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author:("belisle, S.")
1.  Thermodynamic Basis For Engineering High Affinity, High Specificity Binding-Induced DNA Clamp Nanoswitches 
ACS nano  2013;7(12):10863-10869.
Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼ 0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide, however, an additional specificity check that increases the discrimination efficiency towards a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the “specificity window” of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.
doi:10.1021/nn404305e
PMCID: PMC4281346  PMID: 24219761
Clamp-Mechanism; Triplex; DNA Nanomachines; Biomolecular Switch; Molecular Beacons; Specificity; Ligand-Induced Fit
2.  Balancing Energy Budget in a Central-Place Forager: Which Habitat to Select in a Heterogeneous Environment? 
PLoS ONE  2014;9(7):e102162.
Foraging animals are influenced by the distribution of food resources and predation risk that both vary in space and time. These constraints likely shape trade-offs involving time, energy, nutrition, and predator avoidance leading to a sequence of locations visited by individuals. According to the marginal-value theorem (MVT), a central-place forager must either increase load size or energy content when foraging farther from their central place. Although such a decision rule has the potential to shape movement and habitat selection patterns, few studies have addressed the mechanisms underlying habitat use at the landscape scale. Our objective was therefore to determine how Ring-billed gulls (Larus delawarensis) select their foraging habitats while nesting in a colony located in a heterogeneous landscape. Based on locations obtained by fine-scale GPS tracking, we used resource selection functions (RSFs) and residence time analyses to identify habitats selected by gulls for foraging during the incubation and brood rearing periods. We then combined this information to gull survey data, feeding rates, stomach contents, and calorimetric analyses to assess potential trade-offs. Throughout the breeding season, gulls selected landfills and transhipment sites that provided higher mean energy intake than agricultural lands or riparian habitats. They used landfills located farther from the colony where no deterrence program had been implemented but avoided those located closer where deterrence measures took place. On the other hand, gulls selected intensively cultured lands located relatively close to the colony during incubation. The number of gulls was then greater in fields covered by bare soil and peaked during soil preparation and seed sowing, which greatly increase food availability. Breeding Ring-billed gulls thus select habitats according to both their foraging profitability and distance from their nest while accounting for predation risk. This supports the predictions of the MVT for central-place foraging over large spatial scales.
doi:10.1371/journal.pone.0102162
PMCID: PMC4100874  PMID: 25029498
3.  Resource defense and monopolization in a marked population of ruby-throated hummingbirds (Archilochus colubris) 
Ecology and Evolution  2014;4(6):776-793.
Resource defense behavior is often explained by the spatial and temporal distribution of resources. However, factors such as competition, habitat complexity, and individual space use may also affect the capacity of individuals to defend and monopolize resources. Yet, studies frequently focus on one or two factors, overlooking the complexity found in natural settings. Here, we addressed defense and monopolization of nectar feeders in a population of free-ranging ruby-throated hummingbirds marked with passive integrated transponder (PIT tags). Our study system consisted of a 44 ha systematic grid of 45 feeders equipped with PIT tag detectors recording every visit made at feeders. We modeled the number of visits by competitors (NVC) at feeders in response to space use by a focal individual potentially defending a feeder, number of competitors, nectar sucrose concentration, and habitat visibility. Individuals who were more concentrated at certain feeders on a given day and who were more stable in their use of the grid throughout the season gained higher exclusivity in the use of those feeders on that day, especially for males competing against males. The level of spatial concentration at feeders and its negative effect on NVC was, however, highly variable among individuals, suggesting a continuum in resource defense strategies. Although the apparent capacity to defend feeders was not affected by competition or nectar sucrose concentration, the level of monopolization decreased with increasing number of competitors and higher nectar quality. Defense was enhanced by visibility near feeders, but only in forested habitats. The reverse effect of visibility in open habitats was more difficult to interpret as it was probably confounded by perch availability, from which a bird can defend its feeder. Our study is among the first to quantify the joint use of food resource by overlapping individuals unconstrained in their use of space. Our results show the importance of accounting for variation in space use among individuals as it translated into varying levels of defense and monopolization of feeders regardless of food resource distribution.
doi:10.1002/ece3.972
PMCID: PMC3967903  PMID: 24683460
Archilochus colubris; habitat visibility; hummingbirds; intrusion rate; Quebec; resource defense; resource monopolization; space use; territoriality
4.  Using distal site mutations and allosteric inhibition to tune, extend and narrow the useful dynamic range of aptamer-based sensors 
Journal of the American Chemical Society  2012;134(51):10.1021/ja310585e.
