD experiments further suggest that, besides intercalation, groove binding is also operative at low DNA base pair (bp)/dye ratios (data not shown and [421]).
4.1.3. Specific Aims
These dyes owe their ability to operate as DNA probes to the fact that they are essentially non-fluorescent when free in aqueous solution and Figure 4.3. NMR structure of TOTO-1 (grey) bisintercalated into dsDNA (white). Drawn from 108D.pdb [424].
highly fluorescent when bound to DNA. This huge enhancement of the fluorescence quantum yield upon binding to DNA was assumed to origi-nate only in the loss of mobility around the methine bridge due to the constrictive DNA environment [425, 426]. In the free form, the excited dyes can undergo ultrafast non-radiative deactivation through large ampli-tude motion around this bridge. On the other hand, this non-radiative deactivation pathway is no longer operative in DNA where this motion is hindered. This non-radiative decay mechanism, which governs the ex-cited-state lifetime of the free dye, is a common feature of cyanine dyes and has been intensively investigated [427-432]. On the basis of the fluo-rescence quantum yields of the free and bound forms and the radiative lifetime of YOYO-1, this process was predicted to take place with a time constant of 1–5 ps [426]. However, this ultrashort excited-state lifetime of free YOYO-1 had never been confirmed by direct measurements. More-over, the question of the nature of the fluorescence of the free dye has been widely ignored, even for the best-known intercalators such as TO [433, 434].
In the next two sections of this chapter, the excited-state dynamics of several homodimeric (section 4.2) and monomeric (section 4.3) oxazole yellow derivatives are reported. Ultrafast isomerisation will be confirmed as an excited-state decay mechanism and disruption of the excitonic inter-action in molecular aggregates of these dyes upon binding to DNA will be revealed as an additional important fluorescence contrast mechanism.
The potential of these dyes as local environmental probes will further be established. Section 4.4 describes how the fluorescence contrast between the free and bound form of the oxazole yellow and thiazole orange dye families could be optimised by fine-tuning the dye molecular structure. In section 4.5, investigations of the sensitivity of this class of dyes to local changes in their environment is extended to different nucleic acid con-formations and short DNA strands of known sequence and the strength of fluorescence anisotropy experiments with YOYO-1 to gain local struc-tural information is demonstrated. Finally, section 4.6 reports on a new chiral dye which has a structure suitable for an interaction with DNA and the photophysical properties of which are explored with and without DNA. Unless specified, the DNA used throughout the experiments is
salmon sperm double-stranded DNA (simply termed DNA) of heteroge-neous length and unknown sequence.
The general aims of the investigations described in this chapter are to get a detailed account of the photophysics of the dyes that are used and to explore how nucleic acids, in particular DNA, may influence the photo-physics. With this, it is hoped to strengthen the power of the combined use of local environmental probes and time-resolved spectroscopic tech-niques, in particular fluorescence, to monitor local variations in the dye structure and environment which can directly be related to changes in the geometry of the nucleic acid-dye complex.