Equilibrium Collapse and the Kinetic 'foldability' of Proteins

Biochemistry. Jan, 2002  |  Pubmed ID: 11772031

An important element of protein folding theory has been the identification of equilibrium parameters that might uniquely distinguish rapidly folding polypeptide sequences from those that fold slowly. One such parameter, termed sigma, is a dimensionless, equilibrium measure of the coincidence of chain compaction and folding that is predicted to be an important determinant of relative folding kinetics. To test this prediction and improve our understanding of the putative relationship between nonspecific compaction of the unfolded state and protein folding kinetics, we have used small-angle X-ray scattering and circular dichroism spectroscopy to measure the sigma of five well-characterized proteins. Consistent with theoretical predictions, we find that near-perfect coincidence of the unfolded state contraction and folding (sigma approximately 0) is associated with the rapid kinetics of these naturally occurring proteins. We do not, however, observe any significant correlation between sigma and either the relative folding rates of these proteins or the presence or absence of well-populated kinetic intermediates. Thus, while sigma approximately 0 may be a necessary condition to ensure rapid folding, differences in sigma do not account for the wide range of rates and mechanisms with which naturally occurring proteins fold.

Residues Participating in the Protein Folding Nucleus Do Not Exhibit Preferential Evolutionary Conservation

Journal of Molecular Biology. Feb, 2002  |  Pubmed ID: 11851333

To what extent does natural selection act to optimize the details of protein folding kinetics? In an effort to address this question, the relationship between an amino acid's evolutionary conservation and its role in protein folding kinetics has been investigated intensively. Despite this effort, no consensus has been reached regarding the degree to which residues involved in native-like transition state structure (the folding nucleus) are conserved. Here we report the results of an exhaustive, systematic study of sequence conservation among residues known to participate in the experimentally (Phi-value) defined folding nuclei of all of the appropriately characterized proteins reported to date. We observe no significant evidence that these residues exhibit any anomalous sequence conservation. We do observe, however, a significant bias in the existing kinetic data: the mean sequence conservation of the residues that have been the subject of kinetic characterization is greater than the mean sequence conservation of all residues in 13 of 14 proteins studied. This systematic experimental bias gives rise to the previous observation that the median conservation of residues reported to participate in the folding nucleus is greater than the median conservation of all of the residues in a protein. When this bias is corrected (by comparing, for example, the conservation of residues known to participate in the folding nucleus with that of other, kinetically characterized residues) the previously reported preferential conservation is effectively eliminated. In contrast to well-established theoretical expectations, both poorly and highly conserved residues are apparently equally likely to participate in the protein-folding nucleus.

How the Folding Rate Constant of Simple, Single-domain Proteins Depends on the Number of Native Contacts

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2002  |  Pubmed ID: 11904417

Experiments have shown that the folding rate constants of two dozen structurally unrelated, small, single-domain proteins can be expressed in terms of one quantity (the contact order) that depends exclusively on the topology of the folded state. Such dependence is unique in chemical kinetics. Here we investigate its physical origin and derive the approximate formula ln(k) = ln(N) + a + bN, were N is the number of contacts in the folded state, and a and b are constants whose physical meaning is understood. This formula fits well the experimentally determined folding rate constants of the 24 proteins, with single values for a and b.

High-efficiency Fluorescence Quenching of Conjugated Polymers by Proteins

Journal of the American Chemical Society. May, 2002  |  Pubmed ID: 12010029

The fluorescence of the water-soluble anionic conjugated polymer, poly[lithium 5-methoxy-2-(4-sulfobutoxy)-1,4-phenylenevinylene] (MBL-PPV), is quenched in dilute aqueous solution by cytochrome c, a small, naturally occurring electron-transfer protein. The large value obtained for the Stern-Volmer constant (K(sv) = 3.2 x 10(8) at pH 7.4, and approximately 10(9) in acidic solutions) is attributed to a combination of two factors: (1) facile ET between the luminescent semiconducting polymer and the protein and (2) the Columb attraction between the oppositely charged polyelectrolytes. This system shows significant potential for biosensor applications.

The Backbone Conformational Entropy of Protein Folding: Experimental Measures from Atomic Force Microscopy

Journal of Molecular Biology. Sep, 2002  |  Pubmed ID: 12225756

The energy dissipated during the atomic force microscopy-based mechanical unfolding and extension of proteins is typically an order of magnitude greater than their folding free energy. The vast majority of the "excess" energy dissipated is thought to arise due to backbone conformational entropy losses as the solvated, random-coil unfolded state is stretched into an extended, low-entropy conformation. We have investigated this hypothesis in light of recent measurements of the energy dissipated during the mechanical unfolding of "polyproteins" comprised of multiple, homogeneous domains. Given the assumption that backbone conformational entropy losses account for the vast majority of the energy dissipated (an assumption supported by numerous lines of experimental evidence), we estimate that approximately 19(+/-2)J/(mol K residue) of entropy is lost during the extension of three mechanically stable beta-sheet polyproteins. If, as suggested by measured peak-to-peak extension distances, pulling proceeds to near completion, this estimate corresponds to the absolute backbone conformational entropy of the unfolded state. As such, it is exceedingly close to previous theoretical and semi-empirical estimates that place this value at approximately 20J/(mol K residue). The estimated backbone conformational entropy lost during the extension of two helical polyproteins, which, in contrast to the mechanically stable beta-sheet polyproteins, rupture at very low applied forces, is three- to sixfold less. Either previous estimates of the backbone conformational entropy are significantly in error, or the reduced mechanical strength of the helical proteins leads to the rupture of a subsequent domain before full extension (and thus complete entropy loss) is achieved.

Toward a Taxonomy of the Denatured State: Small Angle Scattering Studies of Unfolded Proteins

Advances in Protein Chemistry. 2002  |  Pubmed ID: 12418105

The Topomer Search Model: A Simple, Quantitative Theory of Two-state Protein Folding Kinetics

Protein Science : a Publication of the Protein Society. Jan, 2003  |  Pubmed ID: 12493824

Most small, single-domain proteins fold with the uncomplicated, single-exponential kinetics expected for diffusion on a smooth energy landscape. Despite this energetic smoothness, the folding rates of these two-state proteins span a remarkable million-fold range. Here, we review the evidence in favor of a simple, mechanistic description, the topomer search model, which quantitatively accounts for the broad scope of observed two-state folding rates. The model, which stipulates that the search for those unfolded conformations with a grossly correct topology is the rate-limiting step in folding, fits observed rates with a correlation coefficient of approximately 0.9 using just two free parameters. The fitted values of these parameters, the pre-exponential attempt frequency and a measure of the difficulty of ordering an unfolded chain, are consistent with previously reported experimental constraints. These results suggest that the topomer search process may dominate the relative barrier heights of two-state protein-folding reactions.

Cooperativity, Smooth Energy Landscapes and the Origins of Topology-dependent Protein Folding Rates

Journal of Molecular Biology. Feb, 2003  |  Pubmed ID: 12547206

The relative folding rates of simple, single-domain proteins, proteins whose folding energy landscapes are smooth, are highly dispersed and strongly correlated with native-state topology. In contrast, the relative folding rates of small, Gō-potential lattice polymers, which also exhibit smooth energy landscapes, are poorly dispersed and insignificantly correlated with native-state topology. Here, we investigate this discrepancy in light of a recent, quantitative theory of two-state folding kinetics, the topomer search model. This model stipulates that the topology-dependence of two-state folding rates is a direct consequence of the extraordinarily cooperative equilibrium folding of simple proteins. We demonstrate that traditional Gō polymers lack the extreme cooperativity that characterizes the folding of naturally occurring, two-state proteins and confirm that the folding rates of a diverse set of Gō 27-mers are poorly dispersed and effectively uncorrelated with native state topology. Upon modestly increasing the cooperativity of the Gō-potential, however, significantly increased dispersion and strongly topology-dependent kinetics are observed. These results support previous arguments that the cooperative folding of simple, single-domain proteins gives rise to their topology-dependent folding rates. We speculate that this cooperativity, and thus, indirectly, the topology-rate relationship, may have arisen in order to generate the smooth energetic landscapes upon which rapid folding can occur.

Beyond Superquenching: Hyper-efficient Energy Transfer from Conjugated Polymers to Gold Nanoparticles

Proceedings of the National Academy of Sciences of the United States of America. May, 2003  |  Pubmed ID: 12750470

Gold nanoparticles quench the fluorescence of cationic polyfluorene with Stern-Volmer constants (KSV) approaching 1011 M-1, several orders of magnitude larger than any previously reported conjugated polymer-quencher pair and 9-10 orders of magnitude larger than small molecule dye-quencher pairs. The dependence of KSV on ionic strength, charge and conjugation length of the polymer, and the dimensions (and thus optical properties) of the nanoparticles suggests that three factors account for this extraordinary efficiency: (i) amplification of the quenching via rapid internal energy or electron transfer, (ii) electrostatic interactions between the cationic polymer and anionic nanoparticles, and (iii) the ability of gold nanoparticles to quench via efficient energy transfer. As a result of this extraordinarily high KSV, quenching can be observed even at subpicomolar concentrations of nanoparticles, suggesting that the combination of conjugated polymers with these nanomaterials can potentially lead to improved sensitivity in optical biosensors.

Comparison of the Folding Processes of Distantly Related Proteins. Importance of Hydrophobic Content in Folding

Journal of Molecular Biology. Jul, 2003  |  Pubmed ID: 12842473

The N-terminal domain of HypF from Escherichia coli (HypF-N) is a 91 residue protein module sharing the same folding topology and a significant sequence identity with two extensively studied human proteins, muscle and common-type acylphosphatases (mAcP and ctAcP). With the aim of learning fundamental aspects of protein folding from the close comparison of so similar proteins, the folding process of HypF-N has been studied using stopped-flow fluorescence. While mAcP and ctAcP fold in a two-state fashion, HypF-N was found to collapse into a partially folded intermediate before reaching the fully folded conformation. Formation of a burst-phase intermediate is indicated by the roll over in the Chevron plot at low urea concentrations and by the large jump of intrinsic and 8-anilino-1-naphtalenesulphonic acid-derived fluorescence immediately after removal of denaturant. Furthermore, HypF-N was found to fold rapidly with a rate constant that is approximately two and three orders of magnitudes faster than ctAcP and mAcP, respectively. Differences between the bacterial protein and the two human counterparts were also found as to the involvement of proline isomerism in their respective folding processes. The results clearly indicate that features that are often thought to be relevant in protein folding are not highly conserved in the evolution of the acylphosphatase superfamily. The large difference in folding rate between mAcP and HypF-N cannot be entirely accounted for by the difference in relative contact order or related topological metrics. The analysis shows that the higher folding rate of HypF-N is in part due to the relatively high hydrophobic content of this protein. This conclusion, which is also supported by the highly significant correlation found between folding rate and hydrophobic content within a group of proteins displaying the topology of HypF-N and AcPs, suggests that the average hydrophobicity of a protein sequence is an important determinant of its folding rate.

NMR and Temperature-jump Measurements of De Novo Designed Proteins Demonstrate Rapid Folding in the Absence of Explicit Selection for Kinetics

Journal of Molecular Biology. Jul, 2003  |  Pubmed ID: 12850149

We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1. These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog. Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog. Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date.

Electrochemical Interrogation of Conformational Changes As a Reagentless Method for the Sequence-specific Detection of DNA

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2003  |  Pubmed ID: 12867594

We report a strategy for the reagentless transduction of DNA hybridization into a readily detectable electrochemical signal by means of a conformational change analogous to the optical molecular beacon approach. The strategy involves an electroactive, ferrocene-tagged DNA stem-loop structure that self-assembles onto a gold electrode by means of facile gold-thiol chemistry. Hybridization induces a large conformational change in this surface-confined DNA structure, which in turn significantly alters the electron-transfer tunneling distance between the electrode and the redoxable label. The resulting change in electron transfer efficiency is readily measured by cyclic voltammetry at target DNA concentrations as low as 10 pM. In contrast to existing optical approaches, an electrochemical DNA (E-DNA) sensor built on this strategy can detect femtomoles of target DNA without employing cumbersome and expensive optics, light sources, or photodetectors. In contrast to previously reported electrochemical approaches, the E-DNA sensor achieves this impressive sensitivity without the use of exogenous reagents and without sacrificing selectivity or reusability. The E-DNA sensor thus offers the promise of convenient, reusable detection of picomolar DNA.

