Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Abstract

Double‐stranded DNA offers multiple binding sites to DNA stains. Measurements of noncovalently bound dye–nucleic acid complexes are, necessarily, measurements of an ensemble of chromophores. Thus, it is difficult to assign fluorescence properties to base‐pair‐specific binding modes of cyanine dyes or, vice versa, to obtain information about the local environment of cyanines in nucleic acids by using optical spectroscopy. The feasibility to stain DNA and simultaneously probe local perturbations by optical spectroscopy would be a valuable asset to nucleic acid research. So‐called FIT probes (forced intercalation probes) were used to pinpoint the location of the DNA stain thiazole orange (TO) in PNA⋅DNA duplexes. A detailed analysis of the base‐pair dependence of optical properties is provided and enforced binding of TO is compared with “classical” binding of free TO‐PRO1. UV‐visible absorbance, circular dichroism (CD) and fluorescence spectroscopy, and melting‐curve analyses confirmed site‐specific TO intercalation. Thiazole orange exhibited base‐specific responses that are not observed in noncovalent dye–nucleic acid complexes, such as an extraordinary dependence of the TO extinction coefficient (±60 % variation of the averaged εmax of 57 000 M−1 cm−1) on nearest‐neighbor base pairs. TO signals hybridization, as shown by increases in the steady‐state fluorescence emission. Studies of TO fluorescence lifetimes in FIT–PNA and in DNA⋅DNA and PNA⋅DNA complexes highlighted four different fluorescence‐decay processes that may be closed or opened in response to matched or single‐mismatched hybridization. A very fast decay process (0.04–0.07 ns) and a slow decay process (2.33–3.95 ns) provide reliable monitors of hybridization, and the opening of a fast decay channel (0.22–0.48 ns) that resulted in an attenuation of the fluorescence emission is observed upon the formation of mismatched base pairs.

Structure–Fluorescence Contrast Relationship in Cyanine DNA Intercalators: Toward Rational Dye Design

“…[29][30][31] YO and TO have also been covalently linked to oligonucleotides and inserted into peptide nucleic acids constructs, which become fluorescent upon hybridisation of the light-up probe to a specific complementary strand. [32][33][34][35][36][37][38] The origin of the very high contrast in emission between the free and the bound form of the dyes has long been thought to be due to an ultrafast decay of the excited state of the free form through a large-amplitude torsional motion around the monomethine bond connecting the benzoxazole, benzothiazole and quinoline moieties, respectively. [39,40] This isomerisation mechanism is blocked upon intercalation of the dyes into DNA and, as a consequence, they light-up by three orders of magnitude.…”

Comparing Agent‐Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

“…As a result, the brightness of TO emission is higher in the DNA–RNA duplex formed upon hybridization of FIT‐DNA than that in the PNA–RNA duplex formed with FIT‐PNA. This also is in agreement with previous studies, which have shown that the nonconjugated TO dye is a rather modest stain of PNA–DNA duplexes …”

Enhanced Binding Affinity of siRNA Overhang‐Binding Fluorescent Probes by Conjugation with Cationic Oligopeptides for Improved Analysis of the siRNA Delivery Process

“…The excitation wavelength of the probes was set at the absorption maximum wavelength of the TO unit in the absence of target siRNAs. Fluorescence quantum yield ( Φ ) was determined relative to fluorescein, for which the probe in free or siGL2‐bound states was examined ([probe]=200 n m , [siGL2]=2.0 μ m ).…”