A Microfluidic Device for Structural Studies of Nucleic Acids

We assembled a microfluidic device that enables highresolution structural studies of nucleic acids. The device contains a mineral matrix capable of rapidly generating hydroxyl radicals to cleave the solvent accessible backbone of nucleic acids. Protection from radical cleavage (footprinting) can identify protein binding sites or the presence of tertiary structure. Here we report the fabrication of a microfluidic prototype containing micron-sized particles of pyrite that generate enough radicals within 20 ms to cleave DNA sufficiently for footprinting. This prototype enables the development of high-throughput and/or rapid reaction devices with which to probe nucleic acids. Summary of Research: Protection analysis (footprinting) with hydroxyl radicals (•OH) has long been a valuable tool for the study of DNA and RNA structure and complexes of nucleic acids with proteins [1-4]. Our approach exploits the advantages of microfluidic systems: small sample volumes, short reaction times and the potential for multiplexed and/or high throughput applications. Iron sulfide (fool’s gold, or pyrite) micro-particles are captured by a constriction in a micro-channel. Hydrogen peroxide (H2O2) flowing through the pyrite matrix generates sufficient quantities of •OH to cleave co-flowing nucleic acid Figure 1 (adapted from [5]): Device schematic and microscopy result. Top) Full-length DNA and H2O2 are pushed into the device and after interacting with the pyrite region, the DNA backbone is cleaved at different locations. Bottom) Experimental data from a confocal microscope, with dye flowing from left to right. Prior to interaction with the pyrite (left side), the dye emission is brighter than in the post-pyrite region (right side). The shape and packing of the pyrite is observable in the center of the image. (Figure 1). We demonstrate the efficacy of this device for footprinting on the millisecond time scale, and discuss how it can be incorporated into mixers and/or parallelized for highthroughput sample analysis [5]. The microfluidic device consists of a single linear channel with a constriction in the middle to trap the pyrite particles (Figure 2). The device is fabricated from the thermoplastic Zeonor. Positive Si wafer masters of our device channel geometry are made using standard photolithography and plasma etching techniques, and we use the wafer to emboss the devices on a hotpress [6, 7]. Inlet and outlet holes at the ends of the channels are punched with a custom die set, and tubing is sealed to the device with TorrSeal epoxy. Because we work with radioactively labeled DNA, the devices are potted in PDMS as an added leak-prevention safety measure. Figure 2 (adapted from [5]): Device and pyrite at different lengthscales. The Zeonor is cut square to a slightly larger dimension than the 1 cm channel length, visible in the top-left image, and the entire device is potted in PDMS. Gold-colored pyrite is readily visible in the top-right image; channel width is 200 μm. Aggregates of the pyrite, and an individual particle are shown in the bottom panels. (See full color version on inside front cover.) BIOLOGICAL APPLICATIONS 7 2011-2012 CNF RESEARCH ACCOMPLISHMENTS B I O The devices are sealed by applying a piece of clear onesided polyester pressure sensitive tape (ARseal 90697) [8]. The resulting device has excellent optical clarity and high resistance to chemical decomposition. The use of tape enables more efficient positioning of the pyrite particles, if the particles get stuck in the adhesive at an undesirable location or clog, that region can be surgically removed and “patched” with another piece of tape. Using this technique, embossed devices can be sealed with nearly 100% efficiency about one hundred times faster than with curable silicones. This powerful nucleic acid analytical technique has applications to biological questions including identifying and studying the binding of proteins to DNA and RNA, and monitoring folding and structural transitions of DNA and RNA, e.g. Schlatterer and Brenowitz [9]. We are able to show significant fragmentation (12%), yielding good signal to background for clear fragment detection. Not surprisingly, DNA fragmentation is affected by solution flow rate and the amount of pyrite in the device. As expected, the amount of fragmentation increases as more pyrite is added or as the flow rates decreases (Figure 3). Based on the performance of our prototype, we anticipate achieving single digit millisecond time resolution for the footprinting reaction. The time resolution is sharp, clearly defined, and readily calibrated. Our device could be integrated with lab-on-a-chip modules that conduct the post-exposure processing necessary to visualize •OH reaction products. Our versatile and simple-toimplement approach is readily combined with other fluidic elements, bringing a powerful new analytical tool to the chip.