DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel‐based assay

Abstract

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.

Energetics, Conformation, and Recognition of DNA Duplexes Modified by Monodentate RuII Complexes Containing Terphenyl Arenes

“…A plausible explanation of this observation is that the intercalators thermodynamically stabilize DNA because they lengthen and unwind DNA, increasing the phosphate spacing along the helix axis. [42,43] Also consistent with the intercalation of 1 and associated smaller thermodynamic destabilization of duplexCAL by the adduct of this complex is the observation that a heat capacity change for formation of the duplexCAL (DC p ) that contained an adduct of 1 was markedly more negative than that for formation of the duplexCAL that contained an adduct of 2 ((À632 AE 29) vs.…”

Capillary gel electrophoresis of therapeutic oligonucleotides – Analysis of single‐ and double‐stranded forms

“…At ambient temperature, for ds ODNs of 20-30 base pairs, the relaxation time of the melting/association reaction is in the order of seconds to minutes for the slow relaxation processes investigated so far, which gives the main contribution to the melting/association reaction [32,34]. This indicates that the relaxation time of reaction is in the time scale of separation.…”

“…The transition process between the ss and ds forms of DNA was studied extensively by applying different physicochemical and spectroscopic techniques [31][32][33][34]. As a firstorder approximation one may treat the association/melting process between the two different single strands and the double strand with a two state model ''all or none'' [31], following first-order kinetics [33,34] and neglecting the high complexity of the process due to several annealing configuration of the ss forms [29,35], as follows:…”

“…Horváth in the early 1990s [30]. The dimensionless Damköhler number (Da) of the dsDNA follows a first-order reversible reaction [31][32][33][34] similar to [30]. A schematic presentation of the interplay between the Da and the interference degree of the interconverting species for the dsDS during separation is shown in Fig.…”

Causes of bimodal melting curve:Asymmetric guanine-cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes

“…Considering Blake and Delcourt (1998) findings that the thermodynamic data used in the simulations for base pair stabilities or base stacking are based on empirical data determined for specific salt concentrations in the absence of any intercalating dyes, the difference between the experimental and predicted Tm can be expected. Based on the nature of the interaction between intercalating dyes and dsDNA (Bjorndal and Fygenson, 2002), it would be expected that intercalating dyes are likely to increase the Tm (Rasmussen et al, 2007).…”