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The analysis of drug traces on banknotes with different validated techniques can provide important information about the types of substances that are used in a geographical region. The literature search yielded 88 publications; 9 were included in our review. The concentration of cocaine ranged from 0. Benzoylecgonine was indentified only in three studies with a range from 0. Other indentified drugs were: amphetamine derivatives, opiates, benzodiazepines. Circulating banknotes could be used to indicate substances used in a population, and those recently introduced in a geographical macro-area. The identification of very high amounts of cocaine can provide important information for the identification of banknotes used in illegal trafficking. Drugs abuse and especially cocaine consumption in Western countries are an increasing social problem. The most used drugs can be also detected in the environment, so several studies had proposed the direct measure of drugs from environmental samples, for example from the analysis of the wastewaters, 2 , 3 or from alternative biological samples, such as keratin matrix. In literature there are different studies demonstrating that a significant proportion of banknotes in worldwide circulation are contaminated with cocaine and, to a lesser extent, with other controlled substances. Unfortunately, prevalence measures that can be gained from drug analysis on banknotes are not entirely reliable. First of all, it has been shown that the insertion of banknotes in automated teller machines ATM 9 , 10 provides a source of contamination between notes. In addition there are drugs that are sold in forms that allow greater retention in the banknote tissue, such as cocaine, and substances that can hardly be found, such as cannabis. The purpose of this study was to review the literature about the contamination of the banknotes in the world by cocaine, and by its principle metabolite benzoylecgonine—BEG. An additional aim was to describe the possible presence of other drugs on banknotes. We collected evidences dating from till Studies concerning the contamination of banknotes by cocaine were collected and analyzed. A manual search of reference lists of included studies and review articles was successively performed. All references of the retrieved studies were also reviewed to avoid missing relevant publications. After full screening of titles, abstracts and full texts, the selection of included studies was based on the availability of information regarding the contamination of banknotes by cocaine. A manual search of the reference lists of included studies and review articles was successively performed. All references of the retrieved studies were also reviewed to avoid overlooking relevant publications. Studies were selected in a 2-stage process. Titles and abstracts from electronic searches were scrutinized by two reviewers independently M. The level of agreement between the reviewers was high. We considered eligible articles in English, that we decided to include only if they reported clear and statistically evaluable data about the percentage of contamination by cocaine and the concentration of found cocaine. Additional data about contamination by other drugs were recorded. We excluded all studies that provided ambiguous or insufficient data on every item mentioned above. The literature search yielded 88 publications. The titles of these manuscripts were screened, resulting in 23 studies considered potentially eligible to be included in the review: in fact 33 resulted to be duplicate, 29 were not in line with the aim of the study, 3 were in other languages. Of the total of 23 relevant manuscripts identified, 6 studies were excluded after the examination of the abstracts and 17 studies were further evaluated by retrieving the full text. Finally, after the exclusion of eight studies we identified nine manuscripts figure 1. Flow diagram for identifying studies on the cocaine contamination of banknotes. As shown in table 1 , the percentage of positivity was not reported in all the studies included in the review: in the six studies that showed this datum, the percentage ranged from 2. As shown in table 1 , BEG was indentified only in three studies with a range 0. Other drugs indentified on banknotes were: amphetamine derivatives, opiates, benzodiazepines. In almost all the papers, the analyzed banknote samples came from countries, one from an island. All these techniques are able to highlight traces of cocaine on banknotes but with different sensitivity and specificity. We deduced that data deriving from analysis carried out with different methods cannot be perfectly superimposed. Analytical methods proposed for the determination of cocaine contamination on banknotes can be divided into two different groups: those that use separation techniques, such as GC, LC or capillary electrophoresis CE that allow the quantification of analytes with accuracy and precision, and, those that directly analyze banknotes using immunoassays, TD-MS2 or MS, ELISA. Chromatographic