Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0

Tuesday, March 1, 2016 increasing speed but by focusing measurements on the interesting areas based on the incoming data. Here we describe work on combining this approach with novel, high speed-actuators to yield ultra-high speed imaging. A dualstage actuator is used to drive the tip around a small-radius circle and the measurements used in real time to both center that circle on the biopolymer and to scan the circle along the sample. A proof-of-concept instrument is expected to hit 30 frames per second with the final design aiming for 100 frames per second. 2469-Pos Board B613 High Resolution Magnetic Tweezers to Probe Single Molecule Dynamics Bob M. Lansdorp, Omar A. Saleh. Materials, UCSB, Santa Barbara, CA, USA. The magnetic tweezer is a single molecule manipulation instrument ideally suited to measuring biophysical systems at a constant applied force. The development of a magnetic tweezers with a high-speed camera and GPU-accelerated particle tracking has allowed for the measurement of molecular events at the millisecond time scale. However, as the spatial resolution of the instrument is improved, previously neglected sources of noise start to become limiting, such as: mechanical stability of the sample stage, and coherent light artifacts such as speckle. Here, we isolate the various sources of noise in an attempt to determine the fundamental limit to magnetic tweezer resolution. We use a state-of-the-art high spatial and temporal resolution magnetic tweezer to measure the dynamics of model systems such as DNA hairpins. 2470-Pos Board B614 Multiplexed Mechanochemistry Assay - A Tool for Multiplexed Single Molecule Bond Rupture Force Studies Bhavik Nathwani1,2, Darren Yang3, Wesley Wong2,4, William M. Shih1,2. 1 Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA, 2 Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA, 3Harvard University, Cambridge, MA, USA, 4 Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA. Studies in mechanochemistry have greatly benefited from advances in single molecule force spectroscopy (SMFS) techniques. Despite success, conventional SMFS techniques are inherently low throughput. For example, Atomic Force Microscopy (AFM) and optical tweezers are serial techniques. In recent years, considerable effort has been made to develop massively parallel force spectroscopy methods such as the centrifugal force microscope (CFM), which applies centrifugal force to tethered beads. The capability to apply centrifugal force on a large number of beads opens new multiplexing possibilities. Here, we report the development of a novel technique — Multiplexed Mechanochemistry Assay (MMA) — enabling large-scale study of single molecule chemical bond rupture events. Briefly, in a bead surface assay configuration, we designed a centrifugal system for the application of pre-calibrated forces on up to 320 samples simultaneously. Our system enables two-dimensional multiplexing, (1) number of beads being studied simultaneously, and (2) number of experimental conditions being probed. The force range afforded by our equipment ranges from a fraction of a pN to ~30 nN. This unprecedented level of ultraplexing afforded by our approach, hundreds of experimental conditions tested against orders of magnitude variation in force, provides a dramatic improvement in throughput of individual experiments over conventional SMFS techniques. As system validation, we report a case study on studying bond rupture of covalent bonds. We believe this simple tool will greatly increase the rate of progress in probing the relation between force-lifetime and chemistry in single molecular bonds. 2471-Pos Board B615 Label-Free Intramolecular Chemical Microscopy of a Protein-RNA Complex Duckhoe Kim1, Zhenghan Gao2, Ozgur Sahin1,2. 1 biological sciences, Columbia University, New York, NY, USA, 2Physics, Columbia University, New York, NY, USA. Obtaining structural information from single biomolecules using imaging methods is challenging due to resolution limitations and difficulties in labelling specific sites of biomolecules. Atomic force microscopy based imaging of interaction forces between a specially designed probe molecule and a biomolecular complex of interest can, in principle, allow determining the locations of various regions within the complex, if the probe is designed to exhibit affinity to these regions. Here we demonstrate this concept with protein-RNA complex by targeting specific segments of the RNA with a DNA probe having sequence complementarity with these segments. To

