Molecular Assembly of Superquenchers in Signaling Molecular Interactions

Abstract

We have designed a novel molecular assembly of quencher molecules to form Superquenchers with excellent quenching efficiency. The Superquencher can be engineered as desired by assembling different types and different numbers of quencher molecules. By labeling a Superquencher to a molecular beacon, a 320 folds enhancement of fluorescent signal was achieved, compared to about 14-fold from a molecular beacon prepared with the same monomer quencher. Our molecular assembly approach can effectively improve the sensitivity of a variety of fluorescent assays and can be widely useful for molecular interaction studiesWe have designed a molecular assembly of superquenchers (SQs) for molecular interaction studies and for ultrasensitive bioanalysis. Signaling biomolecular interactions such as DNA/ RNA hybridization and protein interactions is critically important in areas such as medical diagnosis, disease prevention, and drug discovery. While selectivity in biomolecular recognition can be achieved by capitalizing on highly selective molecular interactions such as antibody-antigen binding and DNA base paring, the effectiveness of a molecular probe is highly dependent on the scheme and the ability to transduce a target recognition event into a measurable signal. Of many approaches, fluorescence is one of the most effective ways to signal molecular interaction and recognition. For instance, fluorogenic probes, such as Taqman, 1 molecular beacons (MB), 2;3 and protease probes 4 have been widely used for biotechnological research and development. The interaction of these molecular probes with target molecules renders unquenching of the fluorophores, yielding detectable fluorescence signal change. While exploited in broad areas such as real-time PCR monitoring, DNA assays, and protein studies, these probes have limited increment of signal change upon interacting with their targets, mainly due to an unquenched high background signal from the probe itself. Strategies for improving signal-to-background ratio of molecular probes promise higher assay sensitivity as well as better reproducibility. There has been encouraging progress in attempts at introducing novel signaling schemes, 5;6 exploring nanocomposites 7;8 , as well as making better quenchers using rational molecular design coupled with sophisticated synthesis. 9-11 Herein, we have molecularly assembled an array of quencher molecules to produce SQs for use in engineering fluorogenic molecular probes with high sensitivity and specificity. We believe that using multiple quenchers to pair with one fluorophore provides better quenching efficiency because of the improved absorption efficiency and the increased probability of dipole-dipole coupling between the quenchers and the fluorophore because of a collective quenching effect of these multiple quenchers. MB was used as a model here to test the performance of the SQs. An excellent example of fluorogenic probe, a MB 2;3;12 is a hairpin shaped DNA with a self-complementary stem that brings terminal labeled fluorophore and ...

Molecular Beacon Lighting up on Graphene Oxide

“…For the quencher role, superquenchers and metal nanoparticles have been tested. [9][10][11] In addition to improving photophysical properties, using nanomaterials as MB components offers several other advantages. First, such MBs might be easily separated, allowing for operations unique to heterogeneous assays such as washing and signal amplification.…”

Triplex Molecular Beacons as Modular Probes for DNA Detection

“…Furthermore, the modular assembly [5] facilitates the additional incorporation of functionalities that can be easily introduced via the sfO. In an example of this concept, we prepared "superquenched" [6] tMBs by appending additional quencher moieties to the sfO.…”

“…As an example, we constructed "superquenched" [6] tMBs by attaching additional Dabcyl quencher moieties to sfO 7 a (7 a* and 7 a**, Figure 4 A). The aim was to increase the fluorescence enhancement upon target addition by reducing the fluorescence of the closed tMB.…”

Efficient Quenching of Oligomeric Fluorophores on a DNA Backbone

“…8,9,13 Subsequent experiments showed that efficient quenching is not specific to oligomers of pyrene, and can be observed with other π-stacking fluorophores. Sequences containing benzopyrene and perylene in addition to pyrene (6,7) demonstrated fluorescent excited-state complexes (exciplexes) that were also highly sensitive to quenching by MV (Table 1). Significantly, in cases where both monomer and exciplex emission was present, the delocalized exciplex emission was much more efficiently quenched.…”