Contact Order Revisited: Influence of Protein Size on the Folding Rate

Protein Science : a Publication of the Protein Society. Sep, 2003  |  Pubmed ID: 12931003

Guided by the recent success of empirical model predicting the folding rates of small two-state folding proteins from the relative contact order (CO) of their native structures, by a theoretical model of protein folding that predicts that logarithm of the folding rate decreases with the protein chain length L as L(2/3), and by the finding that the folding rates of multistate folding proteins strongly correlate with their sizes and have very bad correlation with CO, we reexamined the dependence of folding rate on CO and L in attempt to find a structural parameter that determines folding rates for the totality of proteins. We show that the Abs_CO = CO x L, is able to predict rather accurately folding rates for both two-state and multistate folding proteins, as well as short peptides, and that this Abs_CO scales with the protein chain length as L(0.70 +/- 0.07) for the totality of studied single-domain proteins and peptides.

Terahertz Circular Dichroism Spectroscopy: a Potential Approach to the in Situ Detection of Life's Metabolic and Genetic Machinery

Astrobiology. 2003  |  Pubmed ID: 14678660

We propose a terahertz (far-infrared) circular dichroism-based life-detection technology that may provide a universal and unequivocal spectroscopic signature of living systems regardless of their genesis. We argue that, irrespective of the specifics of their chemistry, all life forms will employ well-structured, chiral, stereochemically pure macromolecules (>500 atoms) as the catalysts with which they perform their metabolic and replicative functions. We also argue that nearly all such macromolecules will absorb strongly at terahertz frequencies and exhibit significant circular dichroism, and that this circular dichroism unambiguously distinguishes biological from abiological materials. Lastly, we describe several approaches to the fabrication of a terahertz circular dichroism spectrometer and provide preliminary experimental indications of their feasibility. Because terahertz circular dichroism signals arise from the molecular machinery necessary to carry out life's metabolic and genetic processes, this life-detection method differs fundamentally from more well-established approaches based on the detection of isotopic fractionation, "signature" carbon compounds, disequilibria, or other by-products of metabolism. Moreover, terahertz circular dichroism spectroscopy detects this machinery in a manner that makes few, if any, assumptions as to its chemical nature or the processes that it performs.

Using Protein Folding Rates to Test Protein Folding Theories

Annual Review of Biochemistry. 2004  |  Pubmed ID: 15189160

The fastest simple, kinetically two-state protein folds a million times more rapidly than the slowest. Here we review many recent theories of protein folding kinetics in terms of their ability to qualitatively rationalize, if not quantitatively predict, this fundamental experimental observation.

Random-coil Behavior and the Dimensions of Chemically Unfolded Proteins

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2004  |  Pubmed ID: 15314214

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

Apparent Debye-Huckel Electrostatic Effects in the Folding of a Simple, Single Domain Protein

Biochemistry. Feb, 2005  |  Pubmed ID: 15667218

We have monitored the effects of salts and denaturants on the folding of the simple, two-state protein FynSH3. As predicted by Debye-Huckel limiting law, both the stability and (log) folding rate of FynSH3 increase nearly perfectly linearly (r(2)> 0.99) with the square root of ionic strength upon increasing concentrations of the relatively nonchaotropic salt sodium chloride. The stability of FynSH3 is also linear in square root ionic strength when the relatively nonchaotropic salts sodium bromide, potassium bromide, and potassium chloride are employed. Comparison of the kinetic and equilibrium effects of sodium chloride suggests that the electrostatic interactions formed in the folding transition state are approximately 50% as destabilizing as those formed in the native state, presumably reflecting the more compact nature of the latter. In contrast, the relationship between concentration and folding kinetics is more complex when the highly chaotropic salt guanidine hydrochloride (GuHCl) is employed. At moderate to high GuHCl concentrations the net effect of the linear, presumably chaotrope-induced deceleration and the presumed, square root-dependent ionic strength-induced acceleration is well approximated as linear, thus accounting for the observation of "chevron behavior" (log folding rate linear in denaturant concentration) typically reported for the folding of single domain proteins. At very low GuHCl concentrations, however, significant kinetic rollover is observed. This rollover is reasonably well fitted as a sum of a linear, presumably chaotropic effect and a square root-dependent, presumably electrostatic effect. These results thus not only provide insight into the nature of the folding transition state but also suggest that caution is in order when extrapolating GuHCl-based chevrons to estimate folding rates in the absence of denaturant and in interpreting deviations from chevron linearity as evidence for non-two-state kinetics.

Protein Folding: Defining a "standard" Set of Experimental Conditions and a Preliminary Kinetic Data Set of Two-state Proteins

Protein Science : a Publication of the Protein Society. Mar, 2005  |  Pubmed ID: 15689503

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a "consensus" set of experimental conditions (25 degrees C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two-state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self-consistent data set that will aid ongoing efforts to understand the folding process.

The Protein Folding Transition State: What Are Phi-values Really Telling Us?

Protein and Peptide Letters. Feb, 2005  |  Pubmed ID: 15723637

Protein engineering-based studies of the folding transition state have accelerated significantly in the last decade, and more than a half dozen proteins have been subjected to extensive Phi-value analysis. A general picture is emerging from these studies of a transition state in which the large majority of experimentally characterized side chains participate in relatively homogeneous and energetically weak interactions playing only a relatively small role in defining relative folding rates.

Biosensors Based on Binding-modulated Donor-acceptor Distances

Trends in Biotechnology. Apr, 2005  |  Pubmed ID: 15780710

The promising recognition characteristics exhibited by biomolecules have caused significant interest in biomolecule-based sensor strategies. Here we review several emerging biosensor designs that use modulated electron or energy transfer to a bio-specific ligand as the signaling mechanism. The efficiencies of both electron transfer and energy transfer are strongly dependent on donor-acceptor distance. When coupled with the large conformational changes sometimes associated with biomolecular recognition, these distance-dependent processes provide a robust means for generating optical and electronic signals.

Label-free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-based Sensor

Angewandte Chemie (International Ed. in English). Aug, 2005  |  Pubmed ID: 16044476

Engineering a Signal Transduction Mechanism for Protein-based Biosensors

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2005  |  Pubmed ID: 16046542

Hybridization-induced conformational changes have been successfully used in biosensors for the transduction of DNA-binding events into readily observable optical or electronic signals. Similar signal transduction has not, however, proven of equal utility in protein-based biosensors. The discrepancy arises because, unlike ssDNA, most proteins do not undergo significant conformational changes upon ligand binding. Here, we describe a solution to this problem. We show that an arbitrarily selected, normally well folded protein can be rationally engineered such that it undergoes ligand-induced folding. The engineered protein responds rapidly (milliseconds) and selectively to its target, and it couples recognition with the largest possible conformational change: folding. These traits suggest that ligand-induced folding could serve as an ideal signal-transduction mechanism. Consistent with this claim, we demonstrate a label-free optical biosensor based on the effect that is sufficiently selective to detect its target even in complex, contaminant-ridden samples such as blood serum.

Site-specific Dimensions Across a Highly Denatured Protein; a Single Molecule Study

Journal of Molecular Biology. Sep, 2005  |  Pubmed ID: 16095607

Do highly denatured proteins adopt random coil configurations? Here, we address this question by measuring residue-to-residue separations across the denatured FynSH3 domain. Using single-molecule Forster resonance energy transfer techniques, we have collected transfer efficiency probability distributions for dye-labeled, denatured protein. Applying maximum likelihood analysis to the interpretation of these distributions, we have determined the through-space distance between five residue pairs in the protein's guanidine hydrochloride-unfolded and trifluoroethanol-unfolded states. We find that, while the dimensions of the guanidine hydrochloride -unfolded molecule generally coincide with the dimensions predicted for a random coil ensemble, potentially statistically significant deviations from random coil behavior are also evident. These small, site-specific deviations may provide a means of reconciling earlier, scattering-based evidence for the random coil nature of the unfolded state with more site-specific spectroscopic evidence suggesting residual structure. We have also studied the unfolded ensemble populated in 50% trifluoroethanol, a denaturant that induces a highly helical unfolded state. We find that the size and shape of the unfolded ensemble under these conditions is effectively indistinguishable from that populated in guanidinium hydrochloride solutions, suggesting that the gross structure of the denatured state is, perhaps surprisingly, independent of the chemistry of the cosolvent.

Is There or Isn't There? The Case for (and Against) Residual Structure in Chemically Denatured Proteins

Critical Reviews in Biochemistry and Molecular Biology. Jul-Aug, 2005  |  Pubmed ID: 16126485

First raised some 60 years ago, the question of whether chemically denatured proteins are fully unfolded has, in recent years, seen significantly renewed interest. This increased attention has been spurred, in large part, by new spectroscopic and computational approaches that suggest even the most highly denatured polypeptides contain significant residual structure. In contrast, the most recent scattering results uphold the long-standing view that chemically denatured proteins adopt random coil configurations. Here we review the evidence both for and against residual structure in chemically denatured proteins, and attempt to reconcile these seemingly contradictory observations.

Experimental Investigation of the Frequency and Substitution Dependence of Negative Phi-values in Two-state Proteins

Biochemistry. Sep, 2005  |  Pubmed ID: 16142914

Negative phi-values, which arise, for example, when a mutation stabilizes the folding transition state while destabilizing the native state, have been the focus of significant theoretical interest. Here we survey the experimental folding kinetics literature to ascertain the frequency with which negative phi-values occur in two-state proteins and describe the detailed experimental characterization of a negative phi-value previously reported to be among the most statistically significant. We find that, while almost 9% of more than 500 reported phi-values (from a set of 16, well-characterized two-state proteins) fall below zero, many of these do not represent statistically significant observations. For example, only 6% of the phi-values for which estimates of precision are available fall even one reported "error bar" below zero, and only 4% are simultaneously negative, significant at this level and associated with free energy changes at or above 2.5 kJ/mol (below which phi-value analysis is widely considered unreliable). Moreover, given the asymmetric distribution of phi-values around zero and given that reported error bars may significantly underestimate true confidence intervals, the actual number of negative phi-values may be much smaller still. We have also performed detailed characterization of one of the most statistically significant negative phi-values reported in the literature to date, the V55F mutant of FynSH3. We find that substitution of the wild-type valine to other hydrophobic residues often increases folding rates without significantly altering folding free energy. This in turn leads to poorly defined phi-values, some of which are formally negative but only one or two of which fall statistically significantly below zero. In contrast, substitution to polar residues significantly destabilizes both the transition and native states, generally producing small but statistically significant positive phi-values of approximately 0.1. Thus, unlike other previously characterized phi-values, the negative phi-value associated with position 55 of the FynSH3 domain appears to be strongly dependent on the substitution employed to measure it, suggesting that subtlety will be required in order to develop a theoretical model of such behavior.

A Reagentless Signal-on Architecture for Electronic, Aptamer-based Sensors Via Target-induced Strand Displacement

Journal of the American Chemical Society. Dec, 2005  |  Pubmed ID: 16366535

Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.

Absorption Spectra of Liquid Water and Aqueous Buffers Between 0.3 and 3.72 THz

The Journal of Chemical Physics. Jan, 2006  |  Pubmed ID: 16438614

We have developed a terahertz absorption spectrometer suitable for strongly absorbing liquids such as water, and have precisely measured the absorption spectrum of water between 0.3 and 3.72 THz (10-124 cm(-1)). We have also examined the absorption spectra of aqueous 50 mM potassium phosphate buffers at pH 3 and 8, and find that they do not differ significantly from pure distilled de-ionized water.

