Nanopores in solid-state membranes engineered for single molecule detection.

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

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 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.

Nanoelectromechanics of methylated DNA in a synthetic nanopore.

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.

Detecting SNPs using a synthetic nanopore.

We have discovered a voltage threshold for permeation through a synthetic nanopore of dsDNA bound to a restriction enzyme that depends on the sequence. Molecular dynamic simulations reveal that the threshold is associated with a nanonewton force required to rupture the DNA-protein complex. A single mutation in the recognition site for the restriction enzyme, i.e., a single nucleotide polymorphism (SNP), can easily be detected as a change in the threshold voltage. Consequently, by measuring the threshold voltage in a synthetic nanopore, it may be possible to discriminate between two variants of the same gene (alleles) that differ in one base.

Laser-guided assembly of heterotypic three-dimensional living cell microarrays.

We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation <400 nm. In particular, we have assembled networks of Swiss 3T3 fibroblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power <2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.