procedures represent the best validated methods for the quantification of cocaine on banknotes. Detection with MS2 provides a fingerprint that unequivocally identifies drugs in the analyzed solutions. For these reasons, it is necessary to perform banknote analysis on a large sample, with statistical studies that can detect money certainly associated with illegal trafficking. In addition to these techniques we remember that Termal direct desorption TD-MS2 is a rapid technique that combined with MS2 or IMS, and represents a selective method that allows rapid sample preparation but requires more energy to extract the analyte. Both methods are regarded as semi-quantitative approaches to problems related to particles trapped within the matrix of banknotes and those present in the extracted solution. Ion-mobility spectrometry IMS can be adopted as a screening method able to detect nanogram amounts of drugs, especially useful when great amount of money are seized. Moreover the GLORIA test gold-labeled, optically read, rapid immunoassay , has been applied to the detection of drugs on banknotes. It is capable to detect very low quantities nanograms of analyte and lasts a few minutes. The limits of this test are the lack of specificity and sensibility that can cause false positives and false negatives. As it can be easily inferred from the table, the analysis performed in order to detect cocaine on banknotes has results considerably different as regards both the percentage of positive samples both the concentration of the substance. In fact, the percentage of cocaine detection ranged from 2. Looking at the percentages of cocaine contamination detected on the Euro banknotes, the percentages vary from 2. These percentages are slightly indicative of direct contamination. It is well known that there is high contamination that occurs between the banknotes when these are placed in contact with each other. There are many theories that have been postulated for the retention of controlled substances by banknotes. It has been suggested that the ink on currency never really dries, and could provide a sticky surface onto which human oils, environmental dirt and grime including narcotics can be attached. In addition, counting machines may contribute to the wide dissemination of drugs on currency. Indeed, counting machines at the Bank of England have been shown to be contaminated with cocaine, 24 probably resulting from the myriad banknotes counted daily. Evidently it is not possible to say how traces of drugs originated or how long they have been present. In fact, deposits on banknotes could derive from direct contact with the drug itself, by a contaminated hand, or another contaminated item. With the rapid exchange of money it is possible for an individual who has no personal involvement with drugs to be in possession of banknotes bearing detectable amounts of controlled substances. The upper limit of the general background quantity for cocaine on currency in the United States is 13 ng per banknote, 5 whereas 13— ng amounts indicate closer contact with the original contamination. In , Carter et al. The presence of large amounts of cocaine can prove evidence of close contact with the drug. The proportion of banknotes contaminated with a particular compound and the extent of contamination need to be considered and separated from money from general circulation using statistical evidences. In addition several studies have detected the presence of benzoylecgonine on banknotes, with a concentration ranging from 0. Benzoylecgonine is a metabolite derived from the hydrolysis of cocaine. It can be therefore generated in conditions of humidity and presence of water. The same reasoning is conceivable for the detection of 6-Monoacetylmorphine 6-MAM and morphine which are heroin metabolites. These products can still result from a synthesis error and may be already present in the powder which is sold. Moreover some authors underlined the correlation between the value of the banknotes and the concentration of cocaine detected on them. Mackuak et al. Wimmer and Almeida 13 , 15 have detected that banknotes of smaller size contain a higher amount of drug, perhaps also in relation to their surface that appears rougher and is able to retain a greater quantity of substance. Looking at the other drugs, small amount of other substances were detected on analyzed banknotes. These data need to be critically commented. It is known that although amphetamine is also frequently administered in powder form, the crystal size is bigger than that of cocaine, and retention by banknotes appears less effective. Because of the method of preparation and usage, combined with the small doses used, it is unusual to encounter a detectable amount of these drugs. From our literature review on the presence of cocaine and its derivatives on banknotes, with references to the other types of substances, it is possible to say that this kind of investigation can provide important information for the identification of banknotes used in illegal trafficking. Compounds and solvents used in drug