Differential Labeling of Closely Spaced Biosensor Electrodes Via Electrochemical Lithography

Langmuir : the ACS Journal of Surfaces and Colloids. Feb, 2006  |  Pubmed ID: 16460130

Electrochemical biosensors offer the promise of exceptional scalability and parallelizability. To achieve this promise, however, will require the development of new methods for the differential labeling of closely spaced electrodes with specific biomolecules such as DNA or proteins. Here we report a simple, highly selective method for passivating and differentially labeling closely separated gold electrodes with oligonucleotides or other biomolecules. Analogous to photolithography, where a light-sensitive resist is selectively removed to expose specific surfaces to further modification, we passivate gold electrodes with a self-assembled alkanethiol monolayer that protects them from modification. The monolayer is then electrochemically desorbed at relatively low potentials, allowing for the subsequent labeling of the now exposed array element with a specific sensing biomolecule. The observed passivation is highly efficient: using a C11-OH monolayer as the passivating agent, we do not observe any detectable cross-contamination of adjacent electrodes (95 microm separation) upon labeling with a stem-loop DNA probe. Critically, the conditions employed are sufficiently gentle that depassivation reduces the DNA load on adjacent electrodes by only approximately 1%, allowing for the sequential labeling of multiple, closely spaced electrodes. This technology paves the way for labeling multiple array elements sequentially without observable cross-contamination in a fast and controlled manner.

On the Precision of Experimentally Determined Protein Folding Rates and Phi-values

Protein Science : a Publication of the Protein Society. Mar, 2006  |  Pubmed ID: 16501226

Phi-values, a relatively direct probe of transition-state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which phi-values can be determined experimentally: Because phi is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within- and between-laboratory variability, we have determined the precision with which folding rates and phi-values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of phi-values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (mf and mu) are invariant upon mutation, the precision of our estimates of phi is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature.

An Electronic, Aptamer-based Small-molecule Sensor for the Rapid, Label-free Detection of Cocaine in Adulterated Samples and Biological Fluids

Journal of the American Chemical Society. Mar, 2006  |  Pubmed ID: 16522082

Whereas spectroscopic and chromatographic techniques for the detection of small organic molecules have achieved impressive results, these methods are generally slow and cumbersome, and thus the development of a general means for the real-time, electronic detection of such targets remains a compelling goal. Here we demonstrate a potentially general, label-free electronic method for the detection of small-molecule targets by building a rapid, reagentless biosensor for the detection of cocaine. The sensor, based on the electrochemical interrogation of a structure-switching aptamer, specifically detects micromolar cocaine in seconds. Because signal generation is based on binding-induced folding, the sensor is highly selective and works directly in blood serum and in the presence of commonly employed interferents and cutting agents, and because all of the sensor components are covalently attached to the electrode surface, the sensor is also reusable: we achieve >99% signal regeneration upon a brief, room temperature aqueous wash. Given recent advances in the generation of highly specific aptamers, this detection platform may be readily adapted for the detection of other small molecules of a wide range of clinically and environmentally relevant small molecules.

Rapid, Sequence-specific Detection of Unpurified PCR Amplicons Via a Reusable, Electrochemical Sensor

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2006  |  Pubmed ID: 16537478

We report an electrochemical method for the sequence-specific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium. Using an asymmetric PCR and the electrochemical E-DNA detection scheme, single-stranded amplicons were produced from as few as 90 gene copies and, without subsequent purification, rapidly identified. The detection is specific; the sensor does not respond when challenged with control oligonucleotides based on the gyrB genes of either Escherichia coli or various Shigella species. In contrast to existing sequence-specific optical- and capillary electrophoresis-based detection methods, the E-DNA sensor is fully electronic and requires neither cumbersome, expensive optics nor high voltage power supplies. Given these advantages, E-DNA sensors appear well suited for implementation in portable PCR microdevices directed at, for example, the rapid detection of pathogens.

Probing the Collective Vibrational Dynamics of a Protein in Liquid Water by Terahertz Absorption Spectroscopy

Protein Science : a Publication of the Protein Society. May, 2006  |  Pubmed ID: 16641490

Biological polymers are expected to exhibit functionally relevant, global, and subglobal collective modes in the terahertz (THz) frequency range (i.e., picosecond timescale). In an effort to monitor these collective motions, we have experimentally determined the absorption spectrum of solvated bovine serum albumin (BSA) from 0.3 to 3.72 THz (10-124 cm(-1)). We successfully extract the terahertz molar absorption of the solvated BSA from the much stronger attenuation of water and observe in the solvated protein a dense, overlapping spectrum of vibrational modes that increases monotonically with increasing frequency. We see no evidence of distinct, strong, spectral features, suggesting that no specific collective vibrations dominate the protein's spectrum of motions, consistent with the predictions of molecular dynamics simulations and normal mode analyses of a range of small proteins. The shape of the observed spectrum resembles the ideal quadratic spectral density expected for a disordered ionic solid, indicating that the terahertz normal mode density of the solvated BSA may be modeled, to first order, as that of a three-dimensional elastic nanoparticle with an aperiodic charge distribution. Nevertheless, there are important detailed departures from that of a disordered inorganic solid or the normal mode densities predicted for several smaller proteins. These departures are presumably the spectral features arising from the unique molecular details of the solvated BSA. The techniques used here and measurements have the potential to experimentally confront theoretical calculations on a frequency scale that is important for macromolecular motions in a biologically relevant water environment.

Cooperativity and the Origins of Rapid, Single-exponential Kinetics in Protein Folding

Protein Science : a Publication of the Protein Society. Jul, 2006  |  Pubmed ID: 16815915

The folding of naturally occurring, single-domain proteins is usually well described as a simple, single-exponential process lacking significant trapped states. Here we further explore the hypothesis that the smooth energy landscape this implies, and the rapid kinetics it engenders, arises due to the extraordinary thermodynamic cooperativity of protein folding. Studying Miyazawa-Jernigan lattice polymers, we find that, even under conditions where the folding energy landscape is relatively optimized (designed sequences folding at their temperature of maximum folding rate), the folding of protein-like heteropolymers is accelerated when their thermodynamic cooperativity is enhanced by enhancing the nonadditivity of their energy potentials. At lower temperatures, where kinetic traps presumably play a more significant role in defining folding rates, we observe still greater cooperativity-induced acceleration. Consistent with these observations, we find that the folding kinetics of our computational models more closely approximates single-exponential behavior as their cooperativity approaches optimal levels. These observations suggest that the rapid folding of naturally occurring proteins is, in part, a consequence of their remarkably cooperative folding.

Sequence-specific, Electronic Detection of Oligonucleotides in Blood, Soil, and Foodstuffs with the Reagentless, Reusable E-DNA Sensor

Analytical Chemistry. Aug, 2006  |  Pubmed ID: 16906710

The ability to detect specific oligonucleotides in complex, contaminant-ridden samples, without the use of exogenous reagents and using a reusable, fully electronic platform could revolutionize the detection of pathogens in the clinic and in the field. Here, we characterize a label-free, electronic sensor, termed E-DNA, for its ability to simultaneously meet these challenging demands. We find that because signal generation is coupled to a hybridization-linked conformational change, rather than to only adsorption to the sensor surface, E-DNA is selective enough to detect oligonucleotides in complex, multicomponent samples, such as blood serum and soil. Moreover, E-DNA signaling is monotonically related to target complementarity, allowing the sensor to discriminate between mismatched targets: we readily detect the complementary 17-base target against a 50 000-fold excess of genomic DNA, can distinguish a three-base mismatch from perfect target directly in blood serum, and under ideal conditions, observe statistically significant differences between single-base mismatches. Finally, because the sensing components are linked to the electrode surface, E-DNA is reusable: a 30-s room temperature wash recovers >99% of the sensor signal. This work further supports the utility of E-DNA as a rapid, specific, and convenient method for the detection of DNA and RNA sequences.

Methods for the Accurate Estimation of Confidence Intervals on Protein Folding Phi-values

Protein Science : a Publication of the Protein Society. Oct, 2006  |  Pubmed ID: 17008714

Phi-values provide an important benchmark for the comparison of experimental protein folding studies to computer simulations and theories of the folding process. Despite the growing importance of phi measurements, however, formulas to quantify the precision with which phi is measured have seen little significant discussion. Moreover, a commonly employed method for the determination of standard errors on phi estimates assumes that estimates of the changes in free energy of the transition and folded states are independent. Here we demonstrate that this assumption is usually incorrect and that this typically leads to the underestimation of phi precision. We derive an analytical expression for the precision of phi estimates (assuming linear chevron behavior) that explicitly takes this dependence into account. We also describe an alternative method that implicitly corrects for the effect. By simulating experimental chevron data, we show that both methods accurately estimate phi confidence intervals. We also explore the effects of the commonly employed techniques of calculating phi from kinetics estimated at non-zero denaturant concentrations and via the assumption of parallel chevron arms. We find that these approaches can produce significantly different estimates for phi (again, even for truly linear chevron behavior), indicating that they are not equivalent, interchangeable measures of transition state structure. Lastly, we describe a Web-based implementation of the above algorithms for general use by the protein folding community.

Excimer-based Peptide Beacons: a Convenient Experimental Approach for Monitoring Polypeptide-protein and Polypeptide-oligonucleotide Interactions

Journal of the American Chemical Society. Nov, 2006  |  Pubmed ID: 17061871

While protein-polypeptide and nucleic acid-polypeptide interactions are of significant experimental interest, quantitative methods for the characterization of such interactions are often cumbersome. Here we described a relatively simple means of optically monitoring such interactions using excimer-based peptide beacons (PBs). The design of PBs is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed with macromolecular targets. Using this binding-induced folding to segregate two pyrene moieties and therefore inhibit excimer formation, we have produced PBs directed against both anti-HIV antibodies and the retroviral transactive response (TAR) RNA hairpin. For both polypeptides, target recognition is accompanied by a roughly 2-fold decrease in excimer emission, thus allowing the detection of their respective targets at concentrations of a few nanomolar. Because excimer emission requires the formation of a tight, precisely oriented pyrene dimer, even relatively trivial binding-induced segregation reduces fluorescence significantly. This suggests that the PB approach will be suitable for monitoring a wide range of peptide-macromolecule recognition events. Moreover, the synthesis of excimer-based PBs utilizes commercially available modified pyrenes in a simple and well-established protocol, making the approach well suited for routine laboratory applications.

Single-step Electronic Detection of Femtomolar DNA by Target-induced Strand Displacement in an Electrode-bound Duplex

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2006  |  Pubmed ID: 17065320

We report a signal-on, electronic DNA (E-DNA) sensor that is label-free and achieves a subpicomolar detection limit. The sensor, which is based on a target-induced strand displacement mechanism, is composed of a "capture probe" attached by its 5' terminus to a gold electrode and a 5' methylene blue-modified "signaling probe" that is complementary at both its 3' and 5' termini to the capture probe. In the absence of target, hybridization between the capture and signaling probes minimizes contact between the methylene blue and electrode surface, limiting the observed redox current. Target hybridization displaces the 5' end of the signaling probe, generating a short, flexible single-stranded DNA element and producing up to a 7-fold increase in redox current. The observed signal gain is sufficient to achieve a demonstrated (not extrapolated) detection limit of 400 fM, which is among the best reported for single-step electronic DNA detection. Moreover, because sensor fabrication is straightforward, the approach appears to provide a ready alternative to the more cumbersome femtomolar electrochemical assays described to date.

