The Light-up Probe
Doctoral thesis, 2000
The work presented in this thesis concerns the development of light-up probes and their potential applications in PCR based molecular diagnostics. The light-up probe is a nucleic acid analog (here PNA) oligomer linked to an asymmetric cyanine dye (here thiazole orange) and it combines the excellent hybridization properties of PNA with the large fluorescence enhancement of the dye upon binding to DNA. Upon hybridization of the probe to denatured target DNA the dye binds to the formed complex, which results in up to 40-fold enhancement in fluorescence intensity. Thus, light-up probes report the presence of specific targets in homogeneous solution. Such constructs are valuable in nucleic acid based diagnostics because they eliminate the need for separation of probed targets from any unbound probes or arbitrary nucleic acids.
In Paper I we study, in detail, the interactions between thiazole orange and various single and double-stranded DNA polymers. The binding properties are studied by recording absorption spectra at various ionic strengths and the spectra are analyzed by a chemometric method to reveal the spectra of free and bound dye as well as the binding constants as functions of the ionic strength. We find that the binding constants and spectroscopic properties vary substantially among the polymers. In Paper II we present thiazole orange based light-up probes and show that these specifically hybridizes to, and report the presence of, single-stranded oligonucleotide targets in homogeneous solution. Eight light-up probes, with various sequences and/or linkers are studied and we find that the fluorescence enhancement upon hybridization varies substantially among the probes. This is primarily due to large variations in fluorescence of the probes in free state, which is mainly determined by the base composition of the PNA oligomer. In general, homopyrimidine probes have very low free-probe fluorescence while some mixed sequence probes have high free-probe fluorescence. This brings about a detailed study of the spectroscopic properties of the probes in free state, which is presented in Paper III. We find that the free-probe fluorescence is due to the fact that the dye interacts intramolecularly with the PNA and that the variation in free-probe fluorescence derives from variations in the extent of 'back-binding', and in the fluorescence quantum yield of the probe in back-bound conformation. Furthermore, we find that the free-probe fluorescence decreases substantially with increasing temperature due to a shift in the equilibrium towards the open conformation and a decrease in the quantum yield of the probe in back-bound conformation. In Paper IV we demonstrate that light-up probes can be used to detect specific PCR products, which eliminates tedious post-PCR analysis using for example gel electrophoresis. In Paper V we also demonstrate that light-up probes can be used to monitor PCR product formation in real-time by designing the probe to bind at the PCR annealing temperature but dissociate during primer extension. This allows for quantification of the initial number of template molecules over a large dynamic range and with high reproducibility.
asymmetric cyanine dyes
fluorescent hybridization probes
homogeneous hybridization assays