preparation, are part of a class of chemicals that are placed under tight controls, generally in each country. These chemical products may contain impurities which differ from country to country or from laboratory to laboratory. The identification of money definitely related to drug trafficking can lead to the possibility to carry on analyses for the detection of these impurities that can represent the fingerprints of laboratories involved in production and trafficking of drugs. The analysis of drugs on banknotes for epidemiological purposes and to perform consumption statistical analysis does not appear equally compelling. First of all, the contamination that occurs between banknotes coming in contact one with the other, and in this way between drug money and money normally present in circulation is really significant, in a so large extent that all the latest studies demonstrated that almost all of the banknotes present traces of drugs and in particular of cocaine. It is conceivable that not all the banknotes have been directly used in the exchange of drugs or for snorting. Therefore, with a rate of contamination which affects almost all of the analyzed samples it is not possible to deduce information about the real consumption of a substance in the general population. Moreover notes easily move from one subject to another, are counted in ATM and therefore cannot indicate the history of consumption of its owner at any given time. In addition, a banknote like Euro that circulates in an entire continent can be contaminated in the transition between different countries. It is evident that the presence of traces of drugs in the tissue may refer to a use made throughout the euro-zone, remembering the flow of tourists and the easy travels on the European continent. The same can be asserted for the other types of banknotes. We conclude that at the present state of knowledge it is possible to believe that banknotes circulating recently can only indicate what types of substances are used in a given population. Therefore, the analysis of drug residues on banknotes may represent an useful tool to evaluate and study the prevalence of new substances used in a given geographical macro-area. Key points. Our review showed that banknotes contamination with drugs is a very common phenomenon. The identification contaminated money could be useful to detect the areas at risk of drugs trafficking. Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay. Talanta ; : — Google Scholar. Estimating community drug abuse by wastewater analysis. Environ Health Persp ; : — Drugs and psychoactive substances in the Tiber River. Aust J Forensic Sci ; doi: The analysis of the keratin matrix as a new tool to evaluate the epidemiology of drug use in Perugia Italy : a cross sectional study. A 10 year post-mortem analysis of keratin matrix in Perugia Italy : focus on cocaine involvement in several types of death. Drugs on money. Anal Chem ; 72 : A — A. Validated, non-destructive and environmentally friendly determination of cocaine in euro bank notes. J Chromatogr A ; : — 5. Occurrence of contamination by controlled substances in Euro banknotes from the Spanish archipelago of the Canary Islands. J Forensic Sci ; 56 : — The distribution of controlled drugs on banknotes via counting machines. Forensic Sci Int ; : — Factors influencing the contamination of UK banknotes with drugs of abuse. Analyst ; : — Quim Nova ; 30 : — 7. Wimmer K , Schneider S. Forensic Sci Int ; : — 7. An accurate and non destructive GC method for determination of cocaine on US paper currency. J Sep Sci ; Forensic Sci Int ; : 50 — 5. Mackuak S. Determination of illicit drugs and their metabolites contamination on banknotes. Monatsh Chem ; : 39 — Armenta S , de la Guardia M. Analytical methods to determine cocaine contamination of banknotes from around the world. Trends Anal. Chem ; 27 : — Determination of a trace amount of cocaine on a bank note by gas-chromatography positive-ion chemical-ionization mass-spectrometry. Chromatogr ; : — Ion mobility spectrometry. Anal Chem ; 62 : A — 9A. Google Preview. Visher CA. Comparison of urinalysis technologies for drug testing in criminal justice. In: Justice NIo, editor. Washington Paradis D. ElSohly MA. Urinalysis and casual handling of Marijuana and Cocaine. J Anal Toxicol ; 15 : Burton IAF. Bristol, UK. Sweat testing to monitor drug exposure. Ann Clin Lab Sci ; 43 : 22 — Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Advertisement intended for healthcare professionals. Sign in through your institution. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Journal Article. Cocaine contamination of banknotes: a review. Gianmarco Troiano , Gianmarco Troiano. Oxford Academic. Isabella Mercurio. Marco Golfera. Nicola Nante. Paola Melai. Massimo Lancia. Mauro Bacci. Select Format Select format. Permissions Icon Permissions. Abstract Background. Figure 1. Open in new tab Download slide. Table 1 Open in new tab. Author, year. N samples. Cocaine concentration. BEG concentration. Other substances. Google Scholar Crossref. Search ADS. Google Scholar PubMed. All rights reserved. Download all slides. Comments 0. Add comment Close comment form modal. I agree to the terms and conditions. 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How can I buy cocaine online in De Panne

