New hairpin-structured DNA probes: alternatives to classical molecular beacons

Abstract

In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.

Detection of Several Homologous MicroRNAs by a Single Smart Probe System Consisting of Linear Nucleic Acid Blockers

“…If a nucleic acid sequence has a single-base mutation in it, the SP can differentiate between this mismatch and the target sequence by providing a relatively lower fluorescence signal compared to that of the perfect target, which indicates a less stable hybrid between the SP and the mismatch sequence. This exquisite signaling property makes SPs effective analytical tools in various applications, such as monitoring polymerase chain reactions (PCR), DNA polymerase fidelity, T4 polynucleotide kinase activity, adenosine triphosphate (ATP) detection, detection of UV-induced nucleic acid photodamage, genetic testing, and biomedical diagnostics [8,9,10,11,12,13]. SPs are structurally similar to molecular beacons (MBs), except that guanine bases are used in SPs to replace an extrinsic quencher label used in MBs.…”

Design and Characterization of a Singly Labeled Fluorescent Smart Probe for In Vitro Detection of miR-21

“…Smart probes are nucleic acid hairpin-shaped probes that possess inherent signaling property due to their hairpin conformation. [25][26][27][28][29][30] They consist of a stem-loop structure, with a fluorophore on one end of their nucleic acid sequence and a quencher on the other. The structure of the SP is analogous to molecular beacon hairpins (MBs), 31,32 but the quencher in MBs is replaced with multiple guanine residues.…”

“…The structure of the SP is analogous to molecular beacon hairpins (MBs), 31,32 but the quencher in MBs is replaced with multiple guanine residues. [25][26][27][28][29][30] Thus, in contrast to MBs, SPs only have one extrinsic label (the fluorophore) on one end of the stem, while quenching is achieved using intramolecular guanine residues on the other end of the stem hybrid. 25,26 One major advantage of SP over MB is that probes that are inadvertently left unlabeled with extrinsic quencher (as is possible in the case of MB) do not contribute to the measured signal.…”