Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths.

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.

DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by τclosed. Under Vmax conditions, the average time of τclosed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by τopen, which includes molecular recognition of the incoming dNTP. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair.

Observing lysozyme's closing and opening motions by high-resolution single-molecule enzymology.

Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 µs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 µs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 µs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.

Electrochemical charge-transfer resistance in carbon nanotube composites.

Using a model system of single, isolated carbon nanotubes loaded with high-capacitance metal-oxide films, we have quantitatively investigated electrochemical composites on the single-nanotube scale. Electrochemical charging and discharging of a model MnO2 storage material was used to probe interfacial charge transfer and surface impedances at the nanotube interface. We found that one single-walled carbon nanotube has an apparent surface resistivity of 30 mΩ cm(2), approximately 4 times smaller than for a multiwalled carbon nanotube and 50 times smaller than the 1.5 Ω cm(2) resistivity of Pt or graphite films. The improvement originates in the electrochemical-transport properties of microelectrodes shrunk to a nanotube's dimensions rather than any unique nanotube property like curvature, bandstructure, or surface chemistry. In explaining the enhanced performance of certain nanotube-containing composites, the results overturn widely held assumptions about nanotubes' roles while also providing guidelines for optimizing effective composites.

Electronic measurements of single-molecule catalysis by cAMP-dependent protein kinase A.

Single-molecule studies of enzymes open a window into their dynamics and kinetics. A single molecule of the catalytic domain of cAMP-dependent protein kinase A (PKA) was attached to a single-walled carbon nanotube device for long-duration monitoring. The electronic recording clearly resolves substrate binding, ATP binding, and cooperative formation of PKA's catalytically functional, ternary complex. Using recordings of a single PKA molecule extending over 10 min and tens of thousands of binding events, we determine the full transition probability matrix and conversion rates governing formation of the apo, intermediate, and closed enzyme configurations. We also observe kinetic rates varying over 2 orders of magnitude from one second to another. Anti-correlation of the on and off rates for PKA binding to the peptide substrate, but not ATP, demonstrates that regulation of enzyme activity results from altering the stability of the PKA-substrate complex, not its binding to ATP. The results depict a highly dynamic enzyme offering dramatic possibilities for regulated activity, an attribute useful for an enzyme with crucial roles in cell signaling.

Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment).

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.

Dissecting single-molecule signal transduction in carbon nanotube circuits with protein engineering.

Single-molecule experimental methods have provided new insights into biomolecular function, dynamic disorder, and transient states that are all invisible to conventional measurements. A novel, nonfluorescent single-molecule technique involves attaching single molecules to single-walled carbon nanotube field-effective transistors (SWNT FETs). These ultrasensitive electronic devices provide long-duration, label-free monitoring of biomolecules and their dynamic motions. However, generalization of the SWNT FET technique first requires design rules that can predict the success and applicability of these devices. Here, we report on the transduction mechanism linking enzymatic processivity to electrical signal generation by a SWNT FET. The interaction between SWNT FETs and the enzyme lysozyme was systematically dissected using eight different lysozyme variants synthesized by protein engineering. The data prove that effective signal generation can be accomplished using a single charged amino acid, when appropriately located, providing a foundation to widely apply SWNT FET sensitivity to other biomolecular systems.

Single-molecule lysozyme dynamics monitored by an electronic circuit.

Tethering a single lysozyme molecule to a carbon nanotube field-effect transistor produced a stable, high-bandwidth transducer for protein motion. Electronic monitoring during 10-minute periods extended well beyond the limitations of fluorescence techniques to uncover dynamic disorder within a single molecule and establish lysozyme as a processive enzyme. On average, 100 chemical bonds are processively hydrolyzed, at 15-hertz rates, before lysozyme returns to its nonproductive, 330-hertz hinge motion. Statistical analysis differentiated single-step hinge closure from enzyme opening, which requires two steps. Seven independent time scales governing lysozyme's activity were observed. The pH dependence of lysozyme activity arises not from changes to its processive kinetics but rather from increasing time spent in either nonproductive rapid motions or an inactive, closed conformation.

Single-molecule dynamics of lysozyme processing distinguishes linear and cross-linked peptidoglycan substrates.

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate.