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Single-molecule fluorescence imaging of DNA origami-based transporters showed shorter run lengths in dence layouts of kinesin motors.

Misfolding and aggregation of proteins are characteristic features in the progression of neurodegenerative diseases. While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that a widespread family of heat-resistant obscure (Hero) proteins may also play a similar role. However, it is unclear how such electrostatically charged intrinsically disordered proteins protect client proteins from denaturation and aggregation. Here we utilize single-molecule FRET to monitor the conformations of an aggregation-prone protein, TDP-43. While we observed high conformational heterogeneity of wild-type TDP-43, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, a canonical passive chaperone, DNAJA2, and a Hero protein, Hero11, both of which prevent TDP-43 aggregation in cells, promoted extended conformations. Our results link single-molecule effects on the TDP-43 conformation to macro effects on bulk aggregation propensity, where a Hero protein, like a canonical chaperone, maintains the conformational integrity of a client protein to prevent its aggregation.

Co-translational protein folding is one of the central topics in molecular biology. In Escherichia coli, trigger factor (TF) is a primary chaperone that facilitates co-translational folding by directly interacting with nascent polypeptide chains on translating ribosomes. In this study, we applied fluorescence correlation spectroscopy (FCS), which can analyze the diffusion properties of fluorescent molecules by measuring the fluctuations of the fluorescent intensity, to investigate the interaction between TF and a nascent chain on translating ribosomes both in vitro and in vivo. The FCS analysis with a reconstituted cell-free translation system revealed that the interaction of fluorescently labeled TF with a nascent chain depended on the emergence of the nascent chain from the ribosome exit tunnel, and this interaction was not inhibited by excess amounts of other chaperones. Furthermore, the translation-dependent interaction between GFP-fused TFs and nascent chains was also observed in living E. coli cells. The FCS-based approach established here could be an effective method to investigate the dynamics of other ribosome-associated chaperones besides TF.

Single-molecule imaging is a powerful method for unveiling precise molecular mechanisms. Particularly, single-molecule analysis with total internal reflection fluorescence (TIRF ) microscopy has been successfully applied to the characterization of molecular mechanisms in ncRNA studies. Tracing interactions at the single-molecule level have elucidated the intermediate states of the reaction, which are hidden by ensemble averaging in combinational biochemical approaches, and clarified the key steps of the interaction. However, applying a single-molecule technique to ncRNA analysis still remains a challenge, requiring laborious trial and error to identify a suitable glass surface passivation method. In this chapter, we revisit the major glass surface passivation methods using polyethylene glycol (PEG) treatment and summarize a detailed protocol for single-molecule analysis of the dicing process of Dcr-2, which may apply piRNA studies in the future.

Co-translational protein folding is one of the central topics in molecular biology. In

Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein’s oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct—processive or distributive—modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2’s cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated., Fly Dicer-2 is thought to use two distinct – processive or distributive – modes of cleavage by distinguishing the terminal structures of double-stranded RNA (dsRNA) substrates with the help of its cofactor LoquaciousPD (Loqs-PD). Here the authors show by single-molecule imaging that dsRNA terminal structures and Loqs-PD change the probability for Dicer to initiate processive cleavage but not the mode of cleavage action per se.

Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts’ RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)—the core of the RNA-induced silencing complex (RISC)—in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions.

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition.