Collective Dynamics of Lysozyme in Water: Terahertz Absorption Spectroscopy and Comparison with Theory

The Journal of Physical Chemistry. B. Nov, 2006  |  Pubmed ID: 17125398

To directly measure the low-frequency vibrational modes of proteins in biologically relevant water environment rather than previously explored dry or slightly hydrated phase, we have developed a broadband terahertz spectrometer suitable for strongly attenuating protein solutions. Radiation is provided by harmonic multipliers (up to 0.21 THz), a Gunn oscillator (at 0.139 THz), and the UCSB free-electron lasers (up to 4.8 THz). Our spectrometer combines these intense sources with a sensitive cryogenic detector and a variable path length sample cell to detect radiation after it is attenuated by more than 7 orders of magnitudes by the aqueous sample. Using this spectrometer, we have measured the molar extinction of solvated lysozyme between 0.075 and 3.72 THz (2.5-124 cm(-1)), and we made direct comparison to several published theoretical models based on molecular dynamics simulations and normal-mode analysis. We confirm the existence of dense, overlapping normal modes in the terahertz frequency range. Our observed spectrum, while in rough qualitative agreement with these models, differs in detail. Further, we observe a low-frequency cutoff in terahertz dynamics between 0.2 and 0.3 THz, and we see no evidence of a predicted normal mode at approximately 0.09 THz for the protein.

Comparison of the Signaling and Stability of Electrochemical DNA Sensors Fabricated from 6- or 11-carbon Self-assembled Monolayers

Langmuir : the ACS Journal of Surfaces and Colloids. Dec, 2006  |  Pubmed ID: 17129062

We have characterized the solution-phase and dry storage stability of electrochemical E-DNA sensors fabricated using mixed self-assembled monolayers (SAMs) composed of 6- or 11-carbon (C6 and C11, respectively) alpha,omega-thiol alcohols and the analogous C6- or C11-thiol-terminated stem-loop DNA probe. We find that the solution-phase and dry storage stability of C6-based E-DNA sensors are limited and poorly reproducible. The use of stabilizing agents bovine serum albumin plus either glucose or trehalose significantly improves the dry storage shelf life of such sensors: when using these preservatives, we observe only 7-9% sensor degradation after 1 month of storage in air at room temperature. In comparison, the stability of C11-based E-DNA sensors is significantly greater than that of the C6-based sensors; we observe only minor (5-8%) loss of signal upon storing these sensors for a week under ambient solution conditions or for more than a month in air in the presence of preservatives. Moreover, whereas the electron-transfer rate through C11 SAMs is slower than that observed for C6 SAMs, it is rapid enough to support good sensor performance. It thus appears that C11 SAMs provide a reasonable compromise between electron-transfer efficiency and sensor stability and are well suited for use in electronic DNA-sensing applications.

Aptamer-based Electrochemical Detection of Picomolar Platelet-derived Growth Factor Directly in Blood Serum

Analytical Chemistry. Jan, 2007  |  Pubmed ID: 17194144

We report an electrochemical, aptamer-based (E-AB) sensor for the detection of platelet-derived growth factor (PDGF) directly in blood serum. The E-AB approach employs alternating current voltammetry to monitor target-induced folding in a methylene blue-modified, PDGF-binding aptamer. The sensor is sensitive, highly selective, and essentially reagentless: we readily detect the BB variant of PDGF at 1 nM directly in undiluted, unmodified blood serum and at 50 pM (1.25 ng/mL) in serum-diluted 2-fold with aqueous buffer. The sensitivity and selectivity achieved by this sensor match or significantly exceed those of the best analogous optical approaches. For example, the detection limit attained in 50% serum is achieved against a >25 million-fold excess of contaminating blood proteins and represents a 4 order of magnitude improvement over the most sensitive optical PDGF aptasensor reported to date. Moreover, the E-AB sensor combines these promising attributes in a platform that is reusable, label-free, and electronic. Given these advantages, E-AB sensors appear well suited for implementation in portable microdevices directed at the direct detection of proteins and small molecules in complex, largely unprocessed clinical samples.

Electrochemical Detection of Parts-per-billion Lead Via an Electrode-bound DNAzyme Assembly

Journal of the American Chemical Society. Jan, 2007  |  Pubmed ID: 17212391

Increasing the Resolution of Single Pair Fluorescence Resonance Energy Transfer Measurements in Solution Via Molecular Cytometry

Analytical Chemistry. May, 2007  |  Pubmed ID: 17385843

We report a method to increase the resolution of single pair fluorescence resonance energy transfer (spFRET) measurements in aqueous solutions. Solution-based spFRET measurements of fluorescently labeled biological molecules (proteins, RNA, DNA) are often used to obtain histograms of molecular conformation without resorting to sample immobilization. However, for solution-phase spFRET studies, the number of photons detected from a single molecule as it diffuses through an open confocal volume element are quite limited. An "average" transit may yield on the order of 40 photons. Shot noise on the number of detected photons substantially limits the resolution of the measurement. The method reported here uses a hydrodynamically focused sample stream to ensure molecules traverse the full width of an excitation laser beam. This substantially increases the average number of photons detected per molecular transit (approximately 85 photons/molecule), which increases measurement precision. In addition, this method minimizes another source of heterogeneity present in diffusive measures of spFRET: the distribution of paths taken through the excitation laser beam. We demonstrate here using a FRET labeled protein sample (a FynSH3 domain) that superior resolution (a factor of approximately 2) can be obtained via molecular cytometry compared to spFRET measurements based upon diffusion through an open confocal volume element.

Influence of Local and Residual Structures on the Scaling Behavior and Dimensions of Unfolded Proteins

Biopolymers. Jul, 2007  |  Pubmed ID: 17450572

Although recent spectroscopic studies of chemically denatured proteins hint at significant nonrandom residual structure, the results of extensive small angle X-ray scattering studies suggest random coil behavior, calling for a coherent understanding of these seemingly contradicting observations. Here, we report the results of a Monte Carlo study of the effects of two types of local structures, alpha helix and Polyproline II (PPII) helix, on the dimensions of random coil polyalanine chains viewed as a model of highly denatured proteins. We find that although Flory's power law scaling, long regarded as a signature of random coil behavior, holds for chains containing up to 90% alpha or PPII helix, the absolute magnitude of the chain dimensions is sensitive to helix content. As residual alpha helix content increases, the chain contracts until it reaches a minimum radius at approximately 70% helix, after which the chain dimensions expand rapidly. With an alpha helix content of approximately 20%, corresponding to the Ramachandran probability of being in the helical basin, experimentally observed radii of gyration are recovered. Experimental radii are similarly recovered at an alpha helix content of approximately 87%, providing an explanation for the previously puzzling experimental finding that the dimensions of the highly helical methanol-induced unfolded state are experimentally indistinguishable from those of the helix-poor urea-unfolded state. In contrast, the radius of gyration increases monotonically with increasing PPII content, and is always more expanded than the dimensions observed experimentally. These results suggest that PPII is unlikely the sole, dominant preferred conformation for unfolded proteins.

Peptide Beacons: a New Design for Polypeptide-based Optical Biosensors

Bioconjugate Chemistry. May-Jun, 2007  |  Pubmed ID: 17461545

Both epitope mapping and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here, we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to a macromolecular target. Using this effect to segregate a long-lived fluorophore from an electron transfer based, contact quencher (both covalently attached to the peptide), we have produced a robust optical sensor for anti-HIV antibodies. The binding-induced segregation of the fluorophore-quencher pair produces a 6-fold increase in sensor emission, thus allowing us to readily detect as low as approximately 250 pM of the target antibody. Because the sensor is based on binding-induced folding and a visible-light fluorophore, it is sufficiently selective to work directly in complex, contaminant-ridden samples such as saliva and blood.

Effect of Molecular Crowding on the Response of an Electrochemical DNA Sensor

Langmuir : the ACS Journal of Surfaces and Colloids. Jun, 2007  |  Pubmed ID: 17488132

E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stem-loop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.

Label-free Electrochemical Detection of DNA in Blood Serum Via Target-induced Resolution of an Electrode-bound DNA Pseudoknot

Journal of the American Chemical Society. Oct, 2007  |  Pubmed ID: 17850085

Linear, Redox Modified DNA Probes As Electrochemical DNA Sensors

Chemical Communications (Cambridge, England). Sep, 2007  |  Pubmed ID: 17851622

We show here that hybridization-linked changes in the dynamics of a redox-modified, electrode-bound linear (as opposed to stem-loop) probe DNA produce large changes in Faradaic current, allowing for the ready detection of target oligonucleotides.

Preparation of Electrode-immobilized, Redox-modified Oligonucleotides for Electrochemical DNA and Aptamer-based Sensing

Nature Protocols. 2007  |  Pubmed ID: 18007622

Recent years have seen the development of a number of reagentless, electrochemical sensors based on the target-induced folding or unfolding of electrode-bound oligonucleotides, with examples reported to date, including sensors for the detection of specific nucleic acids, proteins, small molecules and inorganic ions. These devices, which are often termed electrochemical DNA (E-DNA) and E-AB (electrochemical, aptamer-based) sensors, are comprised of an oligonucleotide probe modified with a redox reporter (in this protocol methylene blue) at one terminus and attached to a gold electrode via a thiol-gold bond at the other. Binding of an analyte to the oligonucleotide probe changes its structure and dynamics, which, in turn, influences the efficiency of electron transfer to the interrogating electrode. This class of sensors perform well even when challenged directly with blood serum, soil and other complex, multicomponent sample matrices. This protocol describes the fabrication of E-DNA and E-AB sensors. The protocol can be completed in 12 h.

Chimeric Peptide Beacons: a Direct Polypeptide Analog of DNA Molecular Beacons

Chemical Communications (Cambridge, England). Dec, 2007  |  Pubmed ID: 18361352

We have developed a new biosensor architecture, which is comprised of a polypeptide-peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.

Microfluidic Device Architecture for Electrochemical Patterning and Detection of Multiple DNA Sequences

Langmuir : the ACS Journal of Surfaces and Colloids. Feb, 2008  |  Pubmed ID: 18181654

Electrochemical biosensors pose an attractive solution for point-of-care diagnostics because they require minimal instrumentation and they are scalable and readily integrated with microelectronics. The integration of electrochemical biosensors with microscale devices has, however, proven to be challenging due to significant incompatibilities among biomolecular stability, operation conditions of electrochemical sensors, and microfabrication techniques. Toward a solution to this problem, we have demonstrated here an electrochemical array architecture that supports the following processes in situ, within a self-enclosed microfluidic device: (a) electrode cleaning and preparation, (b) electrochemical addressing, patterning, and immobilization of sensing biomolecules at selected sensor pixels, (c) sequence-specific electrochemical detection from multiple pixels, and (d) regeneration of the sensing pixels. The architecture we have developed is general, and it should be applicable to a wide range of biosensing schemes that utilize gold-thiol self-assembled monolayer chemistry. As a proof-of-principle, we demonstrate the detection and differentiation of polymerase chain reaction (PCR) amplicons diagnostic of human (H1N1) and avian (H5N1) influenza.

Optimization of Electrochemical Aptamer-based Sensors Via Optimization of Probe Packing Density and Surface Chemistry

Langmuir : the ACS Journal of Surfaces and Colloids. Sep, 2008  |  Pubmed ID: 18690727

Electrochemical, aptamer-based (E-AB) sensors, which are comprised of an electrode modified with surface immobilized, redox-tagged DNA aptamers, have emerged as a promising new biosensor platform. In order to further improve this technology we have systematically studied the effects of probe (aptamer) packing density, the AC frequency used to interrogate the sensor, and the nature of the self-assembled monolayer (SAM) used to passivate the electrode on the performance of representative E-AB sensors directed against the small molecule cocaine and the protein thrombin. We find that, by controlling the concentration of aptamer employed during sensor fabrication, we can control the density of probe DNA molecules on the electrode surface over an order of magnitude range. Over this range, the gain of the cocaine sensor varies from 60% to 200%, with maximum gain observed near the lowest probe densities. In contrast, over a similar range, the signal change of the thrombin sensor varies from 16% to 42% and optimal signaling is observed at intermediate densities. Above cut-offs at low hertz frequencies, neither sensor displays any significant dependence on the frequency of the alternating potential employed in their interrogation. Finally, we find that E-AB signal gain is sensitive to the nature of the alkanethiol SAM employed to passivate the interrogating electrode; while thinner SAMs lead to higher absolute sensor currents, reducing the length of the SAM from 6-carbons to 2-carbons reduces the observed signal gain of our cocaine sensor 10-fold. We demonstrate that fabrication and operational parameters can be varied to achieve optimal sensor performance and that these can serve as a basic outline for future sensor fabrication.

Optimization of a Reusable, DNA Pseudoknot-based Electrochemical Sensor for Sequence-specific DNA Detection in Blood Serum

Analytical Chemistry. Jan, 2009  |  Pubmed ID: 19093760

We describe in detail a new electrochemical DNA (E-DNA) sensing platform based on target-induced conformation changes in an electrode-bound DNA pseudoknot. The pseudoknot, a DNA structure containing two stem-loops in which the first stem's loop forms part of the second stem, is modified with a methylene blue redox tag at its 3' terminus and covalently attached to a gold electrode via the 5' terminus. In the absence of a target, the structure of the pseudoknot probe minimizes collisions between the redox tag and the electrode, thus reducing faradaic current. Target binding disrupts the pseudoknot structure, liberating a flexible, single-stranded element that can strike the electrode and efficiently transfer electrons. In this article we report further characterization and optimization of this new E-DNA architecture. We find that optimal signaling is obtained at an intermediate probe density ( approximately 1.8 x 10(13) molecules/cm(2) apparent density), which presumably represents a balance between steric and electrostatic blocking at high probe densities and increased background currents arising from transfer from the pseudoknot probe at lower densities. We also find that optimal 3' stem length, which appears to be 7 base pairs, represents a balance between pseudoknot structural stability and target affinity. Finally, a 3' loop comprised of poly(A) exhibits better mismatch discrimination than the equivalent poly(T) loop, but at the cost of decreased gain. Optimization over this parameter space significantly improves the signaling of the pseudoknot-based E-DNA architecture, leading to the ability to sensitively and specifically detect DNA targets even when challenged in complex, multicomponent samples such as blood serum.

Improving the Stability and Sensing of Electrochemical Biosensors by Employing Trithiol-anchoring Groups in a Six-carbon Self-assembled Monolayer

Analytical Chemistry. Feb, 2009  |  Pubmed ID: 19133790

Alkane thiol self-assembled monolayers (SAMs) have seen widespread utility in the fabrication of electrochemical biosensors. Their utility, however, reflects a potentially significant compromise. While shorter SAMs support efficient electron transfer, they pack poorly and are thus relatively unstable. Longer SAMs are more stable but suffer from less efficient electron transfer, thus degrading sensor performance. Here we use the electrochemical DNA (E-DNA) sensor platform to compare the signaling and stability of biosensors fabricated using a short, six-carbon monothiol with those employing either of two commercially available trihexylthiol anchors (a flexible Letsinger type and a rigid adamantane type). We find that all three anchors support efficient electron transfer and E-DNA signaling, with the gain, specificity, and selectivity of all three being effectively indistinguishable. The stabilities of the three anchors, however, vary significantly. Sensors anchored with the flexible trithiol exhibit enhanced stability, retaining 75% of their original signal and maintaining excellent signaling properties after 50 days storage in buffer. Likewise these sensors exhibit excellent temperature stability and robustness to electrochemical interrogation. The stability of sensors fabricated using the rigid trithiol anchor, by comparison, are similar to those of the monothiol, with both exhibiting significant (>60%) loss of signal upon wet storage or thermocycling. Employing a flexible trithiol anchor in the fabrication of SAM-based electrochemical biosensors may provide a means of improving sensor robustness without sacrificing electron transfer efficiency or otherwise impeding sensor performance.

Protein Complexes: the Evolution of Symmetry

Current Biology : CB. Jan, 2009  |  Pubmed ID: 19138586

Most proteins form symmetric, multimeric complexes. Modeling shows that a strong prevalence for symmetry among stable structures can account for this bias even in the absence of other adaptive advantages.

Beyond Molecular Beacons: Optical Sensors Based on the Binding-induced Folding of Proteins and Polypeptides

Chemistry (Weinheim an Der Bergstrasse, Germany). 2009  |  Pubmed ID: 19191230

Many polypeptides and small proteins can be readily engineered such that they only fold upon binding a specific target ligand. This approach couples target recognition with a considerable change in polymer structure and dynamics. Recent years have seen the development of a number of biosensors that couple these large changes to readily measurable optical (fluorescent) outputs. These sensors afford the detection of a wide variety of macromolecular targets including proteins, polypeptides, and nucleic acids. Here we describe the design of such biosensors, from the first iterations as protein engineering experiments, to the development of biosensors targeting a range of protein and nucleic acid targets.

Reagentless, Electrochemical Approach for the Specific Detection of Double- and Single-stranded DNA Binding Proteins

Analytical Chemistry. Feb, 2009  |  Pubmed ID: 19199570

Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins.

Effects of Probe Length, Probe Geometry, and Redox-tag Placement on the Performance of the Electrochemical E-DNA Sensor

Analytical Chemistry. Mar, 2009  |  Pubmed ID: 19215066

Previous work has described several reagentless, electrochemical DNA (E-DNA) sensing architectures comprised of an electrode-immobilized, redox-tagged probe oligonucleotide. Recent studies suggest that E-DNA signaling is predicated on hybridization-linked changes in probe flexibility, which will alter the efficiency with which the terminal redox tag strikes the electrode. This, in turn, suggests that probe length, probe geometry, and redox-tag placement will affect E-DNA signaling. To test this we have characterized E-DNA sensors comprised of linear or stem-loop probes of various lengths and with redox tags placed either distal to the electrode or internally within the probe sequence (proximal). We find that linear probes produce larger signal changes upon target binding than equivalent stem-loop probes. Likewise, long probes exhibit greater signal changes than short probes provided that the redox tag is placed proximal to the electrode surface. In contrast to their improved signaling, the specificity of long probes is poorer than that of short probes, suggesting that sensor optimization represents a trade off between sensitivity and specificity. Finally, we find that sensor response time and selectivity are only minimally affected by probe geometry or length. The results of this comparative study will help guide future designs and applications of these sensors.

Continuous, Real-time Monitoring of Cocaine in Undiluted Blood Serum Via a Microfluidic, Electrochemical Aptamer-based Sensor

Journal of the American Chemical Society. Apr, 2009  |  Pubmed ID: 19271708

The development of a biosensor system capable of continuous, real-time measurement of small-molecule analytes directly in complex, unprocessed aqueous samples has been a significant challenge, and successful implementation has been achieved for only a limited number of targets. Toward a general solution to this problem, we report here the Microfluidic Electrochemical Aptamer-based Sensor (MECAS) chip wherein we integrate target-specific DNA aptamers that fold, and thus generate an electrochemical signal, in response to the analyte with a microfluidic detection system. As a model, we demonstrate the continuous, real-time (approximately 1 min time resolution) detection of the small-molecule drug cocaine at near physiological, low micromolar concentrations directly in undiluted, otherwise unmodified blood serum. We believe our approach of integrating folding-based electrochemical sensors with miniaturized detection systems may lay the groundwork for the real-time, point-of-care detection of a wide variety of molecular targets.

Surface Chemistry Effects on the Performance of an Electrochemical DNA Sensor

Bioelectrochemistry (Amsterdam, Netherlands). Sep, 2009  |  Pubmed ID: 19362061

E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.

An Electrochemical Sensor for the Detection of Protein-small Molecule Interactions Directly in Serum and Other Complex Matrices

Journal of the American Chemical Society. May, 2009  |  Pubmed ID: 19413316

Here we have demonstrated a general, sensitive, and selective approach for the detection of macromolecules that bind to specific small molecule recognition elements. Our electrochemical approach utilizes a redox-tagged DNA signaling scaffold that is conjugated to a small molecule recognition element and is covalently attached to an interrogating electrode. The binding of a protein to the small molecule recognition element alters the dynamics of the scaffold, increasing or decreasing the efficiency with which the redox tag collides with the electrode and thus altering the observed faradaic current. We optimized the scaffold using a biotin recognition element and streptavidin as a target to determine the variables that define sensor performance before then applying the approach to detection of anti-digoxigenin antibodies using the steroid as the recognition element. We generated streptavidin sensors exhibiting both signal-on (target binding increases the faradaic current) and signal-off behavior, of which only the signal-off approach was generalizable to the detection of antibodies. Sensors for both targets are sensitive (detection limits in the low nanomolar range), rapid (minutes), reusable, and selective enough to function directly in complex matrices including blood serum, soil, and foodstuffs.

High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-molecule Targets in Blood and Other Complex Matrices

Journal of the American Chemical Society. May, 2009  |  Pubmed ID: 19419171

We herein demonstrate a sandwich assay based on single aptamer sequences is suitable for the direct detection of small molecule targets in blood serum and other complex matrices. By splitting an aptamer into two pieces, we convert a single affinity reagent into a two-component system in which the presence of the target drives formation of a complex comprised of the target and the two halves of the aptamer. To demonstrate the utility of this approach we have used single anticocaine and anti-ATP aptamers to fabricate electrochemical sensors directed against the representative small molecules cocaine and ATP. Both targets are detected at low micromolar concentrations, in seconds, and in a convenient, general, readily reusable, electrochemical format. Moreover, both sensors are selective enough to deploy directly in blood, crude cellular lysates and other complex sample matrices.

Fluorescence Detection of Single-nucleotide Polymorphisms with a Single, Self-complementary, Triple-stem DNA Probe

Angewandte Chemie (International Ed. in English). 2009  |  Pubmed ID: 19431180

Singled out for its singularity: In a single-step, single-component, fluorescence-based method for the detection of single-nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self-complementary DNA strand that forms a triple-stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F = fluorophore, Q = quencher).

The Length and Viscosity Dependence of End-to-end Collision Rates in Single-stranded DNA

Biophysical Journal. Jul, 2009  |  Pubmed ID: 19580758

Intramolecular dynamics play an essential role in the folding and function of biomolecules and, increasingly, in the operation of many biomimetic technologies. Thus motivated we have employed both experiment and simulation to characterize the end-to-end collision dynamics of unstructured, single-stranded DNAs ranging from 6 to 26 bases. We find that, because of the size and flexibility of the optical reporters employed experimentally, end-to-end collision dynamics exhibit little length dependence at length scales <11 bases. For longer constructs, however, the end-to-end collision rate exhibits a power-law relationship to polymer length with an exponent of -3.49 +/- 0.13. This represents a significantly stronger length dependence than observed experimentally for unstructured polypeptides or predicted by polymer scaling arguments. Simulations indicate, however, that the larger exponent stems from electrostatic effects that become important over the rather short length scale of these highly charged polymers. Finally, we have found that the end-to-end collision rate also depends linearly on solvent viscosity, with an experimentally significant, nonzero intercept (the extrapolated rate at zero viscosity) that is independent of chain length--n observation that sheds new light on the origins of the "internal friction" observed in the dynamics of many polymer systems.

Thermodynamic Basis for the Optimization of Binding-induced Biomolecular Switches and Structure-switching Biosensors

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2009  |  Pubmed ID: 19666496

Binding-induced biomolecular switches are used throughout nature and, increasingly, throughout biotechnology for the detection of chemical moieties and the subsequent transduction of this detection into useful outputs. Here we show that the thermodynamics of these switches are quantitatively described by a simple 3-state population-shift model, in which the equilibrium between a nonbinding, nonsignaling state and the binding-competent, signaling state is shifted toward the latter upon target binding. Because of this, their performance is determined by the tradeoff inherent to their switching thermodynamics; while a switching equilibrium constant favoring the nonbinding, nonsignaling, conformation ensures a larger signal change (more molecules are poised to respond), it also reduces affinity (binding must overcome a more unfavorable conformational free energy). We then derive and employ the relationship between switching thermodynamics and switch signaling to rationally tune the dynamic range and detection limit of a representative structure-switching biosensor, a molecular beacon, over 4 orders of magnitude. These findings demonstrate that the performance of biomolecular switches can be rationally tuned via mutations that alter their switching thermodynamics and suggest a mechanism by which the performance of naturally occurring switches may have evolved.

