Semiconducting polymer dots (Pdots) represent a new class of ultrabright fluorescent probes for biological imaging. They exhibit several important characteristics for experimentally demanding in ...vitro and in vivo fluorescence studies, such as their high brightness, fast emission rate, excellent photostability, nonblinking, and nontoxic feature. However, controlling the surface chemistry and bioconjugation of Pdots has been a challenging problem that prevented their widespread applications in biological studies. Here, we report a facile yet powerful conjugation method that overcomes this challenge. Our strategy for Pdot functionalization is based on entrapping heterogeneous polymer chains into a single dot, driven by hydrophobic interactions during nanoparticle formation. A small amount of amphiphilic polymer bearing functional groups is co-condensed with the majority of semiconducting polymers to modify and functionalize the nanoparticle surface for subsequent covalent conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG). The Pdot bioconjugates can effectively and specifically label cellular targets, such as cell surface marker in human breast cancer cells, without any detectable nonspecific binding. Single-particle imaging, cellular imaging, and flow cytometry experiments indicate a much higher fluorescence brightness of Pdots compared to those of Alexa dye and quantum dot probes. The successful bioconjugation of these ultrabright nanoparticles presents a novel opportunity to apply versatile semiconducting polymers to various fluorescence measurements in modern biology and biomedicine.
Lighting up brain tumors: Highly fluorescent nanodots that consist of semiconducting polymer blends were attached to the peptide ligand chlorotoxin. The nanodot–chlorotoxin conjugates were ...specifically targeted to malignant brain tumors in a transgenic mouse model, thus proving their potential as in vivo probes for clinical cancer diagnostics (see picture).
All aboard! A facile conjugation method allows covalent linking of functional molecules to semiconducting polymer dots for bioorthogonal labeling of cellular targets. Targeting of the polymer dots to ...newly synthesized proteins and glycoproteins in mammalian cells by click chemistry is highly efficient and specific.
Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric ...complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.
Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is ...initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaA
assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).
Magnetic tweezers (MT) are a powerful tool for the study of DNA-enzyme interactions. Both the magnet-based manipulation and the camera-based detection used in MT are well suited for multiplexed ...measurements. Here, we systematically address challenges related to scaling of multiplexed magnetic tweezers (MMT) towards high levels of parallelization where large numbers of molecules (say 10(3)) are addressed in the same amount of time required by a single-molecule measurement. We apply offline analysis of recorded images and show that this approach provides a scalable solution for parallel tracking of the xyz-positions of many beads simultaneously. We employ a large field-of-view imaging system to address many DNA-bead tethers in parallel. We model the 3D magnetic field generated by the magnets and derive the magnetic force experienced by DNA-bead tethers across the large field of view from first principles. We furthermore experimentally demonstrate that a DNA-bead tether subject to a rotating magnetic field describes a bicircular, Limaçon rotation pattern and that an analysis of this pattern simultaneously yields information about the force angle and the position of attachment of the DNA on the bead. Finally, we apply MMT in the high-throughput investigation of the distribution of the induced magnetic moment, the position of attachment of DNA on the beads, and DNA flexibility. The methods described herein pave the way to kilo-molecule level magnetic tweezers experiments.
Measurements in magnetic tweezers rely upon precise determination of the position of a magnetic microsphere. Fluctuations in the position due to Brownian motion allows calculation of the applied ...force, enabling deduction of the force-extension response function for a single DNA molecule that is attached to the microsphere. The standard approach relies upon using the mean of position fluctuations, which is valid when the microsphere axial position fluctuations obey a normal distribution. However, here we demonstrate that nearby surfaces and the non-linear elasticity of DNA can skew the distribution. Through experiment and simulations, we show that such a skewing leads to inaccurate position measurements which significantly affect the extracted DNA extension and mechanical properties, leading to up to two-fold errors in measured DNA persistence length. We develop a simple, robust and easily implemented method to correct for such mismeasurements.
The cavity enhanced Raman scattering spectrum recorded from an aerosol droplet provides a unique fingerprint of droplet radius and refractive index, assuming that the droplet is homogeneous in ...composition. Aerosol optical tweezers are used in this study to capture a single droplet and a Raman fingerprint is recorded using the trapping laser as the source for the Raman excitation. We report here the retrieval of the real part of the refractive index with an uncertainty of 0.0012 (better than 0.11%), simultaneously measuring the size of the micrometre sized liquid droplet with a precision of better than 1 nm (< 0.05% error). In addition, the equilibrium size of the droplet is shown to depend on the laser irradiance due to optical absorption, which elevates the droplet temperature above that of the ambient gas phase. Modulation of the illuminating laser power leads to a modulation in droplet size as the temperature elevation is altered. By measuring induced size changes of <1 nm, we show that the imaginary part of the refractive index can be retrieved even when less than 10 10
9
with an accuracy of better than 0.5 10
9
. The combination of these measurements allows the complex refractive index of a droplet to be retrieved with high accuracy, with the possibility of making extremely sensitive optical absorption measurements on aerosol samples and the testing of frequently used mixing rules for treating aerosol optical properties. More generally, this method provides an extremely sensitive approach for measuring refractive indices, particularly under solute supersaturation conditions that cannot be accessed by simple bulk-phase measurements.
Sub-nanometre changes in radius induced by optical absorption are used to determine complex refractive index of optically tweezed solution aerosol.
This paper describes a sample digitization method that generates tens of thousands of nanoliter-sized droplets in a high-density array in a matter of minutes. We show that the sample digitization ...depends on both the geometric design of the microfluidic device and the viscoelastic forces between the aqueous sample and a continuous oil phase. Our design avoids sample loss: Samples are split into tens of thousands of discrete volumes with close to 100% efficiency without the need for any expensive valving or pumping systems. We envision this technology will have broad applications that require simple sample digitization within minutes, such as digital polymerase chain reactions and single-cell studies.
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