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Ribavirin, a nucleic acid analog, has been employed as an antiviral agent against RNA and DNA viruses and has become the standard agent used for chronic hepatitis C in combination with interferon-α2a. Furthermore, the potential antitumor efficacy of ribavirin has attracted increasing interest. Recently, we demonstrated a dose-dependent antitumor effect of ribavirin for seven types of malignant glioma cell lines. However, the mechanism underlying the antitumor effect of ribavirin has not yet been fully elucidated. Therefore, the main aim of the present study was to provide further relevant data using two types of malignant glioma cell lines (U-87MG and U-138MG) with different expression of MGMT. Dotted accumulations of γH2AX were found in the nuclei and increased levels of ATM and phosphorylated ATM protein expression were also observed following ribavirin treatment (10 µM of ribavirin, clinical relevant concentration) in both the malignant glioma cells, indicating double-strand breaks as one possible mechanism underlying the antitumor effect of ribavirin. In addition, based on assessements using FACS, ribavirin treatment tended to increase the G0/G1 phase, with a time‑lapse, indicating the induction of G0/G1-phase arrest. Furthermore, an increased phosphorylated p53 and p21 protein expression was confirmed in both glioma cells. Additionally, analysis by FACS indicated that apoptosis was induced following ribavirin treatment and caspase cascade, downstream of the p53 pathway, which indicated the activation of both exogenous and endogenous apoptosis in both malignant glioma cell lines. These findings may provide an experimental basis for the clinical treatment of glioblastomas with ribavirin.

In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit.

Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit.

It has been shown that the genesis of atherosclerotic lesions is resulted from the injury of vascular endothelial cells and the cell damage is triggered by oxygen radicals generated from various tissues. Human vascular endothelial cells can survive and proliferate depending on growth factors such as VEGF or basic FGF and are induced apoptosis by the deprivation of growth factor or serum. It was found that type 1 IFN inhibits the growth factor deprived cell death of human aortic endothelial cells (HAEC) and protects the cells from chemically induced oxidative cytotoxicity. The anti-apoptotic effects of type 1 IFN were certified by flow cytometry using annexin-V-FITC/PI double staining and cell cycle analysis, fluorescence microscopy using Hoechst33342 and PI, colorimetric assay for caspase-3 activity, p53 and bax mRNA expressions, and cell counts. It was considered that IFN-β inhibits the executive late stage apoptosis from the results of annexin-V-FITC/PI double staining and the inhibition of caspase-3 activity, and that the anti-apoptotic effect might be owing to the direct inhibition of the apoptotic pathway mediated by p53 from the transient down-regulation of bax mRNA expression. Whereas, type 1 IFN protected the cells from the oxidative cytotoxicity induced by tertiary butylhydroperoxide (TBH) under the presence of Ca(2+). The effects of IFN-β is more potent inhibitor of cell death than IFN-α. These results indicate that type 1 IFN, especially IFN-β may be useful for the diseases with vascular endothelium damage such as atherosclerosis or restenosis after angioplasty as a medical treatment or a prophylactic.

Trans-translation is an eubacterial quality control system to rescue the stalled ribosome, in which multiple components such as transfer messenger RNA (tmRNA) and Small protein B (SmpB) are involved. However, how these molecules interact with ribosome remains elusive. Here, we report the single molecule analysis of the trans-translation process. We developed a new method to label the functional ribosome, in which a tag protein (the HaloTag protein of 297 amino acids) was fused to the 30S ribosomal protein S2 and covalently labelled with specific ligand (HaloTag ligand), resulting in the stable and specific labelling of ribosome. Ribosomes were anchored onto the glass surface using biotinylated derivative of the Cy3 HaloTag ligand (i.e. biotin-Cy3-ligand), and real-time interactions of Cy5-tmRNA/SmpB/EF-Tu ternary complexes with anchored ribosomes are observed as a model of the trans-translation entry. Statistical analysis revealed that Cy5-tmRNA/SmpB/EF-Tu ternary complexes bind to the anchored ribosome with the second-order rate constant of 2.6 × 10(6) (1/M/s) and tmRNAs undergo multi-modal pathway before release from ribosome. The methods presented here are also applicable to the analysis for general translation processes.

Summary Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Forster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form.

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13-amino-acid chain positioned at the C-terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter-head coordination through the neck linker facilitates long-distance walking.

The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4(-/-), MD2(-/-), MyD88(-/-), and TRIF(-/-) macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively.

