{"id":4090,"date":"2019-09-18T13:16:41","date_gmt":"2019-09-18T04:16:41","guid":{"rendered":"http:\/\/163.180.4.222\/lab\/?p=4090"},"modified":"2019-09-18T13:16:41","modified_gmt":"2019-09-18T04:16:41","slug":"emerging-uses-of-dna-mechanical-devices","status":"publish","type":"post","link":"https:\/\/biochemistry.khu.ac.kr\/lab\/?p=4090","title":{"rendered":"Emerging uses of DNA mechanical devices"},"content":{"rendered":"

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Modern machines, which are composed of force-generating motors, force sensors, and load-bearing structures, enabled the industrial revolution and are foundational to human civilization. Miniature micromachines are used in countless devices including cell phone microphones, implantable biosensors, and car and airplane accelerometers. Further miniaturization to the nanometer scale would enable the design of machines that can manipulate biomolecules and other nanomaterials for applications in medicine, biological research, and material development. Such machines are typically difficult or impossible to build because of their small size. However, a recent boom in the field of DNA nanotechnology, wherein synthetic DNA is used to tailor-make functional nanostructures, has produced extensive insight into the mechanical properties of DNA. This insight has propelled the emergence of a subfield that we call \u201cDNA mechanotechnology,\u201d wherein DNA devices are engineered to generate, transmit, and sense mechanical forces at the nanoscale.<\/p>\n

DNA mechanotechnology is particularly well suited for measuring and controlling piconewton (pN)\u2013scale forces. For context, 10 pN is roughly one-billionth the weight of a paper clip. Mechanical forces on this scale are important in diverse areas including molecular biophysics, immunology, regenerative medicine, materials science, and nanorobotics. Almost all living cells and tissues experience forces that influence biological function. For example, stem cell fate is regulated by the pN forces experienced by molecules studding the cell surface. Engineered DNA sensors are starting to enable interrogation of these forces. Building synthetic nanomachines that generate and respond to mechanical forces is a long-standing goal in nanoengineering. DNA machines that consume chemical energy to create pN forces demonstrate progress toward this goal and are expected to have applications in nanorobotics and active materials.<\/p>\n

Mechanical DNA devices have been enabled by the accessibility, versatility, and precision of DNA-based design. DNA strands with desired sequences and a wide range of useful modifications, such as fluorescent tags and linkage molecules, can be purchased at low (and decreasing) costs. The reliability of DNA base-pairing often enables easy prediction of the three-dimensional (3D) structures and mechanical properties of DNA nanostructures. Furthermore, the DNA origami technique allows for the semiautomated design of tens or hundreds of individual DNA strands that, when mixed together, spontaneously assemble into sophisticated 3D structures with nanometer-level precision. Although the yield of DNA origami can be low, the method is reasonably accessible and has produced hundreds of unique nanodevices, including shape-shifting nanomachines that can precisely reposition nanomaterials (1<\/em><\/a>).<\/p>\n

DNA mechanotechnology tools have played important roles in studies of the fundamental properties of the proteins that sense, transmit, and generate pN forces in cells. These proteins include the motor protein myosin, the cytoskeletal protein actin, and integrin receptors that anchor cells to their surroundings and sense mechanical forces. Studies of the mechanics of these biomolecules are essential to revealing their functions and are often performed using single-molecule force spectroscopy (SMFS) techniques such as optical and magnetic tweezers. In these methods, force is applied to the target molecule by a precisely controlled instrument (e.g., a microbead controlled by magnetism) and the molecule’s conformation is monitored for force-induced changes such as stretching, unfolding, and rupture. SMFS requires building a connection between the target molecule and the instrument, which is achieved using long polymer handles. These handles are highly flexible and thus undergo noise-inducing thermal fluctuations\u2014particularly in the physiologically relevant <10 pN force range. To sidestep this problem, the DNA origami technique was recently used to create rigid handles composed of several DNA helices bundled together (2<\/em><\/a>) (see the figure). These stiff handles exhibit minimal measurement noise, thus enabling direct mechanical interrogation of weak intermolecular bonds such as the stacking forces between neighboring DNA bases.<\/p>\n

In some cases, DNA mechanotechnology is replacing conventional SMFS. For example, the DNA origami force clamp (3<\/em><\/a>) is a mechanically rigid scaffold that binds two ends of a target molecule and holds it at a constant, user-determined tension of \u226420 pN. The target molecule is then monitored using fluorescence microscopy to reveal specific force-function relationships. This approach revealed that the association of a transcription factor, TATA-binding protein, to DNA is inhibited when tension exceeds 10 pN, providing evidence for the importance of mechanical forces in gene regulation. Because fluorescence microscopy can be used to image many clamps simultaneously, this approach has substantially higher throughput than conventional SMFS techniques.<\/p>\n

Another important recent advance in DNA mechanotechnology is the development of tension sensors (4<\/em><\/a>\u20136<\/em><\/a>). Like macroscopic force gauges, DNA tension sensors include two main components: a \u201cspring\u201d that extends under force, and a \u201cruler\u201d that reports on the spring’s extension. In existing DNA tension sensors, a stem-loop hairpin serves as the spring and fluorescence quenching reports on the hairpin’s extension. Each sensor is anchored to a surface at one end; the sensor’s other end presents a ligand that binds to a cell receptor. Receptor force exceeding a threshold (tunable up to 20 pN) unfolds the hairpin, resulting in dequenching of a fluorophore that can be monitored with fluorescence microscopy.<\/p>\n

Because DNA-based tension sensors transduce mechanical events into fluorescence signals, these probes enable live-cell quantification of the magnitude and, recently, the orientation (4<\/em><\/a>) of cellular forces with pN resolution. For example, mouse CD8+<\/sup>\u00a0T cells were found to exert 12 to 19 pN of tension through their T cell receptors (TCRs) on antigen recognition (5<\/em><\/a>), which supports the theory that the TCR uses force as a means of distinguishing between foreign antigens and self-antigens. The ability to measure TCR forces, which are generally infrequent and short-lived, has recently improved exponentially with the development of a new type of DNA tension sensor that can store information related to mechanical events (6<\/em><\/a>). DNA-based tension sensors were also recently used to show that contracting platelets exert pN forces through their integrin receptors at an orientation of \u223c45\u00b0 from the plasma membrane (4<\/em><\/a>). This force geometry may help to mediate clotting by distinguishing between fibrinogen that is immobilized on wounded tissue, which binds to platelet integrins to stimulate clotting, and soluble fibrinogen in the bloodstream, which does not stimulate clotting.<\/p>\n

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