Nanobiotechnology
Research Activities
- Assessment of the USMS measurement needs in Nanobiotechnology. The first U.S. Measurement System (USMS) Workshop on Measurement and Standards Needs in Nanobiotechnology was held on
- Approximately 100 workshop attendees heard introductory talks by Kathleen Matthews (Dean of the Wiess School of Natural Sciences and Stewart Professor of Biochemistry and Cell Biology), S. Ward Casscells (John Edward Tyson Distinguished Professor of Medicine, Cardiology, and Public Health, and Vice President for Biotechnology, the University of Texas Health Science Center at Houston), and Hratch Semerjian (then Deputy Director of NIST).
- Plenary talks on a wide range of topics in nanobiotechnology were given by Scott McNeil (director, Nanotechnology Characterization Laboratory, NCI), David Luzzi (director, Nanotechnology Institute, University of Pennsylvania), Nakissa Sadrieh (associate director for Research Policy and Implementation, Food and Drug Administration), Nigel Walker (National Institute of Environmental Health Sciences, Toxicology Operations) and Vicki Colvin (Director, Center for Biological and Environmental Nanotechnology and Chair of ASTM Committee E56 on Nanotechnology).
- To help identify specific measurement and stan-dards needs in nanobiotechnology, the workshop attendees participated in five concurrent sessions on topics that included Medical/Clinical/Pharmaceutical Issues (drug delivery, targeted therapeutic agents, nanomaterials devices drug discovery), Medical Imaging (nano-based contrast agents, other imaging modalities), manufacturing (nanoparticle handling, manufacturing, safety, scale-up), basic science (structure and dynamics of biomolecules, sensing at the single molecule level), and physical characterization (dimension, composition, purity, surface chemistry, instrumentation). The suggestions obtained from these breakout sessions will aid NIST’s USMS road mapping efforts. The workshop provides direct stakeholder input to the comprehensive USMS assessment process.
- Probing the structure of single nanometer-scale pores with DNA molecular rulers. Over a decade ago, NIST demonstrated that individual molecules of single stranded DNA can be driven electrophoretically through a single Staphylococcus aureus α-hemolysin ion (αHL) channel. Polynucleotides thread through the channel as extended chains, and the polymer-induced ionic current blockades exhibit stable modes during the interactions. Recently, polynucleotides were used to probe structural features of the α-hemolysin nanopore. Specifically, both the pore length and channel aperture profile were estimated by using different length polynucleotides with a large macromolecule tethered to one end. The results are consistent with the channel’s crystal structure and suggest that polymer-based “molecular rulers” may prove useful in deducing the structures of other nanometer-scale pores, including solid-state varietals.
- Electronic readout of information in polymers aided by advanced statistical signal processing. The electronic signatures caused by threading single-stranded DNA through a single nanometer-scale pore are characteristic of the polymer’s type and length (see figure below). A method for characterizing these signals using ergodic, but persistent Hidden Markov Models was developed. Gaussian mixture models were used as output distributions to obtain a maximum likelihood estimate of state sequence and lifetime. The results are consistent with the known structure of the nanopore and suggest an approach to decode information stored in DNA and other polymers. This technology may ultimately provide the basis for rapid DNA sequencing with single nanopores.
- Proof-of-concept for a single molecule mass spectrometer. Preliminary results suggest that a solitary protein nanometer-scale pore provides the basis for single molecule mass spectrometry. The pore conductance is reduced in proportion to the size of individual polymers that enter the pore. Specifically, each molecule that partitions into the pore gives rise to a well-defined conductance state that corresponds to its molecular weight. Because the polymer and nanopore sizes are commensurate, the conductance states caused by differently sized polymers are well separated. With this calibrated solution phase mass spectrometry technique, individual molecules could be further analyzed or sorted. This research is being performed in collaboration with colleagues at the Universidade Federal de Pernambuco in
- Test of a model for the structure of the an-thrax toxin nanopore. Non-electrolyte polymers of poly(ethylene glycol), PEG, are being used to estimate the diameter of the PA63 ion channel. Based on the ability of different molecular weight PEGs to partition into the channel, the PA63 pore appears to be narrower than the one formed by Staphylococcus aureus α-hemolysin. These results are consistent with a recent model for the PA63 channel and the crystal structure of α-hemolysin. The results place a significant constraint on models for this molecule’s mechanism of action.
- Functional reconstitution of protein nanopores in tethered bilayer membranes: structural studies. The crystal structures of many transmembrane proteins remain elusive. In an effort to partially address this issue, neutron reflectance is being used to obtain moderate resolution structural information about pore-forming toxins. The α-hemolysin ion channel was used as a model system because its X-ray structure is known. The technique requires reconstituting the proteins into stable, tethered bilayer membranes on a solid support. Electrochemical Impedance Spectroscopy (EIS) measurements demonstrate that the pores have the same properties as those formed in free-standing lipid bilayer membranes. Neutron reflectometry (NR) shows that the channel’s large protein cap domain is located outside the membrane yet significantly affects both the lipid head group region and the alkyl chains of the outer membrane leaflet. An analysis of the neutron reflectance data, in conjunction with the channel’s X-ray crystal structure, provides high-resolution structural information about the interaction between the channel and the membrane. The results suggest that the combination of EIS and NR could be used to probe the structure of other ion channels.
