The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Voltage-Driven DNA Translocations through a Nanopore We measure current blockade and time distributions for single-stranded DNA polymers during voltagedriven translocations through a single a-hemolysin pore. We use these data to determine the velocity of the polymers in the pore. Our measurements imply that, while polymers longer than the pore are translocated at a constant speed, the velocity of shorter polymers increases with decreasing length. This velocity is nonlinear with the applied field. Based on this data, we estimate the effective diffusion coefficient and the energy penalty for extending a molecule into the pore.

A variety of different DNA polymers were electrophoretically driven through the nanopore of an ␣-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as ϳT ؊2 . A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.T he discovery that a voltage gradient can drive single-stranded RNA or DNA molecules through a 2-nm transmembrane channel, or nanopore, has opened up the possibility of detecting and characterizing unlabeled polynucleotide molecules at low copy number by using single-channel recording techniques. Because an extended molecule of DNA or RNA can occupy, and thus block, much of an otherwise open aqueous channel, the passage of a single polynucleotide can be monitored by recording the translocation duration and blockade current (magnitude of the reduced ionic flow through the pore) (1). Studies with RNAs of differing base composition have begun to suggest how nanopores could be used to discriminate between different nucleic acid polymers (2).We now extend these observations and provide evidence that each of several different DNA polymers can be identified by a unique pattern in ''event diagrams,'' which are plots of translocation duration vs. blockade current for an ensemble of events. Patterns for a given polymer can be characterized uniquely by three statistical parameters representing the most probable translocation current, I P , the most probable translocation duration, t P , and the characteristic dispersion of values for individual translocation durations, T . Because each type of polynucleotide we use gives rise to specific values of these three parameters, these parameters' values can be used to discriminate rapidly between different types of polynucleotides in a mixed sample. Temperature markedly affects these parameters in two ways. The translocation duration scales as T Ϫ2 for all polymers tested, where T is temperature in°C. The other two parameters exhibit a strong temperature dependence for only those polymers that are known to have stable secondary structure. Materials and MethodsSingle channels were formed in a horizontal bilayer of diphytanoyl phosphatidylcholine by using the protein ␣-hemolysin from Staphylococcus aureus. The horizontal bilayer was formed across a ...

Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or ''scaffolds'', are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein -protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.

Chronic stress causes atrophy of the apical dendrites of CA3 pyramidal neurons and deficits in spatial memory. We investigated the effects of chronic stress on hippocampal physiology and long-term potentiation (LTP) in the CA3 and dentate gyrus (DG). Rats were subjected to chronic (21 days, 6 h/day) restraint stress and tested for LTP 48 h following the last stress episode. Control animals were briefly handled each day, similar to the experimental group but without restraint. To eliminate acute stress effects, a second control group of rats was subjected to a single acute (6 h) restraint stress and tested for LTP 48 h later. Field potential recordings were made, under chloropent anesthesia, from the stratum lucidum of CA3, with stimulation of either the mossy fiber or commissural/associational pathways, or in the DG granule-cell layer, with stimulation of the medial perforant pathway. Chronic stress produced a suppression of LTP at 48 h compared to controls in a site-specific manner, namely, significantly lower LTP in the medial perforant input to the DG and also in the commissural/associational input to the CA3, but not in the mossy fiber input to CA3. The animals subjected to acute stress and tested 48 h later did not show a suppression in LTP. High-frequency stimulation (HFS) of the commissural/associational and mossy fiber inputs to CA3 produced epileptic afterdischarges in 56% of acutely stressed animals and in 29% of chronically stressed animals, whereas HFS caused afterdischarges in only 9% of nonstressed controls. No afterdischarges were seen in the medial perforant path input to DG. In order to explore the basis for these changes, we performed paired-pulse inhibition/facilitation (PPI/F) and current-source-density (CSD) analysis in stressed and control animals. For PPI/F, acute stress caused an overall significant enhancement of excitation in the commissural/associational input to CA3 and medial perforant path input to DG. In contrast, chronic stress did not produce significant changes in PPI/F. The CSD analysis revealed significant chronic stress-induced shifts in the current sources and sinks in the apical dendrites and pyramidal cell layers of the CA3 field but not in the DG. These results are consistent with the morphological findings for stress effects upon dendrites of CA3 neurons. Furthermore, they suggest that chronic stress produces changes in the input-output relationship in the hippocampal trisynaptic circuit which could affect information flow through this structure.