In eukaryotic cells, pre-mRNAs are transcribed in the nucleus, processed by 5' capping, 3'-polyadenylation, and splicing, and exported to the cytoplasm for translation. To examine the nuclear mRNA transport mechanism, intron-deficient mRNAs of truncated beta-globin and EGFP were synthesized, fluorescently labeled in vitro, and injected into the nucleus of living Xenopus A6 cells. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2microm(2)/s, about 1/150 of that in water. The slow diffusion could not be explained by simple diffusion obeying the Stokes-Einstein equation, suggesting interactions of the mRNAs with nuclear components. The remaining mRNAs were stationary with an average residence time of about 30s, comparable to the time required for mRNA diffusion from the site of synthesis to nuclear pores.

In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu (ftz) pre-mRNA into the nuclei of HeLa cells. The injected intron-containing ftz pre-mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy-requiring active process. In contrast, the injected intron-less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre-mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, alpha-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells.

The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization-depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments.

We have analyzed the dynamics of the chaperonin (GroEL)-cochaperonin (GroES) interaction at the single-molecule level. In the presence of ATP and non-native protein, binding of GroES to the immobilized GroEL occurred at a rate that is consistent with bulk kinetics measurements. However, the release of GroES from GroEL occurred after a lag period ( approximately 3 s) that was not recognized in earlier bulk-phase studies. This observation suggests a new kinetic intermediate in the GroEL-GroES reaction pathway.

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2-R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2-R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2-R2D2-siRNA complex on Ago2, in a manner dependent on recognition of the 5'-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2-R2D2 and thereby assemble RISC.

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

Speckles are subnuclear domains where pre-mRNA splicing factors accumulate in the interchromatin space. To investigate the dynamics of mRNAs at speckles, fluorescently labeled Drosophila Fushitarazu (ftz) pre-mRNAs were microinjected into the nuclei of Cos7 cells and the dissociation kinetics of pre-mRNAs from speckles was analyzed using photobleaching techniques. The microinjected ftz pre-mRNAs accumulated in speckles in an intron-dependent manner and were spliced and exported to the cytoplasm with a half-time of about 10 min. Dissociation of the accumulated pre-mRNAs in speckles exhibited rapid diffusion and slow-dissociation of about 100 s. The slow-dissociation required metabolic energy of ATP. Two types of splice-defective mutated mRNAs dissociated from the speckle with a time constant similar to that of wild-type mRNA, indicating that slow-dissociation was not coupled to the splicing reaction. Furthermore, some pre-mRNAs shuttled between speckles and nucleoplasm, suggesting that pre-mRNAs repeatedly associated with and dissociated from speckles until introns were removed. Next, endogenous poly(A)+ RNA was visualized by injecting Cy3-labeled 2'O-methyl oligo(U)22 probes. Some poly(A)+ RNA distributed diffusely within the nucleus, but some of them accumulated in speckles and dissociated at time constant of about 100 s.

The selection of effective antisense target sites on a given mRNA molecule is a major problem in the detection of target mRNA in oligonucleotide arrays. In general, antisense oligodeoxynucleotides (asODNs) of about 10-20 nucleotides (nt) in length are used. However, the demand for predicting the sequence of potent asODNs much longer than those mentioned above has been increasing. Here, we prepared 40-nt asODNs directed against fluorescence-labeled green fluorescent protein (GFP) mRNA and quantified their hybridization efficiencies by fluorescence microscopy. We found that the hybridization efficiency depended on the TC content or the minimum free energy of the asODNs. On the basis of these findings, a semiempirical parameter called accessibility score was introduced to predict the potency of asODNs. The results of this study aided in the development of an effective two-step procedure for determining mRNA accessibility, namely, the computer-aided selection of asODN binding sites using an accessibility score followed by an experimental procedure for measuring the hybridization efficiencies between the selected asODNs and the target mRNA by fluorescence microscopy.

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.