Measuring Distances Within Unfolded Biopolymers Using Fluorescence Resonance Energy Transfer: The Effect of Polymer Chain Dynamics on the Observed Fluorescence Resonance Energy Transfer Efficiency

The Journal of Chemical Physics. Aug, 2009  |  Pubmed ID: 19725638

Recent years have seen a number of investigations in which distances within unfolded proteins, polypeptides, and other biopolymers are probed via fluorescence resonance energy transfer, a method that relies on the strong distance dependence of energy transfer between a pair of dyes attached to the molecule of interest. In order to interpret the results of such experiments it is commonly assumed that intramolecular diffusion is negligible during the excited state lifetime. Here we explore the conditions under which this "frozen chain" approximation fails, leading to significantly underestimated donor-acceptor distances, and describe a means of correcting for polymer dynamics in order to estimate these distances more accurately.

Non-sequence-specific Interactions Can Account for the Compaction of Proteins Unfolded Under "native" Conditions

Journal of Molecular Biology. Nov, 2009  |  Pubmed ID: 19751743

Proteins unfolded by high concentrations of chemical denaturants adopt expanded, largely structure-free ensembles of conformations that are well approximated as random coils. In contrast, globular proteins unfolded under less denaturing conditions (via mutations, or transiently unfolded after a rapid jump to native conditions) and molten globules (arising due to mutations or cosolvents) are often compact. Here we explore the origins of this compaction using a truncated equilibrium-unfolded variant of the 57-residue FynSH3 domain. As monitored by far-UV circular dichroism, NMR spectroscopy, and hydrogen-exchange kinetics, CDelta4 (a 4-residue carboxy-terminal deletion variant of FynSH3) appears to be largely unfolded even in the absence of denaturant. Nevertheless, CDelta4 is quite compact under these conditions, with a hydrodynamic radius only slightly larger than that of the native protein. In order to understand the origins of this molten-globule-like compaction, we have characterized a random sequence polypeptide of identical amino acid composition to CDelta4. Notably, we find that the hydrodynamic radius of this random sequence polypeptide also approaches that of the native protein. Thus, while native-like interactions may contribute to the formation of compact "unfolded" states, it appears that non-sequence-specific monomer-monomer interactions can also account for the dramatic compaction observed for molten globules and the "physiological" unfolded state.

The Rate of Intramolecular Loop Formation in DNA and Polypeptides: the Absence of the Diffusion-controlled Limit and Fractional Power-law Viscosity Dependence

The Journal of Physical Chemistry. B. Oct, 2009  |  Pubmed ID: 19780594

The problem of determining the rate of end-to-end collisions for polymer chains has attracted the attention of theorists and experimentalists for more than three decades. The typical theoretical approach to this problem has focused on the case where a collision is defined as any instantaneous fluctuation that brings the chain ends to within a specific capture distance. In this paper, we study the more experimentally relevant case, where the end-to-end collision dynamics are probed by measuring the excited state lifetime of a fluorophore (or other lumiphore) attached to one chain end and quenched by a quencher group attached to the other end. Under this regime, a "contact" is defined not by the chain ends approach to within some sharp cutoff but, instead, typically by an exponentially distance-dependent process. Previous theoretical models predict that, if quenching is sufficiently rapid, a diffusion-controlled limit is attained, where such measurements report on the probe-independent, intrinsic end-to-end collision rate. In contrast, our theoretical considerations, simulations, and an analysis of experimental measurements of loop closure rates in single-stranded DNA molecules all indicate that no such limit exists, and that the measured effective collision rate has a nontrivial, fractional power-law dependence on both the intrinsic quenching rate of the fluorophore and the solvent viscosity. We propose a simple scaling formula describing the effective loop closure rate and its dependence on the viscosity, chain length, and properties of the probes. Previous theoretical results are limiting cases of this more general formula.

An Electrochemical Sensor for Single Nucleotide Polymorphism Detection in Serum Based on a Triple-stem DNA Probe

Journal of the American Chemical Society. Oct, 2009  |  Pubmed ID: 19807078

We report here an electrochemical approach that offers, for the first time, single-step, room-temperature single nucleotide polymorphism (SNP) detection directly in complex samples (such as blood serum) without the need for target modification, postwashing, or the addition of exogenous reagents. This sensor, which is sensitive, stable, and reusable, is comprised of a single, self-complementary, methylene blue-labeled DNA probe possessing a triple-stem structure. This probe takes advantage of the large thermodynamic changes in enthalpy and entropy that result from major conformational rearrangements that occur upon binding a perfectly matched target, resulting in a large-scale change in the faradaic current. As a result, the discrimination capabilities of this sensor greatly exceed those of earlier single- and double-stem electrochemical sensors and support rapid (minutes), single-step, reagentless, room-temperature detection of single nucleotide substitutions. To elucidate the theoretical basis of the sensor's selectivity, we present a comparative thermodynamic analysis among single-, double-, and triple-stem probes.

Comparing the Properties of Electrochemical-based DNA Sensors Employing Different Redox Tags

Analytical Chemistry. Nov, 2009  |  Pubmed ID: 19810694

Many electrochemical biosensor approaches developed in recent years utilize redox-labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported that employ a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers, and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum.

A General Electrochemical Method for Label-free Screening of Protein-small Molecule Interactions

Chemical Communications (Cambridge, England). Nov, 2009  |  Pubmed ID: 19826675

Here we report a versatile method by which the interaction between a protein and a small molecule, and the disruption of that interaction by competition with other small molecules, can be monitored electrochemically directly in complex sample matrices.

On the Signaling of Electrochemical Aptamer-Based Sensors: Collision- and Folding-Based Mechanisms

Electroanalysis. Jun, 2009  |  Pubmed ID: 20436787

Recent years have seen the emergence of a new class of electrochemical sensors predicated on target binding-induced folding of electrode-bound redox-modified aptamers and directed against targets ranging from small molecules to proteins. Previous studies of the relationship between gain and probe-density for these electrochemical, aptamer-based (E-AB) sensors suggest that signal transduction is linked to binding-induced changes in the efficiency with which the attached redox tag strikes the electrode. This, in turn, suggests that even well folded aptamers may support E-AB signaling if target binding sufficiently alters their flexibility. Here we investigate this using a thrombin-binding aptamer that undergoes binding-induced folding at low ionic strength but can be forced to adopt a folded conformation at higher ionic strength even in the absence of its protein target. We find that, under conditions in which the thrombin aptamer is fully folded prior to target binding, we still obtain a ca. 30% change in E-AB signal upon saturated target levels. In contrast, however, under conditions in which the aptamer is unfolded in the absence of target and thus undergoes binding-induced folding the observed signal change is twice as great. The ability of folded aptamers to support E-AB signaling, however, is not universal: a fully folded anti-IgE aptamer, for example, produces only an extremely small, ca. 2.5% signal change in the presence of target despite the larger steric bulk of this protein. Thus, while it appears that binding-induced changes in the dynamics in fully folded aptamers can support E-AB signaling, this signaling mechanism may not be general, and in order to ensure the design of high-gain sensors binding must be linked to a large-scale conformational change.

Engineering New Aptamer Geometries for Electrochemical Aptamer-Based Sensors

Proceedings - Society of Photo-Optical Instrumentation Engineers. 2009  |  Pubmed ID: 20436792

Electrochemical aptamer-based sensors (E-AB sensors) represent a promising new approach to the detection of small molecules. E-AB sensors comprise an aptamer that is attached at one end to an electrode surface. The distal end of the aptamer probed is modified with an electroactive redox marker for signal transduction. Herein we report on the optimization of a cocaine-detecting E-AB sensor via optimization of the geometry of the aptamer. We explore two new aptamer architectures, one in which we concatenate three cocaine aptamers into a poly-aptamer and a second in which we divide the cocaine aptamer into pieces connected via an unstructured, 60-thymine linker. Both of these structures are designed such that the reporting redox tag will be located farther from the electrode in the unfolded, target-free conformation. Consistent with this, we find that signal gains of these two constructs are two to three times higher than that of the original E-AB architecture. Likewise all three architectures are selective enough to deploy directly in complex sample matrices, such as undiluted whole blood, with all three sensors successfully detecting the presence of cocaine. The findings in this ongoing study should be of value in future efforts to optimize the signaling of electrochemical aptamer-based sensors.

Some Recommendations for the Practitioner to Improve the Precision of Experimentally Determined Protein Folding Rates and Phi Values

Proteins. Feb, 2009  |  Pubmed ID: 18655053

The mechanism by which proteins fold from an initially random conformation into a functional, native structure remains a major unsolved question in molecular biology. Of particular interest to the protein folding community is the structure that the protein adopts in the folding transition state (the highest free energy state on the pathway from unfolded to folded), as that state forms the barrier that defines the folding pathway. Unfortunately, however, unlike those of the initial, unfolded state and the final, folded state of the protein, the structure in the transition state cannot be directly assessed via experiment. Instead, experimentalists infer the structure of the transition state, often by estimating changes in its free energy by measuring the effects of amino acid substitutions on folding and unfolding rates (Phi-value analysis). In this article we show how to obtain more efficient estimates of these important quantities via improved experimental designs, and how to avoid common pitfalls in the analysis of kinetic data during the extraction of these parameters.

Universality in the Timescales of Internal Loop Formation in Unfolded Proteins and Single-stranded Oligonucleotides

Biophysical Journal. Dec, 2010  |  Pubmed ID: 21156138

Understanding the rate at which various parts of a molecular chain come together to facilitate the folding of a biopolymer (e.g., a protein or RNA) into its functional form remains an elusive goal. Here we use experiments, simulations, and theory to study the kinetics of internal loop closure in disordered biopolymers such as single-stranded oligonucleotides and unfolded proteins. We present theoretical arguments and computer simulation data to show that the relationship between the timescale of internal loop formation and the positions of the monomers enclosing the loop can be recast in a form of a universal master dependence. We also perform experimental measurements of the loop closure times of single-stranded oligonucleotides and show that both these and previously reported internal loop closure kinetics of unfolded proteins are well described by this theoretically predicted dependence. Finally, we propose that experimental deviations from the master dependence can then be used as a sensitive probe of dynamical and structural order in unfolded proteins and other biopolymers.

Label-free, Dual-analyte Electrochemical Biosensors: a New Class of Molecular-electronic Logic Gates

Journal of the American Chemical Society. Jun, 2010  |  Pubmed ID: 20527878

An "XOR" gate built using label-free, dual-analyte electrochemical sensors and the activation of this logic gate via changing concentrations of cocaine and the relevant cDNA as inputs are described.

Colorimetric Detection of DNA, Small Molecules, Proteins, and Ions Using Unmodified Gold Nanoparticles and Conjugated Polyelectrolytes

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20534499

We have demonstrated a novel sensing strategy employing single-stranded probe DNA, unmodified gold nanoparticles, and a positively charged, water-soluble conjugated polyelectrolyte to detect a broad range of targets including nucleic acid (DNA) sequences, proteins, small molecules, and inorganic ions. This nearly "universal" biosensor approach is based on the observation that, while the conjugated polyelectrolyte specifically inhibits the ability of single-stranded DNA to prevent the aggregation of gold-nanoparticles, no such inhibition is observed with double-stranded or otherwise "folded" DNA structures. Colorimetric assays employing this mechanism for the detection of hybridization are sensitive and convenient--picomolar concentrations of target DNA are readily detected with the naked eye, and the sensor works even when challenged with complex sample matrices such as blood serum. Likewise, by employing the binding-induced folding or association of aptamers we have generalized the approach to the specific and convenient detection of proteins, small molecules, and inorganic ions. Finally, this new biosensor approach is quite straightforward and can be completed in minutes without significant equipment or training overhead.

