Transport Mechanism for Nuclear Localization of Irradiation-Activated EGFR Measured by Single-Molecule Pull-Down Assay.

Nuclear transport of epidermal growth factor receptor (EGFR) is considered to be a key cause of radiation resistance in cancer therapy. Here, we showed that irradiation-activated EGFR binds to the nuclear transport protein karyopherin alpha (KPNA) rather than karyopherin subunit beta 1 (KPNB1), through a single-molecule pull-down assay, which allows measurement of the binding affinity by single proteins in cell lysate without an additional purification step. We also obtained kinetic parameters for the binding between the phosphorylated nuclear localization signal (NLS) peptide of EGFR (645RRRHIVRKRpTLRR657) and KPNA. This observation may help developing small molecules to modulate nuclear transport, which potentially reduces the radiation resistance during irradiation therapy.

Molecular Tension Probes to Quantify Cell-Generated Mechanical Forces.

Living cells generate, sense, and respond to mechanical forces through their interaction with neighboring cells or extracellular matrix, thereby regulating diverse cellular processes such as growth, motility, differentiation, and immune responses. Dysregulation of mechanosensitive signaling pathways is found associated with the development and progression of various diseases such as cancer. Yet, little is known about the mechanisms behind mechano-regulation, largely due to the limited availability of tools to study it at the molecular level. The recent development of molecular tension probes allows measurement of cellular forces exerted by single ligandreceptor interaction, which has helped in revealing the hitherto unknown mechanistic details of various mechanosensitive processes in living cells. Here, we provide an introductory overview of two methods based on molecular tension probes, tension gauge tether (TGT), and molecular tension fluorescence microscopy (MTFM). TGT utilizes the irreversible rupture of double-stranded DNA tether upon application of force in the piconewton (pN) range, whereas MTFM utilizes the reversible extension of molecular springs such as polymer or single-stranded DNA hairpin under applied pN forces. Specifically, the underlying principle of how molecular tension probes measure cell-generated mechanical forces and their applications to mechanosensitive biological processes are described.

CRISPR as a Diagnostic Tool.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

Inhibition of NUPR1-Karyopherin ß1 Binding Increases Anticancer Drug Sensitivity.

BACKGROUND: Nuclear protein-1 (NUPR1, also known as p8/Com-1) is a transcription factor involved in the regulation of cellular stress responses, including serum starvation and drug stimulation. METHODS: We investigated the mechanism of NUPR1 nuclear translocation involving karyopherin ß1 (KPNB1), using a single-molecule binding assay and confocal microscopy. The cellular effects associated with NUPR1-KPNB1 inhibition were investigated by gene expression profiling and cell cycle analysis. RESULTS: The single-molecule binding assay revealed that KPNB1 bound to NUPR1 with a binding affinity of 0.75 nM and that this binding was blocked by the aminothiazole ATZ-502. Following doxorubicin-only treatment, NUPR1 was translocated to the nucleus in more than 90% and NUPR1 translocation was blocked by the ATZ-502 combination treatment in MDA-MB-231 with no change in NUPR1 expression, providing strong evidence that NUPR1 nuclear translocation was directly inhibited by the ATZ-502 treatment. Inhibition of KPNB1 and NUPR1 binding was associated with a synergistic anticancer effect (up to 19.6-fold) in various cancer cell lines. NUPR1-related genes were also downregulated following the doxorubicin-ATZ-502 combination treatment. CONCLUSION: Our current findings clearly demonstrate that NUPR1 translocation into the nucleus requires karyopherin ß1 binding. Inhibition of the KPNB1 and NUPR1 interaction may constitute a new cancer therapeutic approach that can increase the drug efficacy while reducing the side effects.

n-3 Polyunsaturated Fatty Acids Impede the TCR Mobility and the TCR-pMHC Interaction of Anti-Viral CD8+ T Cells.

