Quencher-free Linear Beacon Systems with Dual Fluorene-labeled Deoxyuridines

The identification of natural genetic variation in human populations is arguably one of the most important spin-offs of the effort toward the completion of the Human Genome Project. Single nucleotide polymorphisms (SNPs) are the most abundant natural genetic variations in the human genome. To satisfy the demand for molecular diagnostic assays for identifying SNPs, Tyagi and Kramer developed fluorogenic hairpin-shaped DNA probes, called “molecular beacons in 1996. Conventional molecular beacons are single-stranded oligodeoxynucleotides (ODNs) probes that exist in solution as stable stem-and-loop structures in which a fluorophore is covalently linked to one end and a quencher is covalently linked to the other end. The stem keeps the fluorophore and quencher of the molecular beacon in extremely close proximity to each other, when they are not bound to their targets; this causes the fluorescence of the fluorophore to be quenched via the fluorescence resonance energy transfer. By contrast, the conformational change induced by binding to a target nucleic acid causes the fluorophore and the quencher to move away from each other, thereby restoring the fluorescence. Therefore, the presence of the target nucleic acid is characterized by a sharp increase in fluorescence intensity. Recently, we reported that fluorescent hairpin oligonucleotides can function as molecular beacons even without the attachment of additional quencher moieties. Such probes can be called “quencher-free molecular beacons. A quencher-free molecular beacon consists of only a single fluorophore (2-ethynylfluorene-labeled deoxyuridine, U) positioned in the hairpin loop. When this probe undergoes DNA hybridization, it can discriminate between fully matched and single-base-mismatched sequences by exhibiting a strong dA-selective fluorescence. When the fluorene unit is intercalated within a DNA duplex as a result of mismatched base pairing, the fluorescence of the U unit is quenched as a result of a photoinduced charge transfer originating from interactions with neighboring nucleobases. In particular, the quencher-free molecular beacon probe having C-flanking bases (C-FBs) shows more efficient fluorescence ON/OFF behavior than do any other combinations of FBs. The only drawback of this quencher-free molecular beacon system is its poor detection sensitivity due to its relatively high intrinsic emission intensity. To solve this problem, we recently positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite the U unit to reduce the intrinsic fluorescence on hybridization with a probe. A short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective in reducing the intrinsic fluorescence of a linear beacon. In another effort to eliminate this drawback, we employed an ODN containing dual U units at specific distances (Figure 1). We observed that using this dual-labeled linear beacon system resulted in a clear improvement in the detection sensitivity for the perfectly matched target. The modified nucleotide U was synthesized through Sonogashira coupling of a 2-ethynylfluorene unit at the 5position of a 2-deoxyuridine base. Incorporations of the fluorene-labeled deoxyuridine U into the central position of the ODN 1 and of the two U units into the positions with one deoxycytosine spacer unit were effected using standard protocols of automated DNA synthesis (Figure 2). The fluorescence properties of fluorophore-containing DNA are strongly dependent on the electron injection and transfer properties of the FBs. In particular, fluorescent ODNs having C-FBs produce efficient fluorescence ON/OFF systems and have higher sensitivity as probes than do any other combinations of FBs. Therefore, we positioned two deoxy-