Here we demonstrate multiple, complementary approaches by which to tune, extend or narrow the dynamic range of aptamer-based sensors. Specifically, we have employed both distal site mutations and allosteric control to tune the affinity and dynamic range of a fluorescent aptamer beacon. We show that allosteric control, achieved by using a set of easily designed oligonucleotide inhibitors that competes against the folding of the aptamer, allows to rationally and finely tune the affinity of our model aptamer across three orders of magnitude of target concentration with greater precision than that achieved using mutational approaches. Using these methods we generate sets of aptamers varying significantly in target affinity, which we then combined to recreate several of the mechanisms employed by nature to both narrow and broaden the dynamic range of biological receptors. Such ability to finely control the affinity and dynamic range of aptamers may find many applications in synthetic biology, drug delivery and targeted therapies, fields in which aptamers are of rapidly growing importance.
doi:10.1021/ja310585e
PMCID: PMC3866043  PMID: 23215257
5.  Transcription Factor Beacons for the Quantitative Detection of DNA Binding Activity 
Journal of the American Chemical Society  2011;133(35):10.1021/ja204775k.
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.
doi:10.1021/ja204775k
PMCID: PMC3866037  PMID: 21815647
6.  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  2012;134(4):10.1021/ja208436p.
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, hydroxyl-alkane-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.
doi:10.1021/ja208436p
PMCID: PMC3866038  PMID: 22239220
Thermodynamics; Biosensor; Self Assembled Monolayer
7.  Bio-electrochemical switches for the quantitative detection of antibodies directly in whole blood 
Journal of the American Chemical Society  2012;134(37):10.1021/ja305720w.
The development of rapid, low cost, point-of-care approaches for the quantitative detection of antibodies would drastically impact global health by shortening the delay between sample collection and diagnosis, and by improving the penetration of modern diagnostics into the developing world. Unfortunately, however, current methods for the quantitative detection of antibodies, including ELISAs, western blots and fluorescence polarization assays, are complex, multiple step processes reliant on well-trained technicians working in well-equipped laboratories. In response we describe here a versatile, DNA-based electrochemical “switch” for the rapid, single-step measurement of specific antibodies directly in undiluted, whole blood at clinically relevant, low-nanomolar concentrations.
doi:10.1021/ja305720w
PMCID: PMC3866042  PMID: 22913425
8.  Allosterically tunable, DNA-based switches triggered by heavy metals 
Journal of the American Chemical Society  2013;135(36):10.1021/ja404653q.
Here we demonstrate the rational design of allosterically controllable, metal-ion-triggered molecular switches. Specifically, we designed DNA sequences that adopt two low energy conformations, one of which does not bind to the target ion and the other of which contains mismatches sites serving as specific recognition sites for mercury(II) or silver(I) ions. Both switches contain multiple metal binding sites and thus exhibit homotropic allosteric (cooperative) responses. As heterotropic allosteric effectors we employ single-stranded DNA sequences that either stabilize or destabilize the non-binding state, enabling dynamic range tuning over several orders of magnitude. The ability to rationally introduce these effects into target-responsive switches could be of value in improving the functionality of DNA-based nanomachines.
doi:10.1021/ja404653q
PMCID: PMC3831027  PMID: 23971651
9.  The rational design of allosteric inhibitors and activators using the population-shift model: in vitro validation and application to an artificial biosensor 
Journal of the American Chemical Society  2012;134(37):15177-15180.
