Optical and magnetic tweezers are widely employed to probe the mechanics and activity of individual biomolecular complexes. They rely on micrometer-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 tweezers experiments using multiple particles at a time.

Magnetic tweezers are a wide-spread tool used to study the mechanics and the function of a large variety of biomolecules and biomolecular machines. This tool uses a magnetic particle and a strong magnetic field gradient to apply defined forces to the molecule of interest. Forces are typically quantified by analyzing the lateral fluctuations of the biomolecule-tethered particle in the direction perpendicular to the applied force. Since the magnetic field pins the anisotropy axis of the particle, the lateral fluctuations follow the geometry of a pendulum with a short pendulum length along and a long pendulum length perpendicular to the field lines. Typically, the short pendulum geometry is used for force calibration by power-spectral-density (PSD) analysis, because the movement of the bead in this direction can be approximated by a simple translational motion. Here, we provide a detailed analysis of the fluctuations according to the long pendulum geometry and show that for this direction, both the translational and the rotational motions of the particle have to be considered. We provide analytical formulas for the PSD of this coupled system that agree well with PSDs obtained in experiments and simulations and that finally allow a faithful quantification of the magnetic force for the long pendulum geometry. We furthermore demonstrate that this methodology allows the calibration of much larger forces than the short pendulum geometry in a tether-length-dependent manner. In addition, the accuracy of determination of the absolute force is improved. Our force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.

Production of various structures by self-assembling single stranded DNA molecules is a widely used technology in the filed of DNA nanotechnology. Base sequences of single strands do predict the shape of the resulting nanostructure. Therefore, sequence design is crucial for the successful structure fabrication. This paper presents a sequence design algorithm based on mismatch minimization that can be applied to every desired DNA structure. With this algorithm, junctions, loops, single as well as double stranded regions, and very large structures up to several thousand base pairs can be handled. Thereby, the algorithm is fast for the most structures. Algorithm is Java-implemented. Its implementation is called Seed and is available publicly. As an example for a successful sequence generation, this paper presents the fabrication of DNA chain molecules consisting of double-crossover (DX) tiles as well.

Friction within biomolecules has recently gained increasing interest. Here we present a method that allows to study the friction that occurs during fast, few nm-sized refolding processes of nucleic acids. Branch migration of a homologous Holliday junction serves as an ideal system where such friction can be investigated. In this four-arm DNA junction the opposing arms possess identical sequences with respect to the junction center. In the absence of external constraints the junction is mobile such that one pair of homologous arms can expand at the expense of the other in single base pair diffusive steps. We measure the dynamics of the branch migration process by stretching a torsionally constrained Holliday junction using magnetic tweezers and measuring the length fluctuations of the arms with high-speed videomicroscopy at~3 kHz. Since DNA has a helical structure, branch migration causes twisting of the arms with one turn per helical pitch moved. This constrains the movement of the junction within the tweezers to~10 bp. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Thus they cannot be directly resolved. However, power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Theoretical modeling considering the elastic coupling of DNA bending fluctuations and the junction movement allows to quantitatively determine the stepping rate and thus the friction of the branch migration process. We expect that our method is widely applicable to study local-scale molecular friction in biological systems. 1350-Pos Board B242Accurate Distance and Structure Determination of Three Different RNA Three-Way Junctions via High Precision FRET RNA three-way junctions are important ribosomal structural motifs. They are also widely used as building blocks and functional components in nanotechnology applications. Förster-Resonance-Energy-Transfer (FRET) restrained highprecision structural modeling in combination with molecular dynamics simulations was used to determine the structure of RNA three-way junction (3WJ) without bulges, RNA three-way junction with a small (two unpaired nucleotides) bulge and RNA three way junction with bigger (5 unpaired nucleotides) bulge. In total 81 Donor-Acceptor pairs were measured using singlemolecule multi-parameter fluorescence detection (smMFD). This allows us to observe structural changes of the molecule induced by addition of bulge to the initial structure. Rigid body models for the major conformers were obtained by docking rigid double-stranded A-RNA helices explicitly taking into account dye position distributions. This is done many times (1000 iterations) with random starting conformations, yielding all local minima. Obtained models were then refined by all-atom MD simulations [1,2]. First results indicate the presence of two coaxially stacked helices for 3WJs with additional bulges at the junction, and absence of su...

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