Stem-Loop Oligonucleotides as Hybridization Probes and Their Practical Use in Molecular Biology and Biomedicine

Abstract

As originally designed, stem-loop (SL) hybridization probe is a single-stranded oligonucleotide containing a sequence complementary to the target that is flanked by inverted repeats forming self-complementary termini, and harbors a fluorophore–quencher pair at the 5ʹ- and 3ʹ-ends.When the target is missing, these molecules form closed pan handle-like structures in which the fluorophore and quencher are located in a close proximity, due to 5ʹ- and 3ʹ-termini spatial neighborhood. Such a quencher proximity represses fluorescence. In contrast, in the presence of the target, probe forms a complex with it, which spatially separates the fluorophore from the quencher. Once the fluorophore and quencher are dissociated, the fluoresce increases, thus enabling quantitative measuring of signals with the threshold value evidencing presence of the target. Alternatively, SL probe can be fluorescence resonance energy transfer (FRET)-labeled, with the fluorescence shift message being monitored in the latter case. SL probes are currently in use in a number of applications, primarily for specific nucleic acid motif detection and in real-time polymerase chain reaction (PCR). Also, SL probes can be utilized in protein–DNA interaction studies, and even for specific inorganic ion detection. This quickly evolving group of methods gave rise to at least 40 international patents and is cited in ~50 peer-reviewed papers annually.