The population-shift mechanism can be used to rationally re-engineer structure-switching biosensors to enable their allosteric inhibition and activation. As a proof-of-principle example of this we have introduced distal allosteric sites into molecular beacons, an optical sensor for the detection of specific nucleic acid sequences. The binding of inhibitors and activators to these sites enables the rational modulation of the sensor's target affinity –and thus its useful dynamic range– over three orders of magnitude. The convenience with which this was done suggests that the population-shift mechanism may prove a useful method by which allosteric regulation can be introduced into biosensors, “smart” biomaterials and other artificial biotechnologies.
doi:10.1021/ja304672h
PMCID: PMC3523727  PMID: 22924432
10.  Local assessment of myelin health in a multiple sclerosis mouse model using a 2D Fourier transform approach 
Biomedical Optics Express  2013;4(10):2003-2014.
We present an automated two-dimensional Fourier transform (2D-FT) approach to analyze the local organization of myelinated axons in the spinal cord. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to observe lesions in a commonly used animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). A 2D-FT was applied on the CARS images to find the average orientation and directional anisotropy of the fibers within contiguous image domains. We introduce the corrected correlation parameter (CCP), a measure of the correlation between orientations of adjacent domains. We show that in the EAE animal model of MS, the CCP can be used to quantify the degree of organization/disorganization in the myelin structure. This analysis was applied to a large image dataset from animals at different clinical scores and we show that some descriptors of the CCP probability density function are strongly correlated with the clinical scores. This procedure, compatible with live animal imaging, has been developed to perform local in situ evaluation of myelinated axons afflicted by EAE.
doi:10.1364/BOE.4.002003
PMCID: PMC3799662  PMID: 24156060
(300.6230) Spectroscopy, coherent anti-Stokes Raman scattering; (100.2960) Image analysis; (170.3880) Medical and biological imaging; (180.4315) Nonlinear microscopy; (180.6900) Three-dimensional microscopy; (000.1430) Biology and medicine; (170.4580) Optical diagnostics for medicine
11.  Re-engineering electrochemical biosensors to narrow or extend their useful dynamic range** 
Here we demonstrate two convenient methods to extend and narrow the useful dynamic range of a model electrochemical DNA sensor. We did so by combining DNA probes of different target affinities but with similar specificity on the same electrode. We were able to achieve an extended dynamic response spanning 3 orders of magnitude in target concentration. Using a different strategy we have also narrowed the useful dynamic range of an E-DNA sensor to only an 8-fold range of target concentrations.
doi:10.1002/anie.201202204
PMCID: PMC3482547  PMID: 22674785
electrochemical biosensors; dynamic range; engineering; depletant; pseudo-Hill coefficients
12.  Importance of breeding season and maternal investment in studies of sex-ratio adjustment: a case study using tree swallows 
Biology Letters  2011;8(3):401-404.
The control of primary sex-ratio by vertebrates has become a major focus in biology in recent years. Evolutionary theory predicts that a differential effect of maternal characteristics on the fitness of sons and daughters is an important route, whereby selection is expected to favour a bias towards the production of one sex. However, despite experimental evidence for adaptive brood sex-ratio manipulation, support for this prediction remains a major challenge in vertebrates where inconsistencies between correlative studies are frequently reported. Here, we used a large dataset (2215 nestlings over 3 years) from a wild population of tree swallows (Tachycineta bicolor) and show that variations in breeding conditions affect female sex allocation in this species. Our results also suggest that such variation in sex allocation, owing to breeding season heterogeneity, modifies the relationships between maternal characteristics and maternal investment. Indeed, we detect a positive effect of maternal age on brood sex-ratio when age also affects offspring condition (in a low-quality breeding season). Our results indicate that including measures of both breeding season quality and maternal investment will help to better understand sex allocation patterns.
doi:10.1098/rsbl.2011.1009
PMCID: PMC3367737  PMID: 22130173
birds; breeding season heterogeneity; sex allocation; sex-ratio; tree swallow
13.  Time Variability of C-Reactive Protein: Implications for Clinical Risk Stratification 
PLoS ONE  2013;8(4):e60759.
