Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells

Numerous studies have utilized molecular beacons (MBs) to image RNA expression in living cells; however, there is growing evidence that the sensitivity of RNA detection is significantly hampered by their propensity to emit false-positive signals. To overcome these limitations, we have developed a new RNA imaging probe called ratiometric bimolecular beacon (RBMB), which combines functional elements of both conventional MBs and siRNA. Analogous to MBs, RBMBs elicit a fluorescent reporter signal upon hybridization to complementary RNA. In addition, an siRNA-like double-stranded domain is used to facilitate nuclear export. Accordingly, live-cell fluorescent imaging showed that RBMBs are localized predominantly in the cytoplasm, whereas MBs are sequestered into the nucleus. The retention of RBMBs within the cytoplasmic compartment led to >15-fold reduction in false-positive signals and a significantly higher signal-to-background compared with MBs. The RBMBs were also designed to possess an optically distinct reference fluorophore that remains unquenched regardless of probe confirmation. This reference dye not only provided a means to track RBMB localization, but also allowed single cell measurements of RBMB fluorescence to be corrected for variations in probe delivery. Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells.     

Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers

In order to gain deeper insight into the functions and dynamics of RNA in cells, the development of methods for imaging multiple RNAs simultaneously is of paramount importance. Here, we describe a modular approach to image RNA in living cells using an RNA aptamer that binds to dinitroaniline, an efficient general contact quencher. Dinitroaniline quenches the fluorescence of different fluorophores when directly conjugated to them via ethylene glycol linkers by forming a non-fluorescent intramolecular complex. Since the binding of the RNA aptamer to the quencher destroys the fluorophore-quencher complex, fluorescence increases dramatically upon binding. Using this principle, a series of fluorophores were turned into fluorescent turn-on probes by conjugating them to dinitroaniline. These probes ranged from fluorescein-dinitroaniline (green) to TexasRed-dinitroaniline (red) spanning across the visible spectrum. The dinitroaniline-binding aptamer (DNB) was generated by in vitro selection, and was found to bind all probes, leading to fluorescence increase in vitro and in living cells. When expressed in E. coli, the DNB aptamer could be labelled and visualized with different-coloured fluorophores and therefore it can be used as a genetically encoded tag to image target RNAs. Furthermore, combining contact-quenched fluorogenic probes with orthogonal DNB (the quencher-binding RNA aptamer) and SRB-2 aptamers (a fluorophore-binding RNA aptamer) allowed dual-colour imaging of two different fluorescence-enhancing RNA tags in living cells, opening new avenues for studying RNA co-localization and trafficking.     

Thermodynamic and Kinetic Characterization of Antisense Oligodeoxynucleotide Binding to a Structured mRNA

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit β-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.     

A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization

We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5–11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP–FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA–RNA interactions.     

Formation of the double helix: a mutational study

To investigate the mechanisms by which oligonucleotides hybridize to target molecules, the binding of two oligodeoxynucleotide probes to RNA targets was measured over a broad range of temperatures. Mutations were then scanned across each DNA/RNA hybrid to map, at single base resolution, sequences important for hybridization. Despite being unrelated in sequence, each hybrid formed by a similar mechanism. In the absence of secondary structure, two stretches of bases, termed nucleation regions, cooperated with one another by a looping mechanism to nucleate hybridization. Mutations inside each nucleation region strongly decreased hybridization rates, even at temperatures well below the melting temperature (T_m) of the hybridized duplex. Surprisingly, nucleation regions were detected in a RNA target but not a corresponding DNA target. When either nucleation region was sequestered in secondary structure, the hybridization rate fell and the mechanism of hybridization changed. Single-stranded bases within the nucleation region of the probe and target first collided to form a double helix. If sufficiently G + C rich, the double helix then propagated throughout the oligonucleotide by a strand invasion process. On the basis of these results, general mechanisms for the hybridization of oligonucleotides to complementary and mutant targets are proposed.