Official websites use. Share sensitive information only on official, secure websites. To whom correspondence should be addressed. Present address: Allison H. For commercial re-use, please contact journals. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein—DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters. Recent studies have highlighted the existence of multiple layers of complexity in the modulation of a TF binding to DNA, revealing the effects of the structure and flexibility of the binding site 4 , 5 , the kinetic competition of TFs with nucleosomes 6—8 , cooperativity between TFs 9—13 , the presence of neighboring TF sites 14 , 15 and DNA methylation However, the diversity of mechanisms by which TFs select binding sites in vivo and alter gene expression remain unclear, calling for a mechanistic understanding of all these factors to understand TF function. According to numerous experimental observations, proteins find their specific binding sites on DNA very quickly and efficiently, with search times that are, in some cases, shorter than the ones estimated using three-dimensional 3D diffusion models 14 , 15 , 17 , In particular, the model by Berg, Winter and von Hippel 19—21 postulates that proteins are able to find their targets by coupling 3D diffusion in the solution, with one-dimensional 1D diffusion while bound non-specifically to DNA. Notably, although this theoretical approach is able to explain qualitative features of the facilitated diffusion, the measured diffusion constants and partitioning of time between the solution and the DNA call into question the utilization of this model to describe real biological systems Nonetheless, the sliding of proteins on DNA has been experimentally observed in single molecule experiments 14 , 22—28 , indicating that 1D diffusion on the DNA is an integral part of the search process. This highlights an additional intriguing aspect of the search process: to be able to scan the DNA at high speeds, the protein binding potential must be a smooth function of the position on the DNA, with variations no larger that 1—2 k B T as a function of sequence. However, the required stability of the protein—DNA complex when bound at the specific site requires a tight protein—DNA interaction, i. A proposed solutions to this paradox postulates the existence of two different conformational states and therefore different binding energies : one while the protein diffuses along the DNA, and another one when it probes the DNA for a target sequence 14 , 29 , 32 , However, although the existence of multiple binding conformations has been experimentally demonstrated 28 , 34 , 35 , the two-states model is still controversial, as detailed in ref. Egr-1 also known as zif is a TF responsible for the regulation of a variety of genes, and is induced by various stimulants, such as growth factors 36 , neurotransmitters 37 , hormones 38 , 39 and stress A crystal structure of the protein bound to this motif shows that Egr-1 binds this recognition sequence as a monomer via interactions of its three zinc fingers ZF1, ZF2 and ZF3 , which contact 3 bp of DNA each The wide interface created by the ZFs when bound to DNA provides a strong, stable and specific binding. Hence, in order to activate gene expression and respond to its stimulus, Egr-1 needs to rapidly scan the DNA in search for its response elements. During this search process, Egr-1 is required to discriminate its target site from among billions of bases, within a time frame of minutes. As a result, a tight binding conformation, like the one described above, can prevent the protein from efficiently scanning the genome, since many molecular bonds would need to be constantly created and disrupted. Recent studies using nuclear magnetic resonance spectroscopy helped to resolve it, revealing a surprisingly different protein structure of Egr-1 while bound to non-specific DNA However, binding sites in real, transcribed genes do not exhibit in general the consensus sequence. In fact, many of these sites show gene-specific and evolutionary-conserved deviations from the consensus, indicating that the specific type of interactions Egr-1 makes with its binding elements have a functional importance. Hence, elucidating how the structure and dynamics of the Egr-1 complex is modulated by the binding site sequence and its genomic context is of great interest. Most of these sites, without a regulatory role, are expected to affect the search process, as they can momentarily trap Egr-1 The role of such sites is thought to be negative when these sequences are located relatively far from the target sequence, or positive if they are located in the vicinity of the response element where they help to increase the protein's local concentration 14 , Unfortunately, since most of the experiments have concentrated on the consensus sequence, it is not