Background
C-reactive protein (CRP) is proposed as a screening test for predicting risk and guiding preventive approaches in coronary artery disease (CAD). However, the stability of repeated CRP measurements over time in subjects with and without CAD is not well defined. We sought to determine the stability of serial CRP measurements in stable subjects with distinct CAD manifestations and a group without CAD while carefully controlling for known confounders.
Methods
We prospectively studied 4 groups of 25 stable subjects each 1) a history of recurrent acute coronary events; 2) a single myocardial infarction ≥7 years ago; 3) longstanding CAD (≥7 years) that had never been unstable; 4) no CAD. Fifteen measurements of CRP were obtained to cover 21 time-points: 3 times during one day; 5 consecutive days; 4 consecutive weeks; 4 consecutive months; and every 3 months over the year. CRP risk threshold was set at 2.0 mg/L. We estimated variance across time-points using standard descriptive statistics and Bayesian hierarchical models.
Results
Median CRP values of the 4 groups and their pattern of variability did not differ substantially so all subjects were analyzed together. The median individual standard deviation (SD) CRP values within-day, within-week, between-weeks and between-months were 0.07, 0.19, 0.36 and 0.63 mg/L, respectively. Forty-six percent of subjects changed CRP risk category at least once and 21% had ≥4 weekly and monthly CRP values in both low and high-risk categories.
Conclusions
Considering its large intra-individual variability, it may be problematic to rely on CRP values for CAD risk prediction and therapeutic decision-making in individual subjects.
doi:10.1371/journal.pone.0060759
PMCID: PMC3620269  PMID: 23579782
14.  Ecological immunology in a fluctuating environment: an integrative analysis of tree swallow nestling immune defense 
Ecology and Evolution  2013;3(4):1091-1103.
Evolutionary ecologists have long been interested by the link between different immune defenses and fitness. Given the importance of a proper immune defense for survival, it is important to understand how its numerous components are affected by environmental heterogeneity. Previous studies targeting this question have rarely considered more than two immune markers. In this study, we measured seven immune markers (response to phytohemagglutinin (PHA), hemolysis capacity, hemagglutination capacity, plasma bactericidal capacity, percentage of lymphocytes, percentage of heterophils, and percentage of eosinophils) in tree swallow (Tachycineta bicolor) nestlings raised in two types of agro-ecosystems of contrasted quality and over 2 years. First, we assessed the effect of environmental heterogeneity (spatial and temporal) on the strength and direction of correlations between immune measures. Second, we investigated the effect of an immune score integrating information from several immune markers on individual performance (including growth, mass at fledging and parasite burden). Both a multivariate and a pair-wise approach showed variation in relationships between immune measures across years and habitats. We also found a weak association between the integrated score of nestling immune function and individual performance, but only under certain environmental conditions. We conclude that the ecological context can strongly affect the interpretation of immune defenses in the wild. Given that spatiotemporal variations are likely to affect individual immune defenses, great caution should be used when generalizing conclusions to other study systems.
doi:10.1002/ece3.504
PMCID: PMC3631416  PMID: 23610646
Agricultural intensification; bird; ecological immunology; integrated immune score; performance; tree swallow
15.  Quantification of transcription factor binding in cell extracts using an electrochemical, structure-switching biosensor 
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.
doi:10.1021/ja2115663
PMCID: PMC3523731  PMID: 22313286
16.  Engineering biosensors with extended, narrowed, or arbitrarily edited dynamic range 
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 for 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, we first generated a set of receptor variants displaying similar specificity, but spanning a wide range of target affinities. We then rationally combined sub-sets of these variants to expand the pseudo-log-linear dynamic range of our biosensor to six orders of magnitude. Using other combinations of variants we have also fabricated more elaborate, three-state dose-response sensors that respond sensitively only when the target concentration falls above or below some well-defined intermediate regime. Finally, by combining signaling and non-signaling receptor variants, we have succeeded in both compressing the dynamic range of our biosensor by an order of magnitude, and in rationally tuning its narrowed threshold response to any arbitrarily selected target concentrations. Given their widespread occurrence in nature, it would appear that these same approaches could significantly enhance the performance of many biomolecule-based technologies.
