We examined the voltage-driven movement of single-stranded DNA molecules in a membrane channel or "nanopore". Using single channel recording methods and a statistical analysis of many single molecule events, we determined how voltage influences capture and translocation in the nanopore. We verified that the mean time between capture events follows a simple exponential distribution, whereas the translocation times follow a unique distribution that is partly Gaussian and partly exponential. Measurements of polymer sequence effects demonstrated that translocation duration is heavily influenced by specific or nonspecific purine-channel interactions. The single molecule approach we used revealed molecular interactions that can influence both capture rates and translocation velocities in a manner that enriches naive barrier crossing models.

The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.

We present a new approach for determining optical gradient forces applied by strongly focused laser beams on dielectric particles. We show that when the electromagnetic field is focused to a diffraction limited spot a dipole approximation is valid for any particle size. We derive intuitive predictions for force-displacement curves, maximal trapping forces, and force constants. The theory fits well with recent measurements of particles trapped by laser tweezers. We also discuss effects of radiation pressure and gravity. [S0031-9007(98)06883-5] 05.40. + j, 42.25.Fx The technique of optical tweezers has opened new experimental horizons in the biophysical and colloidal realm. Since the pioneering work of Ashkin [1-3], who first introduced the use of optical gradient forces, researchers have found diverse applications of trapping and manipulating single particles such as dielectric spheres and cellular organelles. Recent work with laser tweezers has allowed quantitative measurements of piconewton forces and nanometer displacements such as those produced by the action of single molecular motors. In addition, laser tweezers are widely used to measure mechanical elastic properties of cellular components such as DNA strands, molecular filaments, and membranes [4-6]. These experimental advances require an understanding and determination of optical gradient forces in a predictive and tractable manner. Theoretical calculations to date that predict the force acting on a general particle are strictly applicable to either small particles (electromagnetic theory) or very large particles (ray optics calculations) [7,8]. In the intermediate regime (typically 1 10 mm), which is often the most interesting one, both theories are incompatible with experimental results [4].In previous work on optical gradient forces two main approaches have been applied, an electromagnetic (EM) approach and a ray optics (RO) approximation. In the EM approach one calculates the Maxwell stress tensor to obtain the force acting on the particle where the EM fields are computed by plane wave Fourier decomposition of the focused beam [7]. For particles of size R much smaller than the wavelength of the light beam, l, the EM approach reduces to a dipole approximation where the particle interacts with the EM field only as an electric dipole (Rayleigh theory). To compute the interaction with a focused beam in the intermediate regime, where R is comparable to l and interference effects are relevant, one needs to sum over many Fourier components and solve the general Mie problem for each plane wave component [7]. This makes the calculation practically intractable and difficult to compare with experiment, yielding predictions of forces which are typically weaker by a factor of 3-5 compared to experimental results [4].For very large particles, R ¿ l, and relatively small optical gradients, one can reduce EM theory to geometrical ray optics approximation. These calculations are based on a vectorial summation of the contributions of single rays, which are reflecte...

We developed an experimental technique which probes the dynamics of a single colloidal particle over many decades in time, with spatial resolution of a few nanometers. By scattering a focused laser beam from a particle observed in an optical microscope, we measure its fluctuations via the temporal autocorrelation function of the scattered intensity g͑t͒. This technique is demonstrated by applying it to a single Brownian particle in an optical trap of force constant k. The decay times of g͑t͒, which are related to the particle position autocorrelation function, scale as k 21 , as expected from theory. [S0031-9007(96)

in a free standing silicon nitride membrane. The translocation of CTPR proteins was measured in KCl solution at pH below and above its isoelectric point (pI), as well as with and without denaturing agent, Guanidine HCl. When a CTPR protein molecule transits through a nanopore driven by an applied voltage, it partially blocks the ions (K þ and Cl -) flow in the nanopore and generates a characteristic electric current blockage signal. The current blockage signal reveals information about the size, conformation, and primary sequence of the CTPR protein molecule. Previous translocation studies carried out with DNA have established that higher bias voltages result in shorter duration current blockages indicating that DNA translocates faster at a stronger electric field. However, CTPR translocation studies presented here show that longer duration current blockades were observed at higher bias voltages. We explain this surprising result by theoretical analysis of CTPR protein translocation in solid state nanopores. We discuss how the inhomogeneous distribution of the primary charge sequence of the CTPR proteins predicts translocation barriers that are proportional to the bias voltage. Larger barriers at higher bias voltages will result in longer translocation times, consistent with our experimental results.

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Solid-state nanopores are an emerging biosensor for nucleic acid and protein characterization. For use in a clinical setting, solid-state nanopore sensing requires sample preparation and purification, fluid handling, a heating element, electrical noise insulators, and an electrical readout detector, all of which hamper its translation to a point-of-care diagnostic device. A stand-alone microfluidicbased nanopore device is described that combines a bioassay reaction/purification chamber with a solid-state nanopore sensor. The microfluidic device is composed of the high-temperature/solvent resistance Zeonex plastic, formed via micro-machining and heat bonding, enabling the use of both a heat regulator and a magnetic controller. Fluid control through the microfluidic channels and chambers is controlled via fluid port selector valves and allows up-to eight different solutions. Electrical noise measurements and DNA translocation experiments demonstrate the integrity of the device, with performance comparable to a conventional stand-alone nanopore setup. However, the microfluidic-nanopore setup is superior in terms of ease of use. To showcase the utility of the device, single molecule detection of a DNA PCR product, after magnetic bead DNA separation, is accomplished on chip.

Most functional transcription factor (TF) binding sites deviate from their "consensus" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used singlemolecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.