Structure-switching Biosensors: Inspired by Nature

Current Opinion in Structural Biology. Aug, 2010  |  Pubmed ID: 20627702

Chemosensing in nature relies on biomolecular switches, biomolecules that undergo binding-induced changes in conformation or oligomerization to transduce chemical information into specific biochemical outputs. Motivated by the impressive performance of these natural 'biosensors,' which support continuous, real-time detection in highly complex environments, significant efforts have gone into the adaptation of such switches into artificial chemical sensors. Ongoing advances in the fields of protein and nucleic acid engineering (e.g. computational protein design, directed evolution, selection strategies and labeling chemistries) have greatly enhanced our ability to design new structure-switching sensors. Coupled with the development of advanced optical readout mechanisms, including genetically encoded fluorophores, and electrochemical readouts supporting detection directly in highly complex sample matrices, switch-based sensors have already seen deployment in applications ranging from real time, in vivo imaging to the continuous monitoring of drugs in blood serum.

Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum Via an Electrochemical, Ribonucleic Acid Aptamer-based Biosensor

Analytical Chemistry. Sep, 2010  |  Pubmed ID: 20687587

Biosensors built using ribonucleic acid (RNA) aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degradation when deployed in clinical samples. To explore methods for protecting RNA-based biosensors from such degradation we have constructed and characterized an electrochemical, aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2'-hydroxyl groups outside of the aptamer's antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degradation-resistant deoxyribonucleic acid (DNA) equivalents. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-molecular-weight-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clinically relevant concentrations using the original RNA-based sensor.

Investigation of an Anomalously Accelerating Substitution in the Folding of a Prototypical Two-state Protein

Journal of Molecular Biology. Oct, 2010  |  Pubmed ID: 20816985

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.

Biomimetic Glass Nanopores Employing Aptamer Gates Responsive to a Small Molecule

Chemical Communications (Cambridge, England). Nov, 2010  |  Pubmed ID: 20865192

We report the preparation of 20 and 65 nm radii glass nanopores whose surface is modified with DNA aptamers controlling the molecular transport through the nanopores in response to small molecule binding.

An Electrochemical Supersandwich Assay for Sensitive and Selective DNA Detection in Complex Matrices

Journal of the American Chemical Society. Oct, 2010  |  Pubmed ID: 20873767

In a traditional sandwich assay, a DNA target hybridizes to a single copy of the signal probe. Here we employ a modified signal probe containing a methylene blue (a redox moiety) label and a "sticky end." When a DNA target hybridizes this signal probe, the sticky end remains free to hybridize another target leading to the creation of a supersandwich structure containing multiple labels. This leads to large signal amplification upon monitoring by voltammetry.

Detection of Telomerase Activity in High Concentration of Cell Lysates Using Primer-modified Gold Nanoparticles

Journal of the American Chemical Society. Nov, 2010  |  Pubmed ID: 20932008

Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its use has been significantly limited when performed directly in complex, interferant-laced samples. In this work, we report a modification of the TRAP assay that allows the detection of high-fidelity amplification of telomerase products directly from concentrated cell lysates. Briefly, we covalently attached 12 nm gold nanoparticles (AuNPs) to the telomere strand (TS) primer, which is used as a substrate for telomerase elongation. These TS-modified AuNPs significantly reduce polymerase chain reaction (PCR) artifacts (such as primer dimers) and improve the yield of amplified telomerase products relative to the traditional TRAP assay when amplification is performed in concentrated cell lysates. Specifically, because the TS-modified AuNPs eliminate most of the primer-dimer artifacts normally visible at the same position as the shortest amplified telomerase PCR product apparent on agarose gels, the AuNP-modified TRAP assay exhibits excellent sensitivity. Consequently, we observed a 10-fold increase in sensitivity for cancer cells diluted 1000-fold with somatic cells. It thus appears that the use of AuNP-modified primers significantly improves the sensitivity and specificity of the traditional TRAP assay and may be an effective method by which PCR can be performed directly in concentrated cell lysates.

Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

Analytical Chemistry. Oct, 2010  |  Pubmed ID: 20936782

We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ∼10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence.

The Art of Writing Science

Protein Science : a Publication of the Protein Society. Dec, 2010  |  Pubmed ID: 20954234

A Mechanistic Study of Electron Transfer from the Distal Termini of Electrode-bound, Single-stranded DNAs

Journal of the American Chemical Society. Nov, 2010  |  Pubmed ID: 20964337

Electrode-bound, redox-reporter-modified oligonucleotides play roles in the functioning of a number of electrochemical biosensors, and thus the question of electron transfer through or from such molecules has proven of significant interest. In response, we have experimentally characterized the rate with which electrons are transferred between a methylene blue moiety on the distal end of a short, single-stranded polythymine DNA to a monolayer-coated gold electrode to which the other end of the DNA is site-specifically attached. We find that this rate scales with oligonucleotide length to the -1.16 ± 0.09 power. This weak, approximately inverse length dependence differs dramatically from the much stronger dependencies observed for the rates of end-to-end collisions in single-stranded DNA and through-oligonucleotide electron hopping. It instead coincides with the expected length dependence of a reaction-limited process in which the overall rate is proportional to the equilibrium probability that the end of the oligonucleotide chain approaches the surface. Studies of the ionic strength and viscosity dependencies of electron transfer further support this "chain-flexibility" mechanism, and studies of the electron transfer rate of methylene blue attached to the hexanethiol monolayer suggest that heterogeneous electron transfer through the monolayer is rate limiting. Thus, under the circumstances we have employed, the flexibility (i.e., the equilibrium statistical properties) of the oligonucleotide chain defines the rate with which an attached redox reporter transfers electrons to an underlying electrode, an observation that may be of utility in the design of new biosensor architectures.

Exploiting Binding-induced Changes in Probe Flexibility for the Optimization of Electrochemical Biosensors

Analytical Chemistry. Jan, 2010  |  Pubmed ID: 20000457

Electrochemical sensors employing redox-tagged, electrode-bound oligonucleotides have emerged as a promising new platform for the reagentless detection of molecular analytes. Signal generation in these sensors is linked to specific, binding-induced changes in the efficiency with which an attached redox tag approaches and exchanges electrons with the interrogating electrode. We present here a straightforward means of optimizing the signal gain of these sensors that exploits this mechanism. Specifically, using square-wave voltammetry, which is exquisitely sensitive to electrode reaction rates, we can tune the frequency of the voltammetric measurements to preferentially enhance the signal associated with either the unbound or target-bound conformations of the probe. This allows us to control not only the magnitude of the signal gain associated with target binding but also the sign of the signal change, generating "signal-on" or "signal-off" sensors. This optimization parameter appears to be quite general: we show here that tuning the square-wave frequency can significantly enhance the gain of the sensors directed against specific oligonucleotide sequences, small molecules, proteins, and protein-small molecule interactions.

On the Binding of Cationic, Water-soluble Conjugated Polymers to DNA: Electrostatic and Hydrophobic Interactions

Journal of the American Chemical Society. Feb, 2010  |  Pubmed ID: 20058862

Water-soluble, cationic conjugated polymer binds single-stranded DNA with higher affinity than it binds double-stranded or otherwise "folded" DNA. This stronger binding results from the greater hydrophobicity of single-stranded DNA. Upon reducing the strength of the hydrophobic interactions, the electrostatic attraction becomes the important interaction that regulates the binding between the water-soluble conjugated polymer and DNA. The different affinities between the cationic conjugated polymer and various forms of DNA (molecular beacons and its open state; single-stranded DNA and double-stranded DNA and single-stranded DNA and complex DNA folds) can be used to design a variety of biosensors.

Sensitive and Selective Amplified Fluorescence DNA Detection Based on Exonuclease III-aided Target Recycling

Journal of the American Chemical Society. Feb, 2010  |  Pubmed ID: 20092337

A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 degrees C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 degrees C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.

Tracking a Molecular Motor with a Nanoscale Optical Encoder

Nano Letters. Mar, 2010  |  Pubmed ID: 20121107

Optical encoders are commonly used in macroscopic machines to make precise measurements of distance and velocity by translating motion into a periodic signal. Here we show how Forster resonance energy transfer can be used to implement this technique at the single-molecule scale. We incorporate a series of acceptor dye molecules into self-assembling DNA, and the periodic signal resulting from unhindered motion of a donor-labeled molecular motor provides nanometer-scale resolution in milliseconds.

Re-engineering Aptamers to Support Reagentless, Self-reporting Electrochemical Sensors

The Analyst. Mar, 2010  |  Pubmed ID: 20174715

Electrochemical aptamer-based (E-AB) sensors have emerged as a promising and versatile new biosensor platform. Combining the generality and specificity of aptamer-ligand interactions with the selectivity and convenience of electrochemical readouts, this approach affords the detection of a wide variety of targets directly in complex, contaminant-ridden samples, such as whole blood, foodstuffs and crude soil extracts, without the need for exogenous reagents or washing steps. Signaling in this class of sensors is predicated on target-induced changes in the conformation of an electrode-bound probe aptamer that, in turn, changes the efficiency with which a covalently attached redox tag exchanges electrons with the interrogating electrode. Aptamer selection strategies, however, typically do not select for the conformation-switching architectures, and as such several approaches have been reported to date by which aptamers can be re-engineered such that they undergo the binding-induced switching required to support efficient E-AB signaling. Here, we systematically compare the merits of these re-engineering approaches using representative aptamers specific to the small molecule adenosine triphosphate and the protein human immunoglobulin E. We find that, while many aptamer architectures support E-AB signaling, the observed signal gain (relative change in signal upon target binding) varies by more than two orders of magnitude across the various constructs we have investigated (e.g., ranging from -10% to 200% for our ATP sensors). Optimization of the switching architecture is thus an important element in achieving maximum E-AB signal gain and we find that this optimal geometry is specific to the aptamer sequence upon which the sensor is built.

Quantitative, Reagentless, Single-step Electrochemical Detection of Anti-DNA Antibodies Directly in Blood Serum

Chemical Communications (Cambridge, England). Mar, 2010  |  Pubmed ID: 20177635

Here we demonstrate the use of redox labeled double- and single-stranded oligonucleotides as recognition probes for the reagentless, single-step, electrochemical detection of anti-DNA antibodies directly in blood serum.

Folding-based Electrochemical Biosensors: the Case for Responsive Nucleic Acid Architectures

Accounts of Chemical Research. Apr, 2010  |  Pubmed ID: 20201486

Biomolecular recognition is versatile, specific, and high affinity, qualities that have motivated decades of research aimed at adapting biomolecules into a general platform for molecular sensing. Despite significant effort, however, so-called "biosensors" have almost entirely failed to achieve their potential as reagentless, real-time analytical devices; the only quantitative, reagentless biosensor to achieve commercial success so far is the home glucose monitor, employed by millions of diabetics. The fundamental stumbling block that has precluded more widespread success of biosensors is the failure of most biomolecules to produce an easily measured signal upon target binding. Antibodies, for example, do not change their shape or dynamics when they bind their recognition partners, nor do they emit light or electrons upon binding. It has thus proven difficult to transduce biomolecular binding events into a measurable output signal, particularly one that is not readily spoofed by the binding of any of the many potentially interfering species in typical biological samples. Analytical approaches based on biomolecular recognition are therefore mostly cumbersome, multistep processes relying on analyte separation and isolation (such as Western blots, ELISA, and other immunochemical methods); these techniques have proven enormously useful, but are limited almost exclusively to laboratory settings. In this Account, we describe how we have refined a potentially general solution to the problem of signal detection in biosensors, one that is based on the binding-induced "folding" of electrode-bound DNA probes. That is, we have developed a broad new class of biosensors that employ electrochemistry to monitor binding-induced changes in the rigidity of a redox-tagged probe DNA that has been site-specifically attached to an interrogating electrode. These folding-based sensors, which have been generalized to a wide range of specific protein, nucleic acid, and small-molecule targets, are rapid (responding in seconds to minutes), sensitive (detecting sub-picomolar to micromolar concentrations), and reagentless. They are also greater than 99% reusable, are supported on micrometer-scale electrodes, and are readily fabricated into densely packed sensor arrays. Finally, and critically, their signaling is linked to a binding-specific change in the physics of the probe DNA, and not simply to adsorption of the target onto the sensor head. Accordingly, they are selective enough to be employed directly in blood, crude soil extracts, cell lysates, and other grossly contaminated clinical and environmental samples. Indeed, we have recently demonstrated the ability to quantitatively monitor a specific small molecule in real-time directly in microliters of flowing, unmodified blood serum. Because of their sensitivity, substantial background suppression, and operational convenience, these folding-based biosensors appear potentially well suited for electronic, on-chip applications in pathogen detection, proteomics, metabolomics, and drug discovery.

