Molecular engineering of DNA: molecular beacons

Abstract

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single-nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal-transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem-loop structure holds the fluorescence-donor and fluorescence-acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein-DNA interaction studies, and protein recognition.

Figure 1

Working mechanism of a molecular beacon. The MB adopts a stem–loop structure and thus holds the fluorophore (orange) and quencher (blue) in close proximity. As a result, the fluorescence emission of the fluorophore is strongly suppressed (in the absence of a target). The target sequence hybridizes with the loop domain of the MB and forces the stem helix to open, whereupon fluorescence is restored because of the spatial separation of the fluorophore from the quencher.

Figure 2

Composition of an LNA-MB. The sugar backbone of the LNA forms a rigid C3′ conformation owing to a 2′-O, 4′-C methylene bridge.

Figure 3

Sticky-end pairing of MBs. Two complementary sticky ends from two MB hybrids can pair to form a short double helix, so that two hybrids are associated at one end. These two MB hybrids can form a closed structure, [MB]₂, through pairing of the other two sticky ends, or polymerize into a larger structure, [MB]_n (n > 3), by pairing with more hybrids. Sticky-end pairing draws the fluorophore and quencher together again and thus causes fluorescence quenching.

Figure 4

Application of MBs in enzymatic studies. a) Real-time monitoring of SSB–DNA binding: The MB binds to the SSB protein whereby its structure is disrupted and its fluorescence is restored. b) Detection of the enzymatic digestion of DNA: The enzyme cleaves the MB and destroys the hairpin structure to restore fluorescence. c) Detection of LDH–DNA interactions: LDH binds the MB and disturbs its structure, whereby fluorescence is enhanced.

Figure 5

Application of MBs in phosphorylation and ligation studies. a) Real-time monitoring of nucleic acid ligation: Two oligonucleotides that are complementary to opposite halves of the MB loop hybridize with the MB, whereby a nick is formed, and the stem may be opened slightly. The DNA ligase binds to the nick and catalyzes the ligation of the two short oligonucleotides to form a longer oligonucleotide. The ligation product hybridizes with the MB to restore fluorescence. b) Monitoring of nucleic acid phosphorylation: Oligonucleotide A is first phosphorylated at the 5′-hydroxy group by the polynucleotide kinase. The nick formed upon the hybridization of oligonucleotide B and phosphorylated oligonucleotide A with MB can be sealed by the DNA ligase, whereupon the stem helix of the MB is opened, and fluorescence is restored.

Figure 6

Simultaneous detection of multiple mRNAs inside a living cell. Time-lapsed fluorescence images of MBs inside a single MDA-MB-231 cell are shown. A: MB for β-actin (green); B: control MB (red); C: MB for MnSOD (blue); D: tris(2,2′-bipyridyl)ruthenium(II) (RuBpy) reference probe (orange).

Figure 7

Living neuron into which MBs with polyU sequences were injected. The green fluorescence is caused by opening of the MBs during neuronal processes.

Figure 8

Superquencher MBs. Signal-to-background ratio of the MBs with one (1-Q-MB), two (2-Q-MB), or three (3-Q-MB) quenchers The MBs have the same sequence and are labeled at the 3′ end with fluorescein as the fluorophore. One, two, or three dabcyl molecules were attached to the 5′ end of the oligonucleotide. The MB containing three quenchers produced a more than 320-fold signal enhancement upon hybridization; a 14-fold signal enhancement was observed with the single-quencher MB.

Figure 9

PPE-MBs Top: Working principle behind. a) a conventional MB and b) a conjugated-polymer-labeled MB. In the conventional MB, only one fluorophore is used to report a target-binding event, whereas in the conjugated-polymer-labeled MB, a fluorescent polymer chain is used. Bottom: Comparison of the fluorescence intensity of different fluorophores. The excitation/emission wavelengths for Cy3, TMR, FAM, ALX488, PPE, and quantum dots are 543/560, 557/581, 488/514, 488/515, 440/520, and 400/520 nm, respectively. The concentration of all dyes is 10 nm

Figure 10

Stern–Volmer plot of the fluorescence quenching of PPE by dabcyl in glycine–HCl buffer (40 mm, pH 2.3). The buffer was used to protonate dabcyl as a counterion to the polymer PPE.