doi:10.1021/ja209850j
PMCID: PMC3522460  PMID: 22239688
17.  Comparison of the dipeptidyl peptidase-4 gene methylation levels between severely obese subjects with and without the metabolic syndrome 
Background
The dipeptidyl peptidase-4 (DPP4) enzyme is a novel adipokine potentially involved in the development of the metabolic syndrome (MetS). Previous observations demonstrated higher visceral adipose tissue (VAT) DPP4 gene expression in non-diabetic severely obese men with (MetS+) vs. without (MetS−) MetS. DPP4 mRNA abundance in VAT correlated also with CpG site methylation levels (%Meth) localized within and near its exon 2 (CpG94 to CpG102) in non-diabetic severely obese women, regardless of their MetS status. The actual study tested whether DPP4 %Meth levels in VAT are different between MetS− and MetS+ non-diabetic severely obese subjects, whether variable metabolic and plasma lipid profiles are observed between DPP4 %Meth quartiles, and whether correlation exists in DPP4 %Meth levels between VAT and white blood cells (WBCs).
Methods
DNA was extracted from the VAT of 26 men (MetS−: n=12, MetS+: n=14) and 79 women (MetS−: n=60; MetS+: n=19), as well as from WBCs in a sub-sample of 17 women (MetS−: n=9; MetS+: n=8). The %Meth levels of CpG94 to CpG102 were assessed by pyrosequencing of sodium bisulfite-treated DNA. ANOVA analyses were used to compare the %Meth of CpGs between MetS− and MetS+ groups, and to compare the metabolic phenotype and plasma lipid levels between methylation quartiles. Pearson correlation coefficient analyses were computed to test the relationship between VAT and WBCs CpG94-102 %Meth levels.
Results
No difference was observed in CpG94-102 %Meth levels between MetS− and MetS+ subjects in VAT (P=0.67), but individuals categorized into CpG94-102 %Meth quartiles had variable plasma total-cholesterol concentrations (P=0.04). The %Meth levels of four CpGs in VAT were significantly correlated with those observed in WBCs (r=0.55−0.59, P≤0.03).
Conclusions
This study demonstrated that %Meth of CpGs localized within and near the exon 2 of the DPP4 gene in VAT are not associated with MetS status. The actual study also revealed an association between the %Meth of this locus with plasma total-cholesterol in severe obesity, which suggests a link between the DPP4 gene and plasma lipid levels.
doi:10.1186/1758-5996-5-4
PMCID: PMC3637825  PMID: 23379505
DNA methylation; Epigenetics; DPP4 gene; Visceral adipose tissue; White blood cells; Plasma cholesterol
18.  LINE-1 methylation in visceral adipose tissue of severely obese individuals is associated with metabolic syndrome status and related phenotypes 
Clinical Epigenetics  2012;4(1):10.
Background
Epigenetic mechanisms may be involved in the regulation of genes found to be differentially expressed in the visceral adipose tissue (VAT) of severely obese subjects with (MetS+) versus without (MetS-) metabolic syndrome (MetS). Long interspersed nuclear element 1 (LINE-1) elements DNA methylation levels (%meth) in blood, a marker of global DNA methylation, have recently been associated with fasting glucose, blood lipids, heart diseases and stroke.
Aim
To test whether LINE-1%meth levels in VAT are associated with MetS phenotypes and whether they can predict MetS risk in severely obese individuals.
Methods
DNA was extracted from VAT of 34 men (MetS-: n = 14, MetS+: n = 20) and 152 premenopausal women (MetS-: n = 84; MetS+: n = 68) undergoing biliopancreatic diversion for the treatment of obesity. LINE-1%meth levels were assessed by pyrosequencing of sodium bisulfite-treated DNA.
