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Fluorescence Resonance Energy Transfer using molecular beacon as a probe, a new approach for in vivo macromolecular interaction study

Sandipan Ganguly
09/16/2015
Modern Research and Educational Topics in Microscopy

Molecular Beacons are useful probes for the visualization of the location of small nuclear ribonucleic acids (snRNA) and fibrillarin proteins in eukaryotic cells.


A molecular beacon is a hairpin-shaped oligonucleotide based hybridization probe. 
Molecular beacons allow for the detection of specific nucleic acid sequences in homogenous solutions. These hairpin shaped molecules contain quenched fluorophors. When the beacon probe binds to the target sequence, the fluorescence of the probe is restored thereby allowing for the detection of the target molecule by monitoring the fluorescence of the hybridization probe.

Confocal microscopy is a powerful optical imaging technique that enables the reconstruction of three-dimensional structures from images. Confocal microscopy is a very useful technique for the study of functional aspects of cell compartments such as the localization and distribution of proteins, DNA, and RNA within cells.

Ganguly et al. in 2004 reported a strategy in which antisense molecular beacons were used to study the interactions of small ribonucleoprotein particle (RNPP) complexes and their formation. The research group applied antisense molecular beacon based Fluorescence Resonance Energy Transfer (FRET) and Flow Cytometric Energy Transfer (FCET) techniques to demonstrate the binding and co-localization of the fibrillarin protein with small nuclear RNA (snRNA) molecules. To study the localization of the fibrillarin protein and snRNA molecules that form the ribonucleoprotein particle (RNPP) complex in Giardia lamblia, confocal microscopy and a flow cytometric energy transfer technique was used. The researchers were able to demonstrate that antisense molecular beacon based FRET and FCET enables in situ detection of RNA-protein complex formation in Giardia lamblia. Their data indicated that snRNA-fibrillarin interact to form a complex during preRNA processing. This processing step is required in the formation of ribosomal RNA. Furthermore, the scientists argued that this technique will be very useful for in situ analysis of RNA-protein interaction in other systems as well.

Fibrillarin is a small nucleolar protein found in eukaryotes that plays an important role in pre-rRNA processing during ribosomal biogenesis. Recent research indicates that fibrillarin plays a critical role in the maintenance of the nuclear shape and cellular growth in eukaryotes.

Molecular beacons are designed as single-stranded oligonucleotides that contain a hairpin structure. The free or not hybridized molecular beacon is not fluorescent. This is because the hairpin stem keeps the fluorophore and the quencher moiety in close proximity. However, the fluorescence is restored after the probe sequence in the hairpin loop hybridizes to the target. The formation of a rigid double helix causes a conformational change that removes the quencher moiety from the vicinity of the fluorophore. The hybridization reaction restores the fluorescence of the fluorophore moiety in the molecular beacon. Several fluorophore/quencher pairs are available for the synthesis of molecular beacons.

Reference

Amin MA, Matsunaga S, Ma N, Takata H, Yokoyama M, Uchiyama S, Fukui K.; Fibrillarin, a nucleolar protein, is required for normal nuclear morphology and cellular growth in HeLa cells. Biochem Biophys Res Commun. 2007 Aug 24;360(2):320-6. Epub 2007 Jun 25.

Ganguly, S., Ghosh, S., Chattopadhyay, D., and Das, P.; Antisense Molecular Beacon Strategy for In Situ Visualization of snRNA and Fibrillarin Protein Interaction in Giardia lambilia. RNA Biol. 2004 May; 1(1): 48-53.