A Protein‐Driven DNA Device That Measures the Excess Binding Energy of Proteins That Distort DNA

Abstract

Nanomechanical devices hold the promise of controlling structure and performing exquisitely fine measurements on the molecular scale. We previously have reported three nanoscale devices that were built from DNA. The first device [1] extrudes or withdraws a portion of a DNA cruciform from a cyclic molecule in response to the presence of an intercalator. The second device is powered by the B-Z transition: [2] it switches a DNA domain from one side of a double-helical shaft to the other in response to the presence or absence of [Co(NH 3 ) 6 ] 3+ in its environment. The third device is sequence-dependent; [3] it changes structure as a function of DNA strands that are in solution with it. All of these devices change their states in response to an external stimulus, but they do not report new information about any other molecular system. Here we describe a new DNA device that changes shape when a DNA-distorting protein is added to the system. In this case, we used E. coli integration host factor (IHF), which bends DNA significantly when it binds to an appropriate recognition site.[4] Thus, we have developed a nanomechanical device for which the stimulus is a protein. However, this device does not merely have a different kind of activation signal. A second part of the device contains a series of nucleotide pairs that must be disrupted when the device is distorted by IHF. Consequently, this is a measuring device that enables us to estimate the amount of additional work that the protein can do when it binds to DNA.Our IHF-activated device is illustrated schematically in Figure 1. This diagram does not illustrate the part of the device involved in measuring the work, but, for simplicity, shows just the way in which the binding of IHF to its target site can distort the relationship between two DNA triple crossover (TX) motifs that flank the binding site. The upper panel shows the DNA molecule in the absence of IHF: two previously characterized TX molecules [5] are connected by a DNA double-helical shaft; each TX molecule creates a stiff domain that rigidly transmits motions of the shaft on one side to the dyes on the other side. The shaft contains the lH1

DNA Nanotechnology: From Biology and Beyond

“…A protein-driven DNA nanodevice was constructed that not only changes its state in response to an external stimulus but also probes the biochemistry of the protein (Shen et al 2004). In this device, an integration host factor (IHF, Rice et al 1996) protein triggers a sharp bend in the device of about 160 , leading to the rupture of several base pairs elsewhere in the device.…”

“…Different motifs can be obtained by executing different numbers of reciprocal exchanges between the strands of double-stranded DNA molecules. This approach has led to the construction of a variety of base motifs, such as the double crossover (DX) (Fu and Seeman 1993), triple crossover (TX) Liu et al 2008), and paranemic crossover (PX) molecules (Shen 1999).…”

Nukleinsäure‐basierte molekulare Werkzeuge

“…Demgemäß werden DNA-Anordnungen dazu gebracht, ihren Zustand als Antwort auf eine Temperaturänderung, [95] Photoisomerisierung, [96][97][98] die Gegenwart verschiedener Ionen [99] -oder deren Verschwinden -und das Binden an Proteine zu verändern. [100,101] Eine besondere Rolle spielen hierbei Konformationsänderungen, die von sequenzspezifischen Hybridisierungsreaktionen bewirkt werdendiese sind hochspezifisch und ermöglichen die präzise Adressierung eines bestimmten Zustands oder Schalters unter anderen. In vielen Fällen schlägt sich die Multistabilität in veränderten Eigenschaften der Anordnung nieder -wie der Fluoreszenz, einem Elektronentransfer, einer Isomerisierung, mechanischen Eigenschaften oder der chemischen Reaktivität, was sich für verschiedene Anwendungen nutzen lässt, wie in Abschnitt 4.7 diskutiert werden wird.…”

“…So haben Seeman und Mitarbeiter DNA-Nanomaschinen entwickelt, die die Bestimmung von Bindungskräften und -energien DNA-bindender Proteine, wie des Integrations-Host-Faktors (IHF) oder des MutS, ermöglichen. [100,101] Hier verursacht die Biegung und Verdrillung einer supramolekularen DNAStruktur das Aufreißen eines Abschnitts von dsDNA und wirkt so als Kraftsensor.…”

From genes to machines: DNA nanomechanical devices

“…A DNA-based molecular device has been developed to establish how much work a DNA-distorting protein can do when it binds to DNA [41]. It was prototyped using integration host factor (IHF), a device that bends DNA 160° [42].…”