Ring-shaped replicative helicase encircles double-stranded DNA during unwinding.

Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26-30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

Mesoporous Polydopamine-Encapsulated Fluorescent Nanodiamonds: A Versatile Platform for Biomedical Applications.

Fluorescent nanodiamonds (FNDs) are versatile nanomaterials with promising properties. However, efficient functionalization of FNDs for biomedical applications remains challenging. In this study, we demonstrate mesoporous polydopamine (mPDA) encapsulation of FNDs. The mPDA shell is generated by sequential formation of micelles via self-assembly of Pluronic F127 (F127) with 1,3,5-trimethyl benzene (TMB) and composite micelles via oxidation and self-polymerization of dopamine hydrochloride (DA). The surface of the mPDA shell can be readily functionalized with thiol-terminated methoxy polyethylene glycol (mPEG-SH), hyperbranched polyglycerol (HPG), and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS). The PEGylated FND@mPDA particles are efficiently taken up by, and employed as a fluorescent imaging probe for, HeLa cells. HPG-functionalized FND@mPDA is conjugated with an amino-terminated oligonucleotide to detect microRNA via hybridization. Finally, the increased surface area of the mPDA shell permits efficient loading of doxorubicin hydrochloride. Further modification with TPGS increases drug delivery efficiency, resulting in high toxicity to cancer cells.

Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader.

In keeping with the rapid expansion of nanoparticle applications, various tools are required to investigate how nanoparticles interact with biological entities. Many assays have been developed to measure the cellular uptake of nanoparticles, but so far most of the methods are laborious and often non-quantitative. Here we developed an easily accessible and robust quantitative measurement method of the level of cellular uptake of fluorescently labeled nanoparticles using a plate reader. In the experimental design, potential issues that could lead to measurement variation were identified and addressed. For example, the variation in fluorescence intensity of samples due to differences in cell number was normalized to optical density, which is a physical value corresponding to the cell number. Number of washings and sample handling temperature were optimized to minimize the interference by residual nanoparticles and possible efflux of nanoparticles from cells, respectively. The developed assay was demonstrated with the lymphocyte cell line Jurkat to measure the cellular uptake of fluorescently labeled 50 nm polystyrene beads, and its applicability was further confirmed with the lung carcinoma cell line A549 and another lymphocyte cell line RPMI8226.

Highly Selective FRET-Aided Single-Molecule Counting of MicroRNAs Labeled by Splinted Ligation.

MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Förster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.

Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy.

Optical and magnetic tweezers are widely employed to probe the mechanics and activity of individual biomolecular complexes. They rely on micrometre-sized particles to detect molecular conformational changes from the particle position. Real-time particle tracking with Ångström accuracy has so far been only achieved using laser detection through photodiodes. Here we demonstrate that camera-based imaging can provide a similar performance for all three dimensions. Particle imaging at kHz rates is combined, with real-time data processing being accelerated by a graphics-processing unit. For particles that are fixed in the sample cell we can detect 3-Å-sized steps that are introduced by cell translations at rates of 10 Hz, while for DNA-tethered particles 5 Å steps at 1 Hz can be resolved. Moreover, 20 particles can be tracked in parallel with comparable accuracy. Our approach provides a simple and robust way for high-resolution tweezer experiments using multiple particles at a time.