Nucleic acids-based detection and quantiWcation methods play an important role in the medical diagnostics and drug discovery. Determining the presence of a speciWc DNA sequence in a homogeneous mixture requires hybridization with Xuorescently labeled oligonucleotides in solution. Recently, hairpin oligonucleotide probes, each of which consists of a Xuorophore at one of the termini and a quencher at the other end, have proven to be eVective in conventional molecular beacons [1]. The molecular beacon possesses a stem–loop-like structure and generally is dual-labeled, with a Xuorophore (a donor dye) at the 5 end and another dye (an acceptor dye that acts as a quencher) at the 3 end of the stem part that may or may not be Xuorescent. The presence of the speciWc target nucleic acid sequences, complementary to the loop part, is detected by a strong increase in the Xuorescence intensity on hybridization, thereby making the homogeneous assay more sensitive and reliable. Thus, the strategy requires labeling at both ends of a single-stranded oligonucleotide probe with speciWc Xuorescent dyes that suVer in overall yield and are expensive. To avoid the limitations that molecular beacons face, hairpin probes have been designed. This kind of probe uses the Xuorescent label at one end and a nonXuorescent molecule (e.g., nucleobase) at the other end and is able to work as an acceptor as well as to quench the Xuorescence intensity of the Xuorophore in the closed-state conformation. For this purpose, a number of unique (rather than traditional) nonXuorescent quenchers, ranging from DNA nucleotides (e.g., guanosine, deazaguanosine) to gold nanoparticles, have already been introduced successfully [2–7]. Furthermore, instead of using interactions between the two extrinsic probes, interactions of Xuorophores with DNA nucleobases or amino acids have potential for the speciWc detection of DNA or RNA sequences and antibodies at the single molecular level [8–17]. In the current study, in contrast to molecular beacons, only the 5 end-labeled hairpin probe (viz. FAM-C3catcAAAAAAAAAAAAAAAAAAAAgatggg-3) was syn