Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

A long-standing question in gene regulation is how remote enhancers communicate with their target promoters, and specifically how chromatin topology dynamically relates to gene activation. Here, we combine genome editing and multi-color live imaging to simultaneously visualize physical enhancer-promoter interaction and transcription at the single-cell level in Drosophila embryos. By examining transcriptional activation of a reporter by the endogenous even-skipped enhancers, which are located 150 kb away, we identify three distinct topological conformation states and measure their transition kinetics. We show that sustained proximity of the enhancer to its target is required for activation. Transcription in turn affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction. Furthermore, the facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects. Our approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates.

It is generally assumed that polymeric micelles, upon administration into the blood stream, carry drug molecules until they are taken up into cells followed by intracellular release. The current work revisits this conventional wisdom. The study using duallabeled micelles containing fluorescently labeled copolymers and hydrophobic fluorescent probes entrapped in the polymeric micelle core showed that cellular uptake of hydrophobic probes was much faster than that of labeled copolymers. This result implies that the hydrophobic probes in the core are released from micelles in the extracellular space. Fö rster resonance energy transfer (FRET) imaging and spectroscopy were used to monitor this process in real time. A FRET pair, DiIC 18(3) and DiOC18(3), was loaded into monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) micelles. By monitoring the FRET efficiency, release of the core-loaded probes to model membranes was demonstrated. During administration of polymeric micelles to tumor cells, a decrease of FRET was observed both on the cell membrane and inside of cells, indicating the release of core-loaded probes to the cell membrane before internalization. The decrease of FRET on the plasma membrane was also observed during administration of paclitaxel-loaded micelles. Taken together, our results suggest a membrane-mediated pathway for cellular uptake of hydrophobic molecules preloaded in polymeric micelles. The plasma membrane provides a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations. A putative role of the PEG shell in the molecular transport from micelle to membrane is discussed. Block copolymer micelles (1-4) have attracted growing interest as carriers to deliver drugs including antitumor reagents to the target site via the enhanced permeability and retention effect (5). Hydrophobic molecules, such as paclitaxel (PTX), can be incorporated into the core of polymeric micelles by physical entrapment. The core composed of hydrophobic chains is expected to regulate the retention and the release of loaded drug molecules on an appropriate time scale (6-8). With micelle-based drug formulations reaching clinical trials (9, 10), the impetus for understanding the pathways involved in micellar drug delivery starts to emerge. It is thought that intact polymeric micelles are taken into cells, followed by intracellular release of drug molecules (8). Kabanov's group (11) reported that pluronic copolymer micelles could be internalized by an endocytic pathway and increase drug absorption by inhibition of P-glycoprotein drug efflux system in cancer cells. Allen et al. (12) suggested that poly(ethylene oxide)-poly(-caprolactone) (PEO-PCL) micelles enter PC12 cells via endocytosis. To directly monitor the micelles, Luo et al. (13) and Savic et al. (14) chemically conjugated a rhodamine molecule to the end of the PCL block and investigated the internalization of PEO-PCL micelles into P19 and PC12 cells. In their study, micelles were reported to en...

Understanding the in vivo behavior of nanoparticles is critical for the translation of nanomedicine from laboratory research to clinical trials. In this work, in vivo Forster resonance energy transfer (FRET) imaging was employed to monitor the release of hydrophobic molecules from circulating poly(ethylene glycol)-poly( D, L-lactic acid) (PEG-PDLLA) micelles. A lipophilic FRET pair (DiIC(18) and DiOC(18)) was physically entrapped into micelle cores by mimicking the loading of hydrophobic drugs. The FRET efficiency was found significantly reduced within 15 min after intravenous injection, implying that DiIC(18) and DiOC(18) quickly escaped from the circulating micelles. FRET spectroscopy studies further demonstrated that alpha- and beta-globulins were major factors for the observed fast release, while gamma-globulins, albumin, and red blood cells played minor roles. These results provide useful information for developing blood-stable micelles to deliver hydrophobic drugs to the target site via prolonged circulation and extravasation from the vascular system.

Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii) occurs independently of endocytic vesicle trafficking along microtubules, and (iv) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.disulfide bond reduction ͉ endosome ͉ folate receptor ͉ drug targeting