Tiny Molecular Beacons: LNA/2'-O-methyl RNA Chimeric Probes for Imaging Dynamic mRNA Processes in Living Cells

New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time. In order to spatially and temporally resolve dynamic steps involving RNA in cells, molecular beacons need to efficiently hybridize to their RNA targets. To expand the repertoire of target sites accessible to molecular beacons, we decreased the length of their probe sequences and altered their backbone by the inclusion of LNA (locked nucleic acid) nucleotides. We named these new LNA/2'-O-methyl RNA chimera oligonucleotides "tiny molecular beacons". We analyzed these tiny molecular beacons and found that the incorporation of just a few LNA nucleotides enables these shorter probes to stably anneal to more structured regions of the RNA than is possible with conventional molecular beacons. The ease of synthesis of tiny molecular beacons and the flexibility to couple them to a large variety of fluorophores and quenchers render them optimal for the detection of less abundant and/or highly structured RNAs. To determine their efficiency to detect endogenous mRNAs in live specimens, we designed tiny molecular beacons that were specific for oskar mRNA and microinjected them into living Drosophila melanogaster oocytes. We then imaged the live oocytes via spinning disk confocal microscopy. The results demonstrate that tiny molecular beacons hybridize to target mRNA at faster rates than classically designed molecular beacons and are able to access previously inaccessible target regions.     

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 FRET molecular beacons for mRNA detection in living cells

The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT–PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.     

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.     

Linear 2′ O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2′ O-Methyl oligoribonucleotides (2′ OMe RNA). Antisense 2′ OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2′ OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA–protein interaction studies.     

Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells

Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)(+)-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25nM to 0.5μM) of poly(A)-RNA, as well as for determination of K(d) values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed.     

Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons

Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 x 10(3.6) 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.     

Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells

Real-time visualization of specific endogenous mRNA expression in vivo has the potential to revolutionize medical diagnosis, drug discovery, developmental and molecular biology. However, conventional liposome- or dendrimer-based cellular delivery of molecular probes is inefficient, slow, and often detrimental to the probes. Here we demonstrate the rapid and sensitive detection of RNA in living cells using peptide-linked molecular beacons that possess self-delivery, targeting and reporting functions. We conjugated the TAT peptide to molecular beacons using three different linkages and demonstrated that, at relatively low concentrations, these molecular beacon constructs were internalized into living cells within 30 min with nearly 100% efficiency. Further, peptide-based delivery did not interfere with either specific targeting by or hybridization-induced fluorescence of the probes. We could therefore detect human GAPDH and survivin mRNAs in living cells fluorescently, revealing intriguing intracellular localization patterns of mRNA. We clearly demonstrated that cellular delivery of molecular beacons using the peptide-based approach has far better performance compared with conventional transfection methods. The peptide-linked molecular beacons approach promises to open new and exciting opportunities in sensitive gene detection and quantification in vivo.     

A molecular beacon assay for monitoring RNA splicing

Small molecule targeting of self-splicing RNAs like group I and II introns has been limited in part by the lack of a universal high-throughput screening platform for studies of splicing inhibition and kinetics. Here, we present the development of a molecular beacon assay for monitoring the accumulation of spliced exons during RNA splicing reactions. In this case, we applied it to the autocatalyzed reaction of the H.c.LSU group II intron found in the mitochondria of the pathogenic dimorphic fungus Histoplasma capsulatum. We find that a molecular beacon with the loop length of 18 nucleotides selectively recognizes ligated exons formed during self-splicing and exhibits high fluorescent signal upon binding of its target. We demonstrate that the fluorescent assay using molecular beacons can be successfully applied to kinetic characterization of the splicing reaction and determination of inhibition constants for small molecules. The results presented herein offer support for a molecular beacon approach to identifying small molecule inhibitors of intron splicing.     

Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells

Imaging products of gene expression in live cells will provide unique insights into the biology of cells. Molecular beacons make attractive probes for imaging mRNA in live cells as they can report the presence of an RNA target by turning on the fluorescence of a quenched fluorophore. However, when oligonucleotide probes are introduced into cells, they are rapidly sequestered in the nucleus, making the detection of cytoplasmic mRNAs difficult. We have shown that if a molecular beacon is linked to a tRNA, it stays in the cytoplasm and permits detection of cytoplasmic mRNAs. Here we describe two methods of linking molecular beacons to tRNA and show how the joint molecules can be used for imaging an mRNA that is normally present in the cytoplasm in live cultured cells. This protocol should take a total of 4 d to complete.     

Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization can provide unprecedented opportunities for biological and disease studies. However, the mRNA detection specificity and sensitivity are critically dependent on the selection of target sequences and their accessibility. We carried out an extensive study of the target accessibility of BMP-4 mRNA using 10 different designs of molecular beacons (MBs), and identified the optimal beacon design. Specifically, for MB design 1 and 8 (MB1 and MB8), the fluorescent intensities from BMP-4 mRNA correlated well with the GFP signal after upregulating BMP-4 and co-expressing GFP using adenovirus, and the knockdown of BMP-4 mRNA using siRNA significantly reduced the beacon signals, demonstrating detection specificity. The beacon specificity was further confirmed using blocking RNA and in situ hybridization. We found that fluorescence signal from MBs depends critically on target sequences; the target sequences corresponding to siRNA sites may not be good sites for beacon-based mRNA detection, and vice versa. Possible beacon design rules are identified and approaches for enhancing target accessibility are discussed. This has significant implications to MB design for live cell mRNA detection.     

Primary and secondary structure of U8 small nuclear RNA

U8 small nuclear RNA is a new, capped, 140 nucleotides long RNA species found in Novikoff hepatoma cells. Its sequence is: m3GpppAmUmCGUCAGGA GGUUAAUCCU UACCUGUCCC UCCUUUCGGA GGGCAGAUAG AAAAUGAUGA UUGGAGCUUG CAUGAUCUGC UGAUUAUAGC AUUUCCGUGU AAUCAGGACC UGACAACAUC CUGAUUGCUU CUAUCUGAUUOH. This RNA is present in approximately 25,000 copies/cell, and it is enriched in nucleolar preparations. Like U1, U2, U4/U6, and U5 RNAs, U8 RNA was also present as a ribonucleoprotein associated with the Sm antigen. The rat U8 RNA was highly homologous (greater than 90%) to a recently characterized 5.4 S RNA from mouse cells infected with spleen focus-forming virus (Kato, N., and Harada, F. (1984) Biochim. Biophys. Acta, 782, 127-131). In addition to the U8 RNA, three other U small nuclear RNAs were found in anti-Sm antibody immunoprecipitates from labeled rat and HeLa cells. Each of these contained a m3GpppAm cap structure; their apparent chain lengths were 60, 130, and 65 nucleotides. These U small nuclear RNAs are designated U7, U9, and U10 RNAs, respectively.     

Molecular beacons: fluorogenic probes for living cell study

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.     

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.     

Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.     

Molecular beacons as probes of RNA unfolding under native conditions

Hybridization of fluorescent molecular beacons provides real-time detection of RNA secondary structure with high specificity. We used molecular beacons to measure folding and unfolding rates of the Tetrahymena group I ribozyme under native conditions. A molecular beacon targeted against 15 nt in the 5′ strand of the P3 helix specifically hybridized with misfolded forms of the ribozyme, without invading the native tertiary structure. The beacon associated with the misfolded ribozyme 300 times more slowly than with an unstructured oligonucleotide containing the same target sequence, suggesting that the misfolded ribozyme core remains structured in the absence of Mg²⁺. The rate of beacon hybridization under native conditions revealed a linear relationship between the free energy of unfolding and Mg²⁺ concentration. A small fraction of the RNA population unfolded very rapidly, suggesting parallel unfolding in one step or through misfolded intermediates.