The immune-suppressive effects of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) on T cells have been observed via multiple in vitro and in vivo models. However, the precise mechanism that causes these effects is still undefined. In this study, we investigated whether n-3 PUFAs regulated T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) interactions. The expansion of anti-viral CD8+ T cells that endogenously synthesize n-3 PUFAs (FAT-1) dramatically decreased upon lymphocytic choriomeningitis virus (LCMV) infection in vivo. This decrease was not caused by the considerable reduction of TCR expression or the impaired chemotactic activity of T cells. Interestingly, a highly inclined and laminated optical sheet (HILO) microscopic analysis revealed that the TCR motility was notably reduced on the surface of the FAT-1 CD8+ T cells compared to the wild type (WT) CD8+ T cells. Importantly, the adhesion strength of the FAT-1 CD8+ T cells to the peptide-MHC was significantly lower than that of the WT CD8+T cells. Consistent with this result, treatment with docosahexaenoic acid (DHA), one type of n-3 PUFA, significantly decreased CD8+ T cell adhesion to the pMHC. Collectively, our results reveal a novel mechanism through which n-3 PUFAs decrease TCR-pMHC interactions by modulating TCR mobility on CD8+ T cell surfaces.

Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations.

FUS is a nuclear RNA-binding protein, and its cytoplasmic aggregation is a pathogenic signature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It remains unknown how the FUS-RNA interactions contribute to phase separation and whether its phase behavior is affected by ALS-linked mutations. Here we demonstrate that wild-type FUS binds single-stranded RNA stoichiometrically in a length-dependent manner and that multimers induce highly dynamic interactions with RNA, giving rise to small and fluid condensates. In contrast, mutations in arginine display a severely altered conformation, static binding to RNA, and formation of large condensates, signifying the role of arginine in driving proper RNA interaction. Glycine mutations undergo rapid loss of fluidity, emphasizing the role of glycine in promoting fluidity. Strikingly, the nuclear import receptor Karyopherin-ß2 reverses the mutant defects and recovers the wild-type FUS behavior. We reveal two distinct mechanisms underpinning potentially disparate pathogenic pathways of ALS-linked FUS mutants.

Positive Identification of DNA Cleavage by CRISPR-Cas9 Using Pyrene Excimer Fluorescence to Detect a Subnanometer Structural Change.

The Cas9 nuclease binds and cleaves DNA through its large-scale structural rearrangements. However, its unique property of not releasing the cleaved DNA has forbidden spectroscopic detection of the cleavage event. Here, we employ a novel fluorescence probe based on pyrene excimer emission to detect a minute structural change not detectable by other methods and demonstrate its applicability to spectroscopic tracking of the Cas9 nuclease activity in time. We show that the intensity of excimer emission depends sensitively on a subtle change in the structural environment of the target nucleic acid, which enables discrimination between cleaved and uncleaved nucleic acids within the DNA/Cas9/gRNA ternary complex. Kinetic parameters were obtained from the temporal evolution of the excimer emission, which revealed that DNA binding is hardly affected by PAM-distal mismatches, whereas the rate of cleavage by Cas9 decreases dramatically even with a 1-bp mismatch. Spectroscopic studies using the pyrene-based probe should be promising for biomolecular systems affected by subnm structural changes.

Single-Cell Imaging Approaches for Studying Small-RNA-Induced Gene Regulation.

RNA interference (RNAi) is a process by which gene expression is downregulated by small interfering RNAs or microRNAs. The quantification of the RNAi efficiency can be performed at both the messenger RNA (mRNA) and the protein level, which is required to assess the potency of small interfering RNAs or microRNAs. Recently, we employed a single-cell mRNA imaging method to study RNAi in which we visualized individual mRNA targets with high precision while resolving the cellular localization and cell-to-cell heterogeneity in addition to RNAi efficiency. In this Biophysical Perspective, we highlight our recent work on quantitative analysis of the RNAi pathway and point out some important future directions. Alongside, we discuss about several single-cell imaging techniques that can be applied to study RNAi. The single-cell imaging techniques discussed here are widely applicable to other gene regulation processes such as the CRISPR-CAS system.

Correlating Transcription Initiation and Conformational Changes by a Single-Subunit RNA Polymerase with Near Base-Pair Resolution.

We provide a comprehensive analysis of transcription in real time by T7 RNA Polymerase (RNAP) using single-molecule fluorescence resonance energy transfer by monitoring the entire life history of transcription initiation, including stepwise RNA synthesis with near base-pair resolution, abortive cycling, and transition into elongation. Kinetically branching pathways were observed for abortive initiation with an RNAP either recycling on the same promoter or exchanging with another RNAP from solution. We detected fast and slow populations of RNAP in their transition into elongation, consistent with the efficient and delayed promoter release, respectively, observed in ensemble studies. Real-time monitoring of abortive cycling using three-probe analysis showed that the initiation events are stochastically branched into productive and failed transcription. The abortive products are generated primarily from initiation events that fail to progress to elongation, and a majority of the productive events transit to elongation without making abortive products.