clear how Egr-1 binds to the variable ones. Interestingly, a thermodynamic additivity model was proposed to predict the affinity of near-consensus sites, but it was found to fail for sequences with four or more substitutions In gonadotrope cells, Egr-1 is induced by the Gonadotropin Releasing Hormone GnRH to regulate the expression of the Lhb gene, and is essential for GnRH-induced gene activation and fertility in mice Interestingly, both Egr-1 binding elements are different from the consensus motif, and they also differ by 4 bp from each other. These variations are conserved among the species Supplementary Figure S1 , suggesting a functional role. Moreover, sequences flanking these sites are evolutionary conserved too, suggesting also that the DNA content surrounding these sites is important. In this work, we used single-molecule DNA unzipping to study how the DNA sequence at the binding sites and their flanking regions alter the structure and affinity of the Egr-1—DNA complex. This approach provides a direct measurement of the position and forces associated with proteins bound to DNA 49—51 , thus overcoming the inherent limitations of averaging in more traditional ensemble methods. We first measured the occupancy and breaking forces associated with Egr-1 binding to each of its response elements in their native context on the Lhb gene promoter, and then compared them with those obtained for the same sequences in other DNA contexts. We also used a novel method to characterize the dissociation of Egr-1 from its underlying DNA under asymmetric perturbations of the DNA. Together, the findings indicate that each of the functional binding sites, and their native flanking sequences, modulate the structure and affinity of the Egr-1—DNA complex in a different way, and suggest a unique functional role for ZF3 contacts with DNA. The protein was expressed and purified as previously described 52 , by cloning it into a pGex2t plasmid GE Healthcare Life Sciences along with a cleavable glutathione S-transferase GST tag. Experiments were performed in a custom-made double-trap optical tweezers apparatus 53 , as previously described 8 , Briefly, the beam from an nm laser TA PRO, Toptica was coupled into a polarization-maintaining single-mode optical fiber. The light is collected by a second, identical objective, the two polarizations separated by a PBS, and imaged onto two position sensitive detectors First Sensor. The position of the beads relative to the center of the trap is determined by back focal plane interferometry Calibration of the setup was done by analysis of the thermal fluctuations of the trapped beads 56 , which were sampled at kHz. The constructs for single-molecule experiments were generated as described previously 8 , with a number of modification. Primers used for the amplification reactions are listed in Supplementary Tables S9 and S The other two primers were designed to contain repeats of three DNA sequences recognized by single strand nicking enzymes: Nt. BbvCI and Nb. BbvCI both from New England Biolabs on the biotin-tagged handle and on the digoxygenin-tagged handle, respectively. The nicking enzymes generated 29 nt complementary overhangs on each handle. Binding segments were ligated to DNA handles using a rapid ligase system Promega in molar ratio, 30 min at room temp. The full construct i. Tether formation was performed in situ inside the experimental chamber by trapping an anti-digoxigenin bead bound by the DNA construct in one trap, trapping a 0. Data were digitized at a Hz sampling rate and saved to a disk. All further processing of the data was done with Matlab Mathworks. Stretching of the tether was used to find the polymer-models parameters under our experimental conditions. From the measured and filtered tether extension and force, the stretching of the dsDNA handles at each time point was subtracted from the measured extension. Then, the extension was divided by the extension of two ssDNA bases calculated from the measured force using the WLC model to result in the number of unzipped bp. To improve the accuracy of the experiments, the alignment DNA segment was used to perform a correlation-based alignment of all traces in a group i. In the presence of a bound protein, the propagation of the unzipping fork is halted and the force increases. At forces of 17—26 pN, the protein-DNA complex is disrupted in a single event that results in complete dissociation of the protein, leaving the DNA in a high-force, out-of-equilibrium state. A segment of DNA then immediately unzips, allowing the system to relax to equilibrium. Binding events were identified by detecting an increase in force of more than 0. Applying the same criteria for the data obtained without Egr-1 resulted in no binding events detected. Events were classified as belonging to a specific site if the breaking event was located within a bp window relative to the expected center of the binding site. Differences in binding probability were checked using a Chi-squared test. Differences were considered statistically significant if the calculated P -value was