Dye selection for live cell imaging of intact siRNA

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.     

FRET-based probing to gain direct information on siRNA sustainability in live cells: Asymmetric degradation of siRNA strands

Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.     

Fluorescence resonance energy transfer-based stoichiometry in living cells

Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photobleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks.     

siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.     

Intracellular stability of 2'-OMe-4'-thioribonucleoside modified siRNA leads to long-term RNAi effect

Chemically modified siRNAs are expected to have resistance toward nuclease degradation and good thermal stability in duplex formation for in vivo applications. We have recently found that 2'-OMe-4'-thioRNA, a hybrid chemical modification based on 2'-OMeRNA and 4'-thioRNA, has high hybridization affinity for complementary RNA and significant resistance toward degradation in human plasma. These results prompted us to develop chemically modified siRNAs using 2'-OMe-4'-thioribonucleosides for therapeutic application. Effective modification patterns were screened with a luciferase reporter assay. The best modification pattern of siRNA, which conferred duration of the gene-silencing effect without loss of RNAi activity, was identified. Quantification of the remaining siRNA in HeLa-luc cells using a Heat-in-Triton (HIT) qRT-PCR revealed that the intracellular stability of the siRNA modified with 2'-OMe-4'-thioribonucleosides contributed significantly to the duration of its RNAi activity.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer

Small catalytic RNAs like the hairpin ribozyme are proving to be useful intracellular tools; however, most attempts to demonstrate trans-cleavage of RNA by ribozymes in cells have been frustrated by rapid cellular degradation of the cleavage products. Here, we describe a fluorescence resonance energy transfer (FRET) assay that directly monitors cleavage of target RNA in tissue-culture cells. An oligoribonucleotide substrate was modified to inhibit cellular ribonuclease degradation without interfering with ribozyme cleavage, and donor (fluorescein) and acceptor (tetramethylrhodamine) fluorophores were introduced at positions flanking the cleavage site. In simple buffers, the intact substrate produces a strong FRET signal that is lost upon cleavage, resulting in a red-to-green shift in dominant fluorescence emission. Hairpin ribozyme and fluorescent substrate were microinjected into murine fibroblasts under conditions in which substrate cleavage can occur only inside the cell. A strong FRET signal was observed by fluorescence microscopy when substrate was injected, but rapid decay of the FRET signal occurred when an active, cognate ribozyme was introduced with the substrate. No acceleration in cleavage rates was observed in control experiments utilizing a noncleavable substrate, inactive ribozyme, or an active ribozyme with altered substrate specificity. Subsequently, the fluorescent substrates were injected into clonal cell lines that expressed cognate or noncognate ribozymes. A decrease in FRET signal was observed only when substrate was microinjected into cells expressing its cognate ribozyme. These results demonstrate trans-cleavage of RNA within mammalian cells, and provide an experimental basis for quantitative analysis of ribozyme activity and specificity within the cell.     

Surveillance of siRNA integrity by FRET imaging

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.     

High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy

Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.     

Improvements in siRNA properties mediated by 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)

RNA interference (RNAi) has emerged recently as an efficient mechanism for specific gene silencing. Short double-stranded small interfering RNAs (siRNAs) are now widely used for cellular or drug target validation; however, their use for silencing clinically relevant genes in a therapeutic setting remains problematic because of their unfavourable metabolic stability and pharmacokinetic properties. To address some of these concerns, we have investigated the properties of siRNA modified with 2'-deoxy-2'-fluoro-beta-d-arabinonucleotide units (araF-N or FANA units). Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA. We also show that the incorporation of FANA units into siRNA duplexes increases activity and substantially enhances serum stability of the siRNA. A fully modified sense 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) strand when hybridized to an antisense RNA (i.e. FANA/RNA hybrid) was shown to be 4-fold more potent and had longer half-life in serum (approximately 6 h) compared with an unmodified siRNA (<15 min). While incorporation of FANA units is well tolerated throughout the sense strand of the duplex, modifications can also be included at the 5' or 3' ends of the antisense strand, in striking contrast to other commonly used chemical modifications. Taken together, these results offer preliminary evidence of the therapeutic potential of FANA modified siRNAs.     

siRNA function in RNAi: a chemical modification analysis

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.     

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNA interference (RNAi). Here, we describe a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinity-tagged siRNAs, we demonstrate that a single-stranded siRNA resides in the RNA-induced silencing complex (RISC) together with eIF2C1 and/or eIF2C2 (human GERp95) Argonaute proteins. RISC is rapidly formed in HeLa cell cytoplasmic extract supplemented with 21 nt siRNA duplexes, but also by adding single-stranded antisense RNAs, which range in size between 19 and 29 nucleotides. Single-stranded antisense siRNAs are also effectively silencing genes in HeLa cells, especially when 5'-phosphorylated, and expand the repertoire of RNA reagents suitable for gene targeting.     

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.     

Fluorescence resonance energy transfer-based technologies in the study of protein-protein interactions at the cell surface

Understanding of the molecular mechanisms of protein-protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed to characterize plasma membrane PPIs. Importantly, these technologies alone - or in concert with complementary methods (i.e. SNAP-tag/TR-FRET, TIRF/FRET) - can become extremely powerful approaches for visualizing cell surface PPIs, even between more than two proteins and also in native tissues. Interestingly, these methods would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Accordingly, herein we provide a thorough assessment on all biotechnological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied in the study of PPIs occurring at the cell surface of living cells.     

Near-infrared fluorescent oligodeoxyribonucleotide reporters for sensing NF-kappaB DNA interactions in vitro

Two types of reporters for optical sensing of NF-kappaB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3'-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-kappaB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease.The incubation of 800CW-Cy reporter in the presence of control or IL-1beta treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor-acceptor fluorochrome pair. NF-kappaB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex.     

Functional comparison of single- and double-stranded siRNAs in mammalian cells

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.     

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.     

Biodistribution of phosphodiester and phosphorothioate siRNA

Short interfering RNAs (siRNAs) are valuable tools for analyzing protein function in mammalian cell culture. This success has led to high expectations for in vivo and therapeutic applications. However, the pharmacokinetic properties of siRNA are not known. Here we report the biodistribution of a phosphodiester (PO) siRNA duplex and examine the effect of phosphorothioate (PS) linkages. Our findings indicate that biodistribution of siRNA is similar to that for single-stranded antisense oligonucleotides and offer insights for use of siRNA in vivo.