- Comparison of theory and experiment for ionic conduction through protein ion channels. In collaboration with researchers at U. Pittsburgh, theoretical current-voltage (I-V) relationships for the Staphylococcus aureus α-hemolysin channel are being obtained via three-dimensional Poisson-Nernst-Planck (PNP) computations. The molecular structure of the channel employed is the X-ray crystal structure obtained by E. Gouaux’s group at
- Mechanism of ionic conductance and ion selectivity of a mesoscopic protein nanopore probed with cysteine scanning mutagenesis. To better understand the structure-function relationship of protein nanopores, the ion conducting properties of channels formed by wild-type and genetically engineered versions of Staphylococcus aureus α-hemolysin (αHL) reconstituted into planar lipid bilayer membranes were studied. The ion selectivities and current-voltage relationships of channels formed with 24 different αHL point cysteine mutants (before and after derivatizing the cysteines with positively and negatively charged sulfhydryl-specific reagents) were measured. Novel negative charges convert the selectivity of the channel from weakly anionic to strongly cationic, and new positive charges increase the anionic selectivity. The extent of these changes depends on the channel radius at the position of the novel charge (predominately affects ion selectivity) or on the location of these charges along the longitudinal axis of the channel (mainly alters the conductance-voltage curve). The results suggest that the net charge of the nanopore wall is responsible for cation-anion selectivity of the αHL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves.
- Electronic measurement of the interaction between anthrax lethal toxins. Previous biochemical assays performed elsewhere demonstrated that full-length anthrax Lethal Factor (LF) binds strongly to the Bacillus anthracis PA63 ion channel. A electronic assay was developed at NIST to rapidly measure the binding constant for the interaction. The binding of LF to the PA63 channel converts the pore’s I-V relationship from slightly nonlinear to strongly rectifying (see right figure). This method also provided the basis for a high-throughput screening assay for therapeutic agents against anthrax toxins. It was also used to demonstrate that the apparent binding constant for the complex is ~ 40 pM at pH 7.2 and is ~ 3-fold weaker when the pH is decreased to a value similar to that in endosomal vesicles. These and similar results suggest that a simple reversible reaction between LF and the PA63 channel is altered by changes in the local electrostatic potentials on the two proteins. This work is a collaborative effort with the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID) and the NCI.
- Characterizing ultra-thin films for the practical use of protein nanopore technology. Conventional planar lipid bilayer membranes permit the functional reconstitution of many transmembrane proteins. However, they are not sufficiently robust for use in practical applications. To address that issue, the ability of α-hemolysin and Bacillus anthracis PA63 to form ionic channels when the membranes were in the fluid phase was demonstrated. In addition, the αHL channel remained functional even after UV-induced lipid polymerization. The ability to stabilize nanopores in robust films could lead to their use in a wide variety of applications including single molecule sensing, rapid DNA sequencing and single molecule mass spectrometry. However, more must be known about the state of the lipid under polymerizing conditions.
- The use of biomimetic membranes on solid supports to characterize enzymatic activity. Using phospholipase as a sensitive amplifier of defect states, the structural order in the surfaces of tethered lipid bilayers on solid substrates was characterized using electrochemical impedance spectroscopy. The defect-mediated enzyme activity on palmitoyloleoylphosphocholine is ~ 2 orders of magnitude larger than on diphytanoyl-phosphocholine surfaces and is consistent with the known mechanism of action of the lipase.
- NIST was awarded a patent on the use of designed protein nanopores as components for biosensors. Based on the work initially conducted at NIST in the early 1990s, and then further developed in collaboration with a research group at
- Personnel. Dr. Brian Nablo (NRC Research Associate) is studying the mechanism of interac-tions between anthrax toxins. He is also develop-ing new methods to probe structural features of the Bacillus anthracis PA63 channel. Dr. Joseph W.F. Robertson (NRC Research Associate) is developing novel technical applications for single nanopore analysis of DNA and other polymeric molecules. He recently demonstrated a new method for single molecule mass spectrometry based on the interaction between individual molecules and a solitary nanopore. Dr. Elaine Chan (NRC Research Associate), who recently joined the Nanobiotechnology Project, is investigating experimentally and theoretically the physical basis of ionic conduction through the anthrax PA63 channel. Dr. Joseph Reiner, formerly a NRC Research Associate in the NIST Physics Laboratory was recently hired in our project. He is developing single molecule optical methods that will complement and extend electronic measurements on single protein and solid-state nanopores. Dr. Martin Misakian, who retired from NIST EEEL and later returned as a Guest Researcher in our project, has been using nonelectrolyte polymers to experimentally test a model for the PA63 channel structure. Dr. Kenneth Rubinson (Guest Researcher) is using his expertise in chemical synthesis and analytical chemistry to further develop new technical applications based on nanopores. Louis Hromada (graduate student) is developing a microfluidic system to automate the formation of lipid bilayer membranes.
