RESUMO
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 Mg(2+) 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.
Assuntos
Proteínas de Ligação a DNA/química , Desoxirribonuclease EcoRI/química , Nanoestruturas/química , Simulação por Computador , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Modelos Moleculares , Nanoestruturas/ultraestrutura , Ligação ProteicaRESUMO
A nanopore is an analytical tool with single molecule sensitivity. For detection, a nanopore relies on the electrical signal that develops when a molecule translocates through it. However, the detection sensitivity can be adversely affected by noise and the frequency response. Here, we report measurements of the frequency and noise performance of nanopores =8 nm in diameter in membranes compatible with semiconductor processing. We find that both the high frequency and noise performance are compromised by parasitic capacitances. From the frequency response we extract the parameters of lumped element models motivated by the physical structure that elucidates the parasitics, and then we explore four strategies for improving the electrical performance. We reduce the parasitic membrane capacitances using: (1) thick Si(3)N(4) membranes; (2) miniaturized composite membranes consisting of Si(3)N(4) and polyimide; (3) miniaturized membranes formed from metal-oxide-semiconductor (MOS) capacitors; and (4) capacitance compensation through external circuitry, which has been used successfully for patch clamping. While capacitance compensation provides a vast improvement in the high frequency performance, mitigation of the parasitic capacitance through miniaturization offers the most promising route to high fidelity electrical discrimination of single molecules.
RESUMO
We have explored the electromechanical properties of DNA by using an electric field to force single hairpin molecules to translocate through a synthetic pore in a silicon nitride membrane. We observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the DNA. The threshold for a diameter 1.5 < d < 2.3 nm is V > 1.5 V, which corresponds to the force required to stretch the stem of the hairpin, according to molecular dynamics simulations. On the other hand, for 1.0 < d < 1.5 nm, the threshold voltage collapses to V < 0.5 V because the stem unzips with a lower force than required for stretching. The data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in DNA.
Assuntos
DNA/química , Membranas Artificiais , Nanoestruturas/ultraestrutura , Transporte Biológico , DNA/metabolismo , Condutividade Elétrica , Modelos Moleculares , Conformação de Ácido Nucleico , Compostos de Silício/químicaRESUMO
Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a <2 nm diameter pore, which we attribute to the stretching transition; this can differ by >1 V/20 nm depending on the methylation level, but not the DNA sequence.
Assuntos
Metilação de DNA , DNA/química , DNA/metabolismo , Membranas Artificiais , Algoritmos , Sequência de Bases , Eletroforese em Gel de Ágar , Genes BRCA1 , Fator de Crescimento Insulin-Like II/genética , Permeabilidade , Reação em Cadeia da Polimerase , Conformação ProteicaRESUMO
We have fabricated and tested the performance of sub-50nm gate nMOSFETs to assess their suitability for mixed signal applications in the super high frequency (SHF) band, i.e. 3-30GHz. For a 30nm×40 µm×2 device, we found f(T) =465GHz at V(ds)=2V, V(g)=0.67V, which is the highest cut-off frequency reported for a MOSFET produced on bulk silicon substrate so far. However, our measurements of f(max) and noise figure indicate that parasitics impose limitations on SHF operation. We also present a high-frequency ac model appropriate to sub-50nm gate length nanotransistors, which incorporates the effects of the parasitics. The model accurately accounts for measurements of the S and Y parameters in the frequency range from 1 to 50GHz.
RESUMO
Capillaries pervade human physiology. The mean intercapillary distance is only about 100 µm in human tissue, which indicates the extent of nutrient diffusion. In engineered tissue the lack of capillaries, along with the associated perfusion, is problematic because it leads to hypoxic stress and necrosis. However, a capillary is not easy to engineer due to its complex cytoarchitecture. Here, it is shown that it is possible to create in vitro, in about 30 min, a tubular microenvironment with an elastic modulus and porosity consistent with human tissue that functionally mimicks a bona fide capillary using "live cell lithography"(LCL) to control the type and position of cells on a composite hydrogel scaffold. Furthermore, it is established that these constructs support the forces associated with blood flow, and produce nutrient gradients similar to those measured in vivo. With LCL, capillaries can be constructed with single cell precision-no other method for tissue engineering offers such precision. Since the time required for assembly scales with the number of cells, this method is likely to be adapted first to create minimal functional units of human tissue that constitute organs, consisting of a heterogeneous population of 100-1000 cells, organized hierarchically to express a predictable function.