Results
The mean LINE-1%meth in VAT was of 75.8% (SD = 3.0%). Multiple linear regression analyses revealed that LINE-1%meth was negatively associated with fasting glucose levels (β = -0.04; P = 0.03), diastolic blood pressure (β =  -0.65; P = 0.03) and MetS status (β = -0.04; P = 0.004) after adjustments for the effects of age, sex, waist circumference (except for MetS status) and smoking. While dividing subjects into quartiles based on their LINE-1%meth (Q1 to Q4: lower %meth to higher %meth levels), greater risk were observed in the first (Q1: odds ratio (OR) = 4.37, P = 0.004) and the second (Q2: OR = 4.76, P = 0.002) quartiles compared to Q4 (1.00) when adjusting for age, sex and smoking.
Conclusions
These results suggest that lower global DNA methylation, assessed by LINE-1 repetitive elements methylation analysis, would be associated with a greater risk for MetS in the presence of obesity.
doi:10.1186/1868-7083-4-10
PMCID: PMC3464682  PMID: 22748066
Blood pressure; Epigenetics; Fasting glucose; Global DNA methylation; LINE-1; Metabolic syndrome; Severe obesity; Visceral adipose tissue
19.  High-Content Neurite Development Study Using Optically Patterned Substrates 
PLoS ONE  2012;7(4):e35911.
The study of neurite guidance in vitro relies on the ability to reproduce the distribution of attractive and repulsive guidance molecules normally expressed in vivo. The identification of subtle variations in the neurite response to changes in the spatial distribution of extracellular molecules can be achieved by monitoring the behavior of cells on protein gradients. To do this, automated high-content screening assays are needed to quantify the morphological changes resulting from growth on gradients of guidance molecules. Here, we present the use of laser-assisted protein adsorption by photobleaching (LAPAP) to allow the fabrication of large-scale substrate-bound laminin-1 gradients to study neurite extension. We produced thousands of gradients of different slopes and analyzed the variations in neurite attraction of neuron-like cells (RGC-5). An image analysis algorithm processed bright field microscopy images, detecting each cell and quantifying the soma centroid and the initiation, terminal and turning angles of the longest neurite.
doi:10.1371/journal.pone.0035911
PMCID: PMC3338543  PMID: 22563416
20.  Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA 
Analytical chemistry  2010;82(21):10.1021/ac1024528.
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.
doi:10.1021/ac1024528
PMCID: PMC3134121  PMID: 20936782
21.  Structure-switching biosensors: inspired by Nature 
Summary
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 read-out mechanisms, including genetically encoded fluorophores, and electrochemical read-outs 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.
doi:10.1016/j.sbi.2010.05.001
PMCID: PMC3249393  PMID: 20627702
nanosensors; nanomachines; structure-switching sensors; molecular switch; allosteric biosensors; synthetic biology; allosteric enzymes; allosteric ribozymes; aptasensors; switch-based sensors; conformational-switching; ligand sensing; signalling aptamer; molecular beacons; calcium sensor; point-of-care
22.  High-Precision, In Vitro Validation of the Sequestration Mechanism for Generating Ultrasensitive Dose-Response Curves in Regulatory Networks 
PLoS Computational Biology  2011;7(10):e1002171.
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.
Author Summary
Here we recreate in vitro the sequestration mechanism thought to underlie the extraordinary sensitivity (the steepness of the input/output function) of a number of genetic networks. We do so first using fluorescent molecular beacons, a well-established, DNA-based biosensor architecture, as our model system. The experimental parameters that define this in vitro model can be controlled with great precision, allowing us to dissect and test a quantitative model of sequestration in unprecedented detail. Following on this we employ 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. Our study thus highlights the versatility with which this approach can be used to improve the performance of both synthetic biological systems and artificial biosensors.
doi:10.1371/journal.pcbi.1002171
PMCID: PMC3188500  PMID: 21998566
23.  Biomimetic glass nanopores employing aptamer gates responsive to a small molecule† 
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.
doi:10.1039/c0cc02649b
PMCID: PMC3073859  PMID: 20865192

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