We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 g/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions.Multivalent cations with a charge of 3ϩ or greater induce the condensation of DNA in aqueous solution (reviewed in Ref. 1). In extremely dilute DNA solutions, one can observe the monomolecular collapse of long chains; with more concentrated DNA solutions (of short or long chains), aggregation sets in. Electrostatic forces appear to be predominant in DNA condensation. For highly charged polyelectrolytes there is a strong electrostatic repulsion between the chains. One expects the addition of multivalent cations to decrease this repulsion. DNA condensation by multivalent cations has been analyzed within the framework of the counterion condensation theory developed by Manning (2). This theory predicts the fraction of the DNA charges neutralized by a given cation: a saturating trivalent cation for instance should neutralize 92% of the DNA charges. It has been shown experimentally that approximately 90% of the DNA charges must be neutralized before DNA condensation can occur (3, 4). In addition, mono-and divalent cations compete with multivalent cations in the condensation process, in agreement with the proposal that the interactions are predominantly electrostatic. It is known, however, that a purely electrostatic model is insufficient to account for the experimental data; cobalthexamine is for instance five times more efficient as a condensing agent than spermidine, although these compounds have the same charge (3ϩ) (4).DNA condensation has been studied with the naturally occurring polyamines spermidine (3ϩ) and spermine (4ϩ), as well as with the inorganic cation cobalthexamine (3ϩ). Experimental data indicate that condensation is usually coupled with an isotropic to an anisotropic transition. In particular, high molecular weight DNA aggregates formed by spermidine, spermine, or cobalthexamine give a strong equatorial reflection when analyzed by x-ray diffraction (5, 6). Based on these data, several types of crystalline and liquid crystalline structures have been suggested for these DNA aggregates (5, 7). We have recently undertaken a study of these DNA aggregates using short (about 150 base pairs long) DNA molecules (8). In the case of the triv...

Author contributions A.O. conceived the project and supervised all ionic current measurements. A.A. conceived and supervised the modeling part of the project and contributed to design of experiments. H.O. carried out experiments and performed data analysis. K.S. performed all MD simulations and SEM calculations and developed the theoretical model. F.P. developed data analysis methods and applied it to experiment results. J.P. contributed to design of the project, suggested the experiment to split suspected cysteine dimers using dithiothreitol, participated to data interpretation and in the general discussion. P.M. participated to the project discussion, suggested an interpretation for the two peaks found for proline and participated in the general discussion and data interpretation, write a draft to answer the referee questions. T.E. with H.O. performed experiments on the high-resolution set-up and analyzed data. J.C.B. conceived and supervised high-resolution recordings, contributed software for data analysis of complex resistive pulses, analyzed data, prepared Supplementary Figs. 12-15 and wrote Supplementary Note 3 and the pertinent part of the Online Methods. A.A. and A.O. wrote the first draft of the manuscript.

We study the electrophoretic blockades due to entries of partially unfolded proteins into a nanopore as a function of the concentration of the denaturing agent. Short and long pore blockades are observed by electrical detection. Short blockades are due to the passage of completely unfolded proteins, their frequency increases as the concentration of the denaturing agent increases, following a sigmoidal denaturation curve. Long blockades reveal partially folded conformations. Their duration increases as the proteins are more folded. The observation of a Vogel-Fulcher law suggests a glassy behavior.

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.

There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.

We report experimentally the dynamic properties of the entry and transport of unfolded and native proteins through a solid-state nanopore as a function of applied voltage, and we discuss the experimental data obtained as compared to theory. We show an exponential increase in the event frequency of current blockades and an exponential decrease in transport times as a function of the electric driving force. The normalized current blockage ratio remains constant or decreases for folded or unfolded proteins, respectively, as a function of the transmembrane potential. The unfolded protein is stretched under the electric driving force. The dwell time of native compact proteins in the pore is almost 1 order of magnitude longer than that of unfolded proteins, and the event frequency for both protein conformations is low. We discuss the possible phenomena hindering the transport of proteins through the pores, which could explain these anomalous dynamics, in particular, electro-osmotic counterflow and protein adsorption on the nanopore wall.

Conditions of formation of DNA aggregates by the addition of spermidine were determined with 146 base pair DNA fragments as a function of spermidine and NaCl concentration. Two different phases of spermidine-DNA complexes are obtained: a cholesteric liquid crystalline phase with a large helical pitch, with interhelix distances ranging from 31.6 to 32.6 A, and a columnar hexagonal phase with a restricted fluidity in which DNA molecules are more closely packed (29.85 +/- 0.05 A). In both phases, the DNA molecule retains its B form. These phases are always observed in equilibrium with the dilute isotropic solution, and their phase diagram is defined for a DNA concentration of 1 mg/ml. DNA liquid crystalline phases induced by spermidine are compared with the DNA mesophases already described in concentrated solutions in the absence of spermidine. We propose that the liquid crystalline character of the spermidine DNA complexes is involved in the stimulation of the functional properties of the DNA reported in numerous experimental articles, and we discuss how the nature of the phase could regulate the degree of activity of the molecule.

We report experimentally the transport of an unfolded protein through a narrow solid-state nanopore of 3 nm diameter as a function of applied voltage. The random coil polypeptide chain is larger than the nanopore. The event frequency dependency of current blockades from 200 to 750 mV follows a van't Hoff-Arrhenius law due to the confinement of the unfolded chain. The protein is an extended conformation inside the pore at high voltage. We observe that the protein dwell time decreases exponentially at medium voltage and is inversely proportional to voltage for higher values. This is consistent with the translocation mechanism where the protein is confined in the pore, creating an entropic barrier, followed by electrophoretic transport. We compare these results to our previous work with a larger pore of 20 nm diameter. Our data suggest that electro-osmotic flow and protein adsorption on the narrowest nanopore wall are minimized. We discuss the experimental data obtained as compared with recent theory for the polyelectrolyte translocation process. This theory reproduces clearly the experimental crossover between the entropic barrier regime with medium voltage and the electrophoretic regime with higher voltage.