Probes for Detection of Specific DNA Sequences at the Single-Molecule Level

Abstract

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.

Probing conformational dynamics in single donor-acceptor synthetic molecules by means of photoinduced reversible electron transfer

“…Hence, the decay of C 2 (t) within tens of milliseconds time scale that we detect in both low and high molecular weight polymers reflects through-bond D-A coupling changes induced by libration. Conformational changes taking place in millisecond time scale and leading to D-A coupling (distance) changes were previously detected in single flavin reductase͞FAD complexes (11). The reversible switch between different donor moieties is a process dictated by the polymer chain motion and contributes to the decaying of C 2 (t) in the seconds time scale.…”

“…However, whereas highly efficient FRET results in fluorescence emitted mainly from the acceptor fluorophore, highly efficient ET usually leads to a strong quenching of the fluorescence of the emitting chromophore. Thus, reports on single-molecule photoinduced ET are rather limited (10)(11)(12)(13)(14), in contrast to reports on singlemolecule FRET. A particular situation of ET refers to the case in which the locally excited state (LES) and the charge-separated state (CSS) are relatively close in energy (Fig.…”

Direct Detection of Mutant DNA in a Mixed Population of Higher Copy Number Wild-Type DNA Based on Ligase Detection Reaction in Conjunction with Fluorescence Resonance Energy Transfer

“…Several fluorescence resonance energy transfer (FRET)-based molecular probes, [8][9][10][11][12][13] whose fluorescence signals change as a result of hybridization or enzymatic reactions, have been developed to enable separation-free detection of DNA. Here, we report a LDR/FRET system capable of detecting mutated DNA in a mixed population of higher copy number wild-type DNA in a separation-free format.…”

High-speed FLIM data acquisition by time-correlated single-photon counting

“…Thus, FLIM is a direct approach to measure all effects that change the quantum efficiency of a fluorophore. Typical examples are mapping of cell parameters such as pH, ion concentrations or oxygen saturation by fluorescence quenching [9][10][11][12] , as well as probing protein or DNA structures by lifetime sensitive dyes 13,14 .…”