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map the interaction forces between the probe and the sample, we used T-shaped cantilevers that allow mapping interaction forces during the tapping mode imaging process [1]. We worked with stem loop binding proteins in complex with RNA [2] and we targeted the flanking RNA regions adjacent to the stem loop. We show that locations and sequence identities of the RNA segments can be imaged with this approach. Repeated measurements of the same protein-RNA complex showed the consistency of the images. Our results show that it is possible obtain chemically-specific structural information from single biomolecules in physiologically relevant conditions without using labels. 1. Kim, D., Sahin, O. Nature Nanotechnology 10, 264-269 (2015). 2. Tan, D., Mazluff, W. F., Dominski, Z., Tong, L. Science 339, 318-321 (2013). 2472-Pos Board B616 Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0 Douwe Kamsma1, Ramon Creyghton2, Gerrit Sitters1, Erwin J.G. Peterman1, Gijs J.L. Wuite1. 1 VU Vrije Universiteit, Amsterdam, Netherlands, 2UvA university of Amsterdam, Amsterdam, Netherlands. AFS is a novel technique that uses an acoustic standing wave in a microfluidic chip to apply forces on single tethers to study the structural and mechanochemical properties of biomolecules. It distinguishes itself by a high experimental throughput, a wide force range and an unmatched range of force loading rates. In its original implementation the method has several limitations: it makes use of an opaque piezo element to generate the acoustic force, the force profile is not constant over the fluid layer and it is not possible to apply forces close to the coverslip side. Here we present innovative solutions to tackle these shortcomings, making AFS a more valuable technique. The use of a transparent piezo element allows transillumination of the sample, greatly improving optical performance and improving compatibility with existing microscopes. We also have developed a model to calculate forces in the sample, which is used to optimize the dimensions of the system, creating the possibility to overstretch a DNA molecule tethered to a 1 mm in diameter silica beads at the coverslip side. In addition, a superposition of standing ultrasound waves can be applied to the sample, which allow modification of the force profile, for example to generate a more constant force across the fluid channel. This approach can also be used to create highly distance-dependent force profiles, transforming AFS in a distance clamp. Finally, we show that AFS is compatible with high NA water or oil immersion objectives, although less high forces can be achieved. AFS already distinguishes itself by its relative simplicity, low cost and compactness, which allow straightforward implementation in lab-on-a-chip devices and these new developments make AFS an even more accessible technique, with a wider field of applications that can be integrated in almost every microscope.

Micro- and Nanotechnology I 2473-Pos Board B617 Characterization of Nucleosomes using DNA Origami Jenny V. Le1,2, Yi Luo1,3, Christopher R. Lucas2, Michael G. Poirier1,3, Carlos E. Castro1,2. 1 Biophysics, The Ohio State University, Columbus, OH, USA, 2Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH, USA, 3Physics, The Ohio State University, Columbus, OH, USA. While biophysical tools such as single molecule fluorescence and force spectroscopy have been used to study the structural dynamics of individual nucleosomes or large chromatin assemblies, it is challenging to probe conformational dynamics of gene regulation in the 10-100nm range. This is the length scale of genomic critical features, including promoter regions that span several nucleosomes. Our work aims to develop DNA origami tools to probe these mesoscale structure and dynamics of nucleosome arrays and chromatin. DNA origami itself is an emerging nanotechnology that enables the selfassembly of precisely designed molecular-scale structures. Here, we implement a DNA origami hinge device with the length scale of ~100nm as a nanocaliper to study the structure and dynamics of a single nucleosome as proof-of-concept. The measurement capabilities of the device were calibrated by integrating DNA duplexes of varying lengths between the hinge arms to demonstrate the use the hinge angular distribution as a readout for the sample size attached between the arms. We further integrated single nucleosomes with varying but symmetric lengths of DNA linkers (i.e. DNA extending out after wrapping around the histone core) to verify our ability to detect structural