Nanoscale pores have proved useful as a means to assay DNA and are actively being developed as the basis of genome sequencing methods. Hairpin DNA (hpDNA), having both double-helical and overhanging coil portions, can be trapped in a nanopore, giving ample time to execute a sequence measurement. In this article, we provide a detailed account of hpDNA interaction with a synthetic nanopore obtained through extensive all-atom molecular dynamics simulations. For synthetic pores with minimum diameters from 1.3 to 2.2 nm, we find that hpDNA can translocate by three modes: unzipping of the double helix and--in two distinct orientations--stretching/distortion of the double helix. Furthermore, each of these modes can be selected by an appropriate choice of the pore size and voltage applied transverse to the membrane. We demonstrate that the presence of hpDNA can dramatically alter the distribution of ions within the pore, substantially affecting the ionic current through it. In experiments and simulations, the ionic current relative to that in the absence of DNA can drop below 10% and rise beyond 200%. Simulations associate the former with the double helix occupying the constriction and the latter with accumulation of DNA that has passed through the constriction.

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.Restriction enzymes are used prevalently in recombinant DNA technology for cleaving doublehelical DNA segments containing a specific target sequence. Another use is genotyping. Because the binding to the target is extraordinarily sequence specific, restriction enzymes can be used to identify single nucleotide polymorphisms (SNPs) that occur when variants of the same gene (alleles) differ in one base.We have discovered a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. When a voltage is applied across a membrane containing a nanopore, polyanionic DNA immersed in electrolyte at the cathode diffuses toward the anode and is driven across the membrane by the electric field in the pore. The force due to the field acting on the strand during the translocation impels DNA to bend and stretch within the pore. 1-4 At low fields ℰ < 500mV/10nm, double-stranded DNA (dsDNA) easily permeates pores with diameters ≥2.4nm because the double helix (~2nm diameter) is smaller than the pore. 5 But the permeability of DNA through the pore changes dramatically if it is bound to a restriction enzyme.To study the binding of a restriction enzyme like EcoRI to DNA, we introduced an excess of the enzyme in solution with DNA without the Mg +2 cofactor that is required for cleaving the nucleic acid. Under these conditions, EcoRI is thought to bind and diffuse along DNA. 6,7 The diffusive motion along the strand is arrested at the cognate site, i.e. -GAATTC-for EcoRI. Bulk measurements of the binding at the cognate site indicate a free energy of formation ΔG =−15.2kcal/mol. 6-9 However, the introduction of any mutation among the cognate sites produces a position-dependent reduction in the binding energy that ranges from 6-13kcal/mol. 8,9 Site-specific DNA-binding proteins also have an affinity for nonspecific DNA. In contrast with site-specific binding or binding to a non-cognate site with a single nucleotide mutation, a nonspecifically bound complex is not localized to a particular site. For EcoRI, sites that differ from the cognate sequence by two or more base-pairs(bps) are considered nonspecific since they are not cleaved and show low binding constants. For a nonspecifically bound EcoRI-DNA complex, the free energy of formation is reduced to −4.8kcal/mol. 8,9We measured the permeability of dsDNA in solution with EcoRI ...

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.

Multiplexing multiple orbital angular momentum (OAM) channels enables high-capacity optical communication. However, optical scattering from ambient microparticles in the atmosphere or mode coupling in optical fibers significantly decreases the orthogonality between OAM channels for demultiplexing and eventually increases crosstalk in communication. Here, we propose a novel scattering-matrix-assisted retrieval technique (SMART) to demultiplex OAM channels from highly scattered optical fields and achieve an experimental crosstalk of –13.8 dB in the parallel sorting of 24 OAM channels after passing through a scattering medium. The SMART is implemented in a self-built data transmission system that employs a digital micromirror device to encode OAM channels and realize reference-free calibration simultaneously, thereby enabling a high tolerance to misalignment. We successfully demonstrate high-fidelity transmission of both gray and color images under scattering conditions at an error rate of <0.08%. This technique might open the door to high-performance optical communication in turbulent environments.

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.