Observing Changes in the Structure and Oligomerization State of a Helical Protein Dimer Using Solid-State Nanopores.

Protein analysis using solid-state nanopores is challenging due to limitations in bandwidth and signal-to-noise ratio. Recent improvements of those two aspects have made feasible the study of small peptides using solid-state nanopores, which have an advantage over biological counterparts in tunability of the pore diameter. Here, we report on the detection and characterization of peptides as small as 33 amino acids. Silicon nitride nanopores with thicknesses less than 10 nm are used to provide signal-to-noise (S/N) levels up to S/N ∼ 10 at 100 kHz. We demonstrate differentiation of monomer and dimer forms of the GCN4-p1 leucine zipper, a coiled-coil structure well studied in molecular biology, and compare with the unstructured 33-residue monomer. GCN4-p1 is sequence segment associated with homodimerization of the transcription factor General Control Nonderepressible 4 (GCN4), which is involved in the control of amino acid synthesis in yeast. The differentiation between two oligomeric forms demonstrates the capabilities of improved solid-state nanopore platforms to extract structural information involving short peptide structures.

Differentiation of short, single-stranded DNA homopolymers in solid-state nanopores.

In the last two decades, new techniques that monitor ionic current modulations as single molecules pass through a nanoscale pore have enabled numerous single-molecule studies. While biological nanopores have recently shown the ability to resolve single nucleotides within individual DNA molecules, similar developments with solid-state nanopores have lagged, due to challenges both in fabricating stable nanopores of similar dimensions as biological nanopores and in achieving sufficiently low-noise and high-bandwidth recordings. Here we show that small silicon nitride nanopores (0.8- to 2-nm diameter in 5- to 8-nm-thick membranes) can resolve differences between ionic current signals produced by short (30 base) ssDNA homopolymers (poly(dA), poly(dC), poly(dT)), when combined with measurement electronics that allow a signal-to-noise ratio of better than 10 to be achieved at 1-MHz bandwidth. While identifying intramolecular DNA sequences with silicon nitride nanopores will require further improvements in nanopore sensitivity and noise levels, homopolymer differentiation represents an important milestone in the development of solid-state nanopores.

Entropy-driven collective interactions in DNA brushes on a biochip.

Cell-free gene expression in localized DNA brushes on a biochip has been shown to depend on gene density and orientation, suggesting that brushes form compartments with partitioned conditions. At high density, the interplay of DNA entropic elasticity, electrostatics, and excluded volume interactions leads to collective conformations that affect the function of DNA-associated proteins. Hence, measuring the collective interactions in dense DNA, free of proteins, is essential for understanding crowded cellular environments and for the design of cell-free synthetic biochips. Here, we assembled dense DNA polymer brushes on a biochip along a density gradient and directly measured the collective extension of DNA using evanescent fluorescence. DNA of 1 kbp in a brush undergoes major conformational changes, from a relaxed random coil to a stretched configuration, following a universal function of density to ionic strength ratio with scaling exponent of 1/3. DNA extends because of the swelling force induced by the osmotic pressure of ions, which are trapped in the brush to maintain local charge neutrality, in competition with the restoring force of DNA entropic elasticity. The measurements reveal in DNA crossover between regimes of osmotic, salted, mushroom, and quasineutral brush. It is surprising to note that, at physiological ionic strength, DNA density does not induce collective stretch despite significant chain overlap, which implies that excluded volume interactions in DNA are weak.

Collective conformations of DNA polymers assembled on surface density gradients.

To study dense double-stranded DNA (dsDNA) polymer phases, we fabricated continuous density gradients of binding sites for assembly on a photochemical interface and measured both dsDNA occupancy and extension using evanescent fluorescence. Despite the abundance of available binding sites, the dsDNA density saturates after occupation of only a fraction of the available sites along the gradient. The spatial position at which the density saturates marks the onset of collective stretching of dsDNA, a direct manifestation of balancing entropic and excluded-volume interactions. The methodology presented here offers a new means to investigate dense dsDNA compartments.

Chirality of silver nanoparticles synthesized on DNA.

Silver nanocrystals grown on a poly(dG)-poly(dC) double stranded DNA scaffold displayed circular dichroism at their surface plasmon excitation band. This chiral plasmon signature was not observed in a control experiment where silver nanocrystals of similar size were produced without the DNA template and adsorbed to the DNA. It is concluded that the DNA templated Ag nanocrystals had a preferred structural handedness.