Sequence-specific, self-reporting hairpin inversion probes

Sequence-specific probes for detecting target nucleic acids are the cornerstone of the genomics revolution (e.g., microarrays) and of molecular diagnostics. Molecular beacons are self-reporting, nucleic acid probes whose structure includes complementary terminal arm sequences and a loop that is complementary to a target sequence; fluorescence detection is by changes in proximity of fluorophore and quencher pairs attached on opposite arms. However, molecular beacon design is not as simple as attaching arbitrary arm sequences onto previously designed linear probes. The stem arms can also interact with flanking target sequences, changing the hybridization specificity; constantly adapting the arms to avoid such interactions, if not desired, increases design complexity. Herein, I report the use of inversion linkages in probe backbones leading to stem arms of sequence polarity opposite to that of the target-binding region, thereby eliminating potential hybridization of the arms with the target. Using two microbial sequence categories, thermal denaturation and target titration analyses demonstrate that these new hairpin inversion probes retain closed-state stability comparable to that of molecular beacons, contain easily designed arm sequences that do not interact with targets, and, therefore, can be used universally with optimized linear probe sequences.