Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore

Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI–DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments

Single molecule DNA synthesis can be detected electronically

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Three Routes To Modulate the Pore Size of the MscL Channel/Nanovalve

MscL is a bacterial mechanosensitive channel that protects cells from lysis upon acute decrease in external osmotic environment. It is one of the best characterized mechanosensors known, thus serving as a paradigm of how such molecules sense and respond to stimuli. In addition, the fact that it can be genetically modified, expressed, isolated, and manipulated has led to its proposed use as a triggered nanovalve for various functions including sensors within microelectronic array chips, as well as vesicular-based targeted drug release. X-ray crystallography reveals a homo-pentameric complex with each subunit containing two transmembrane α-helices (TM1 and TM2) and a single carboxyl terminal α-helix arranging within the complex to form a five-fold cytoplasmic bundle (CB), whose function and stability remain unclear. In this study, we show three routes that throttle the open channel conductance. When the linker between the TM2 and CB domain is shortened by deletions or constrained by either cross linking or heavy metal coordination, the conductance of the channel is reduced; in later two cases, even reversibly. While having implications for the stability of the CB, these data also provide routes for engineering MscL sensors that are more versatile for potential nanotech devices