Probing Dynamic Assembly and Disassembly of Rad51 Tuned by Srs2 Using smFRET.

The integrity of DNA is critical for sustaining the life of any living organism, as DNA is a reservoir of its genetic information. However, DNA is continuously damaged by either normal metabolic pathways or environmental insults such as ultraviolet exposure or chemicals. Double-stranded DNA break is one of the most common types of DNA damage that requires activation of homologous recombination (HR) pathway mediated by Rad51 in eukaryotes (Paques & Haber, 1999; Symington, 2002). Rad51 protein forms a helical nucleoprotein filament on resected DNA to initiate homology search but also can interact with other single-stranded DNA (ssDNA)-binding proteins including Srs2. Srs2, a well-known antirecombinase in HR, is an ATP-dependent 3'-5' DNA helicase in the budding yeast Saccharomyces cerevisiae as well as an ssDNA translocase. It disrupts Rad51 filaments, preventing HR (Krejci et al., 2003; Le Breton et al., 2008; Veaute et al., 2003). In the following text, we provide detailed experimental platforms employed to investigate the activity of Rad51 and Srs2 using single-molecule Forster resonance energy transfer and protein-induced fluorescence enhancement. First, we demonstrate how to detect Rad51 filament formation to address the binding site size binding kinetic of the Rad51, as well as the directionality of the filament formation. Next, we explain how to visualize ATP-dependent translocation and unwinding activities of Srs2 on DNA. Lastly, we demonstrate the filament forming activity by Rad51 which is counteracted by the filament removal activity of Srs2.

RNA stem structure governs coupling of dicing and gene silencing in RNA interference.

PremicroRNAs (premiRNAs) possess secondary structures consisting of a loop and a stem with multiple mismatches. Despite the well-characterized RNAi pathway, how the structural features of premiRNA contribute to dicing and subsequent gene-silencing efficiency remains unclear. Using single-molecule FISH, we demonstrate that cytoplasmic mRNA, but not nuclear mRNA, is reduced during RNAi. The dicing rate and silencing efficiency both increase in a correlated manner as a function of the loop length. In contrast, mismatches in the stem drastically diminish the silencing efficiency without impacting the dicing rate. We show that this decoupling effect is not due to the loading to the RNA-induced silencing complex, RNA uptake, or cellular dicing. We postulate that the stem mismatches perturb the handover of the cleaved miRNAs from Dicer to Argonaute, leading to poor strand selection. Our results imply that the stem structures prevalent in cellular miRNAs have suboptimal silencing efficiency.

Nonclassical Kinetics of Clonal yet Heterogeneous Enzymes.

Enzyme-to-enzyme variation in the catalytic rate is ubiquitous among single enzymes created from the same genetic information, which persists over the lifetimes of living cells. Despite advances in single-enzyme technologies, the lack of an enzyme reaction model accounting for the heterogeneous activity of single enzymes has hindered a quantitative understanding of the nonclassical stochastic outcome of single enzyme systems. Here we present a new statistical kinetics and exactly solvable models for clonal yet heterogeneous enzymes with possibly nonergodic state dynamics and state-dependent reactivity, which enable a quantitative understanding of modern single-enzyme experimental results for the mean and fluctuation in the number of product molecules created by single enzymes. We also propose a new experimental measure of the heterogeneity and nonergodicity for a system of enzymes.

RNA Scanning of a Molecular Machine with a Built-in Ruler.

Advanced single-molecule techniques have enabled tracking of nanometer-scale movements of DNA and RNA motor proteins in real time. Previously, we reported an ATP-independent diffusion of transactivation response RNA binding protein (TRBP) on dsRNA, yet the mechanistic details remain elusive. Using single-molecule fluorescence assays, we demonstrate that the diffusion activity of TRBP is coordinated by an independent movement of two subdomains, dsRBD1 and dsRBD2, in which the diffusion distance is determined by the length of a flexible linker domain that connects the two dsRBDs. When the linker is shortened, the diffusion distance is reduced proportionally, suggesting a ruler-like function of the linker domain. Diffusion stalls upon encountering a physical barrier in the form of an RNA:DNA hybrid segment or bulky secondary structures, indicating a dsRNA scanning mode of TRBP. The results display a plausible mechanism of TRBP in scanning for pre-miRNA or pre-siRNA as proper substrates for the RNAi pathway.

Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform.

Dynamic profiling of double-stranded RNA binding proteins.

Double-stranded (ds) RNA is a key player in numerous biological activities in cells, including RNA interference, anti-viral immunity and mRNA transport. The class of proteins responsible for recognizing dsRNA is termed double-stranded RNA binding proteins (dsRBP). However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 on six dsRNA substrates that vary in length and secondary structure. We combined single molecule pull-down (SiMPull), single molecule protein-induced fluorescence enhancement (smPIFE) and molecular dynamics (MD) simulations to investigate the dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificities and dynamic properties when in contact with different RNA substrates. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to highly structured RNA substrates. Upon interaction with RNA substrates, TRBP and Staufen1 exhibit dynamic sliding whereas two deaminases ADAR1 and ADAD2 mostly remain immobile when bound. MD simulations provide a detailed atomic interaction map that is largely consistent with the affinity differences observed experimentally. Collectively, our study highlights the diverse nature of substrate specificity and mobility exhibited by dsRBPs that may be critical for their cellular function.

Repetitive RNA unwinding by RNA helicase A facilitates RNA annealing.

Helicases contribute to diverse biological processes including replication, transcription and translation. Recent reports suggest that unwinding of some helicases display repetitive activity, yet the functional role of the repetitiveness requires further investigation. Using single-molecule fluorescence assays, we elucidated a unique unwinding mechanism of RNA helicase A (RHA) that entails discrete substeps consisting of binding, activation, unwinding, stalling and reactivation stages. This multi-step process is repeated many times by a single RHA molecule without dissociation, resulting in repetitive unwinding/rewinding cycles. Our kinetic and mutational analysis indicates that the two double stand RNA binding domains at the N-terminus of RHA are responsible for such repetitive unwinding behavior in addition to providing an increased binding affinity to RNA. Further, the repetitive unwinding induces an efficient annealing of a complementary RNA by making the unwound strand more accessible. The complex and unusual mechanism displayed by RHA may help in explaining how the repetitive unwinding of helicases contributes to their biological functions.

Substrate recognition and specificity of double-stranded RNA binding proteins.

Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA-RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2'-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA-RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA-RNA hybrids. Our study reveals the structural and molecular basis of protein-RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins.

Srs2 prevents Rad51 filament formation by repetitive motion on DNA.

Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation.

ATP-independent diffusion of double-stranded RNA binding proteins.

The proteins harboring double-stranded RNA binding domains (dsRBDs) play diverse functional roles such as RNA localization, splicing, editing, export, and translation, yet mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response RNA binding protein (TRBP) consisting of three dsRBDs, which functions in HIV replication, protein kinase R(PKR)-mediated immune response, and RNA silencing. Here we report an ATP-independent diffusion activity of TRBP exclusively on dsRNA in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator and R3D1-L, displayed the same diffusion, implying a universality of the diffusion activity among this protein family. Furthermore, a Dicer-TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity. These results implicate the dsRNA-specific diffusion activity of TRBP that contributes to enhancing siRNA and miRNA processing by Dicer.

Effect of single-base mutation on activity and folding of 10-23 deoxyribozyme studied by three-color single-molecule ALEX FRET.

We investigated the effect of single-base mutation on the RNA-cleaving activity and ion-induced folding of 10-23 deoxyribozyme at the single-molecule level by 3-color ALEX FRET (alternating laser excitation fluorescence resonance energy transfer). We found that substitution or deletion of a single base in the active region of the enzyme leads to a different folding pathway and enzymatic activity for all three mutants studied, but the severity of the effect was dependent on the type of mutation and the mutation site. We suggest that mutation of even a single base may result in a considerably different ionic and hydrogen-bonding interactions. Structural changes of 10-23 deoxyribozyme as it successively binds with Mg(2+) and the substrate were also unambiguously identified by the current single-molecule-detection method.