Switch-based Biosensors: a New Approach Towards Real-time, in Vivo Molecular Detection

Trends in Biotechnology. Jan, 2011  |  Pubmed ID: 21106266

Although the ability to monitor specific molecules in vivo in real-time could revolutionize many aspects of healthcare, the technological challenges that stand in the way of reaching this goal are considerable and are poorly met by most existing analytical approaches. Nature, however, has already solved the problem of real-time molecular detection in complex media by employing biomolecular "switches". That is, protein and nucleic acids that sense chemical cues and, by undergoing specific, binding-induced conformational changes, transduce this recognition into high-gain signal outputs. Here, we argue that devices that employ such switches represent a promising route towards versatile, real-time molecular monitoring in vivo.

CheapStat: an Open-source, "do-it-yourself" Potentiostat for Analytical and Educational Applications

PloS One. 2011  |  Pubmed ID: 21931613

Although potentiostats are the foundation of modern electrochemical research, they have seen relatively little application in resource poor settings, such as undergraduate laboratory courses and the developing world. One reason for the low penetration of potentiostats is their cost, as even the least expensive commercially available laboratory potentiostats sell for more than one thousand dollars. An inexpensive electrochemical workstation could thus prove useful in educational labs, and increase access to electrochemistry-based analytical techniques for food, drug and environmental monitoring. With these motivations in mind, we describe here the CheapStat, an inexpensive (<$80), open-source (software and hardware), hand-held potentiostat that can be constructed by anyone who is proficient at assembling circuits. This device supports a number of potential waveforms necessary to perform cyclic, square wave, linear sweep and anodic stripping voltammetry. As we demonstrate, it is suitable for a wide range of applications ranging from food- and drug-quality testing to environmental monitoring, rapid DNA detection, and educational exercises. The device's schematics, parts lists, circuit board layout files, sample experiments, and detailed assembly instructions are available in the supporting information and are released under an open hardware license.

Electrochemical Biosensors Employing an Internal Electrode Attachment Site and Achieving Reversible, High Gain Detection of Specific Nucleic Acid Sequences

Analytical Chemistry. Dec, 2011  |  Pubmed ID: 21975121

Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ∼800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.

Polarity-switching Electrochemical Sensor for Specific Detection of Single-nucleotide Mismatches

Angewandte Chemie (International Ed. in English). Nov, 2011  |  Pubmed ID: 21976312

High-precision, in Vitro Validation of the Sequestration Mechanism for Generating Ultrasensitive Dose-response Curves in Regulatory Networks

PLoS Computational Biology. Oct, 2011  |  Pubmed ID: 21998566

Our ability to recreate complex biochemical mechanisms in designed, artificial systems provides a stringent test of our understanding of these mechanisms and opens the door to their exploitation in artificial biotechnologies. Motivated by this philosophy, here we have recapitulated in vitro the "target sequestration" mechanism used by nature to improve the sensitivity (the steepness of the input/output curve) of many regulatory cascades. Specifically, we have employed molecular beacons, a commonly employed optical DNA sensor, to recreate the sequestration mechanism and performed an exhaustive, quantitative study of its key determinants (e.g., the relative concentrations and affinities of probe and depletant). We show that, using sequestration, we can narrow the pseudo-linear range of a traditional molecular beacon from 81-fold (i.e., the transition from 10% to 90% target occupancy spans an 81-fold change in target concentration) to just 1.5-fold. This narrowing of the dynamic range improves the sensitivity of molecular beacons to that equivalent of an oligomeric, allosteric receptor with a Hill coefficient greater than 9. Following this we have adapted the sequestration mechanism to steepen the binding-site occupancy curve of a common transcription factor by an order of magnitude over the sensitivity observed in the absence of sequestration. Given the success with which the sequestration mechanism has been employed by nature, we believe that this strategy could dramatically improve the performance of synthetic biological systems and artificial biosensors.

Two-step, PCR-free Telomerase Detection by Using Exonuclease III-aided Target Recycling

Chembiochem : a European Journal of Chemical Biology. Dec, 2011  |  Pubmed ID: 22125097

We report the sensitive detection of telomerase activity by using exonuclease III-aided target recycling to amplify the signal produced by a chimeric LNA-DNA molecular beacon. We demonstrate the specific detection of as few as 30 telomerase-positive breast cancer cells in a single-measurement fluorescence assay that avoids the problematic PCR and gel analysis of the current "gold-standard" assay.

Dielectric Spectroscopy of Proteins As a Quantitative Experimental Test of Computational Models of Their Low-frequency Harmonic Motions

Journal of the American Chemical Society. Jun, 2011  |  Pubmed ID: 21542634

Decades of molecular dynamics and normal mode calculations suggest that the largest-scale collective vibrational modes of proteins span the picosecond to nanosecond time scale. Experimental investigation of these harmonic, low-amplitude motions, however, has proven challenging. In response, we have developed a vector network analyzer-based spectrometer that supports the accurate measurement of both the absorbance and refractive index of solvated biomolecules over the corresponding gigahertz to terahertz frequency regime, thus providing experimental information regarding their largest-scale, lowest frequency harmonic motions. We have used this spectrometer to measure the complex dielectric response of lysozyme solutions over the range 65 to 700 GHz and an effective medium model to separate the dielectric response of the solvated protein from that of its buffer. In doing so, we find that each lysozyme is surrounded by a tightly bound layer of 165 ± 15 water molecules that, in terms of their picosecond dynamics, behave as if they are an integral part of the protein. We also find that existing computational descriptions of the protein's dynamics compare poorly with the results of our experiment. Specifically, published normal mode and molecular dynamics simulations do not explain the measured dielectric response unless we introduce a cutoff frequency of 250 GHz below which the density of vibrational modes drops to zero. This cutoff is physically plausible, given the known size of the protein and the known speed of sound in proteins, raising questions as to why it is not apparent in computational models of the protein's motions.

Transcription Factor Beacons for the Quantitative Detection of DNA Binding Activity

Journal of the American Chemical Society. Sep, 2011  |  Pubmed ID: 21815647

The development of convenient, real-time probes for monitoring protein function in biological samples represents an important challenge of the postgenomic era. In response, we introduce here "transcription factor beacons," binding-activated fluorescent DNA probes that signal the presence of specific DNA-binding activities. As a proof of principle, we present beacons for the rapid, sensitive detection of three transcription factors (TATA Binding Protein, Myc-Max, and NF-κB), and measure binding activity directly in crude nuclear extracts.

Probe Accessibility Effects on the Performance of Electrochemical Biosensors Employing DNA Monolayers

Analytical and Bioanalytical Chemistry. Jan, 2012  |  Pubmed ID: 21928081

Surface-confined DNA probes are increasingly used as recognition elements (or presentation scaffolds) for detection of proteins, enzymes, and other macromolecules. Here we demonstrate that the density of the DNA probe monolayer on the gold electrode is a crucial determinant of the final signalling of such devices. We do so using redox modified single-stranded and double-stranded DNA probes attached to the surface of a gold electrode and measuring the rate of digestion in the presence of a non-specific nuclease enzyme. We demonstrate that accessibility of DNA probes for binding to their macromolecular target is, as expected, improved at lower probe densities. However, with double-stranded DNA probes, even at the lowest densities investigated, a significant fraction of the immobilized probe is inaccessible to nuclease digestion. These results stress the importance of the accessibility issue and of probe density effects when DNA-based sensors are used for detection of macromolecular targets.

Wash-free, Electrochemical Platform for the Quantitative, Multiplexed Detection of Specific Antibodies

Analytical Chemistry. Jan, 2012  |  Pubmed ID: 22145706

The diagnosis, prevention, and treatment of many illnesses, including infectious and autoimmune diseases, would benefit from the ability to measure specific antibodies directly at the point of care. Thus motivated, we designed a wash-free, electrochemical method for the rapid, quantitative detection of specific antibodies directly in undiluted, unprocessed blood serum. Our approach employs short, contiguous polypeptide epitopes coupled to the distal end of an electrode-bound nucleic acid "scaffold" modified with a reporting methylene blue. The binding of the relevant antibody to the epitope reduces the efficiency with which the redox reporter approaches, and thus exchanges electrons with, the underlying sensor electrode, producing readily measurable change in current. To demonstrate the versatility of the approach, we fabricated a set of six such sensors, each aimed at the detection of a different monoclonal antibody. All six sensors are sensitive (subnanomolar detection limits), rapid (equilibration time constants ∼8 min), and specific (no appreciable cross reactivity with the targets of the other five). When deployed in a millimeter-scale, an 18-pixel array with each of the six sensors in triplicate support the simultaneous measurement of the concentrations of multiple antibodies in a single, submilliliter sample volume. The described sensor platform thus appears be a relatively general approach to the rapid and specific quantification of antibodies in clinical materials.

Entropic and Electrostatic Effects on the Folding Free Energy of a Surface-attached Biomolecule: an Experimental and Theoretical Study

Journal of the American Chemical Society. Feb, 2012  |  Pubmed ID: 22239220

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ∼130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.

Engineering Biosensors with Extended, Narrowed, or Arbitrarily Edited Dynamic Range

Journal of the American Chemical Society. Feb, 2012  |  Pubmed ID: 22239688

Biomolecular recognition has long been an important theme in artificial sensing technologies. A current limitation of protein- and nucleic acid-based recognition, however, is that the useful dynamic range of single-site binding typically spans an 81-fold change in target concentration, an effect that limits the utility of biosensors in applications calling for either great sensitivity (a steeper relationship between target concentration and output signal) or the quantification of more wide-ranging concentrations. In response, we have adapted strategies employed by nature to modulate the input-output response of its biorecognition systems to rationally edit the useful dynamic range of an artificial biosensor. By engineering a structure-switching mechanism to tune the affinity of a receptor molecule, we first generated a set of receptor variants displaying similar specificities but different target affinities. Using combinations of these receptor variants (signaling and nonsignaling), we then rationally extended (to 900000-fold), narrowed (to 5-fold), and edited (three-state) the normally 81-fold dynamic range of a representative biosensor. We believe that these strategies may be widely applicable to technologies reliant on biorecognition.

Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor

Journal of the American Chemical Society. Feb, 2012  |  Pubmed ID: 22313286

Transcription factor expression levels, which sensitively reflect cellular development and disease state, are typically monitored via cumbersome, reagent-intensive assays that require relatively large quantities of cells. Here, we demonstrate a simple, quantitative approach to their detection based on a simple, electrochemical sensing platform. This sensor sensitively and quantitatively detects its target transcription factor in complex media (e.g., 250 μg/mL crude nuclear extracts) in a convenient, low-reagent process requiring only 10 μL of sample. Our approach thus appears a promising means of monitoring transcription factor levels.