no larger than 0. DNA was unzipped until reaching an extension corresponding to the Egr-1 consensus binding site. The fluctuations in extension over time were measured for 1 min before the exposure to Egr-1, after which the construct was moved to a region of the laminar flow chamber Lumicks that contains Egr No binding events were detected in the absence of protein Supplementary Figure S8a. The dissociation time was taken as the time lapsed until the reappearance of the fluctuations. Two different methods were used: in the first, following binding the construct was moved back to the channel in which Egr-1 was absent, to prevent re-binding, thus providing a single event of dissociation. In the second Supplementary Figure S8c , multiple binding and dissociation events were observed by keeping the construct at the Egr-1 channel. These sites are remarkably conserved among the species despite a number of introduced substitutions which diverge from the consensus motif Supplementary Figure S1. Then, we subjected the construct to mechanical force by moving one of the traps away from the other. It has been previously shown 49—51 that following the propagation of the unzipping fork allows to measure the position and strength of protein—DNA interactions, as the force required to disrupt these interactions is significantly higher than those needed to disrupt DNA alone. Hence, using a laminar flow system, we exposed our construct to a solution containing the DNA binding domain of Egr-1 referred to as Egr-1 for simplicity , and unzipped the DNA in its presence. The force peaks detected for the three sites were not observed in the absence of Egr-1 Figure 1D and Supplementary Figure S2b , nor for a control DNA sequence that does not harbor any known Egr-1 binding sites. Moreover, although the experiment was performed at a high concentration of protein nM , at which a significant amount of it will be bound to DNA non-specifically, the measured forces in non-specific regions were identical in the presence or absence of the TF, suggesting that the forces required to disrupt the non-specific complexes are significantly lower than those required to disrupt Egr-1 from the specific sites. In addition, to evaluate whether peaks detected in successive unzipping cycle for a single molecule correspond to new binding events following each unzipping cycle, we performed the following control experiment: We first unzipped the DNA, thus disrupting Egr-1 binding, and then moved into a flow channel that does not contain Egr-1, relaxed the tension to allow re-annealing of double stranded DNA dsDNA , and immediately unzipped the DNA again. Single-molecule probing of Egr-1 binding to the Lhb promoter. Egr-1 binding to the construct is initiated in situ inside the experimental chamber. One of the traps is moved to stretch the tethered construct and disrupt protein—DNA interactions. The process is repeated multiple times. Binding to each of the sites -1, -2 and -3 is designated with arrows. E Breaking force for the three Egr-1 binding sites located on the Lhb promoter. Accordingly, unzipping DNA multiple times in the presence of Egr-1, allowed us to calculate the mean breaking force Figure 1E as well as the fraction of successive Egr-1 binding events, out of the total number of unzipping cycles i. Increasing the time between consecutive unzipping cycles did not affect the binding probability, indicating that the system is able to reach thermal equilibrium Supplementary Figure S9. Hence, the binding frequency measured in these experiments should reflect the affinity of Egr-1 for a particular DNA sequence It was previously shown that the affinity of Egr-1 to site -1 is higher than that to site -2 and lower than the affinity of the Egr-1 consensus sequence 59 , which is consistent with our results. Next, to further clarify the differences in affinity between the different sites, we repeated these experiments in the presence of various concentrations of Egr It has been shown that the consensus sequence exhibits the highest affinity of Egr-1 to DNA Therefore, in addition to Lhb DNA, we performed the same experiment with the consensus sequence, introduced into a DNA segment derived from the nucleosome positioning context C1, Supplementary Figure S3. For all the four sites probed, the binding probability is consistent with a hyperbolic saturation curve, as function of protein concentration Supplementary Figure S4a , from which the affinity of Egr-1 for a specific site can be estimated. As expected, the affinity for the consensus sequence was the highest, as compared with the sites located on Lhb , with similar affinities for sites -1 and -3, and lowest for site In addition, the mean breaking force did not show a concentration dependence Supplementary Figure S4b , suggesting that it is an intrinsic property of each site. As a result, differences in breaking force can reflect differences in structure, even if the different structures have similar affinities. Hence, the breaking forces we observed for the three binding sites on Lhb , which are uncorrelated with their binding probability, suggest that Egr-1 binds each of these sequence motifs in a significantly different structural conformation. Previous studies suggested that the presence of an additional binding site proximal to a given site, can reduce its binding probability, as each site will compete with the other for protein binding. To that end we calculated the conditional probability of detecting a protein at a given site, given that there is a protein at the second. When we compared unconditional with conditional binding probability of both sites, we did not observe any significant difference between them Supplementary Figure S5. The observed breaking force and binding probability of each single site was measured in the context of others sites in cis. This made us wonder whether the observed differences are solely due to the identity i. To that end, we introduced the 9 bp motif corresponding to each of the three sites, separately, into the same flanking context of C1 Supplementary Figure S3 and subjected them to multiple rounds of unzipping in presence of Egr-1 Figure 2. The breaking forces for site -1 and site -2 were significantly elevated, while for site -3 it was significantly reduced Figure 2A. Introducing the sites into a different flanking context C2, Supplementary Figure S3 , showed a significantly different set of forces and binding probabilities Figure 2A and B. For example, the binding probability for site -3 in the C2 context is nearly equal to the one in the native Lhb context. DNA sequence flanking the binding sites modulate Egr-1 binding. A Mean breaking force and B binding probability are presented for sites -3, -2, -1 and the consensus motif. The data is analyzed as in Figure 1. The crystal structure of Egr-1 shows that ZF1 can make hydrogen bonds with this base 60 , thus it is possible that the observed changes in binding probability and breaking force upon change of DNA context, are due to the substitution of this specific nucleotide. Remarkably, unzipping experiments using the mutated flanking base at this position led to a mild decrease in breaking force, but showed no significant change in binding probability Figure 3B and C. In contrast, when we mutated the first nucleotide proximal to ZF3 base number 10 , a significant reduction in both breaking force and binding probability was observed Figure 3B and C. The first nucleotide proximal to ZF3 modulates Egr-1 binding. The data for binding probability and breaking force is analyzed and presented as in Figure 1. Previous studies have shown that 3—5 nt surrounding the core binding site have a major effect on binding for some TFs 61 , In addition, a recent theoretical work predicted that the chemical composition near the binding site might influence the dynamics of search by a protein Hence, to check whether the effect of flanking sequences can extend beyond the first nucleotide flanking the binding motif, we gradually mutated 8—11 nt proximal to ZF1 or ZF3 of site -1, by replacing them with repeats of the AT di-nucleotides Supplementary Figure S6. The effect of replacement on breaking force was evident for some of the nucleotide substitutions flanking both ZF1 and ZF3 Figure 4. However, only the change of base number 10, proximal to ZF3, significantly reduced both breaking force and binding probability. Collectively, these results suggest that, at least for naturally occurring Egr-1 binding sites, the flanking sequences proximal to ZF1 affect the structure of the protein—DNA complex, without a strong effect on its affinity, while the first nucleotide proximal to ZF3 controls both structure and affinity of Egr-1—DNA complex. Effect for a gradual change of flanking context on Egr-1 binding. B and C Difference between the breaking force measured for the unmodified C1 context, and constructs in which nucleotides proximal to ZF1 b or ZF3 c were mutated. D and E The corresponding binding probabilities, analyzed and presented as in Figure 1. The observed asymmetric effect of flanking nucleotides suggested that the region proximal to ZF3 plays an important role in Egr-1 binding. Thus, we hypothesized that the dramatic effect observed as a result of the substitution of the nucleotide proximal to the triplet bound by ZF3, may reflect a particular sensitivity of the Egr-1—DNA complex for the local perturbation of ZF3, modulating Egr-1 binding as a whole. If this is the case, we would expect that dissociation of Egr-1 from the DNA will require less force if the perturbation is from the ZF3 direction. Moreover, the force required for disruption of Egr-1 from site -3, that was the highest among the three sites in the forward unzipping, was so dramatically reduced, that we could not detect any force peak in the location near this site Figure 5. This suggest that the sequence of the binding motif can modulate the specific interactions of the ZFs, creating different degrees of asymmetry in the structure for sites -2 and -3, where ZF1 is more tightly associated with DNA than ZF3, as compared to the complex formed on site -1 where the interactions are nearly symmetric. Of note, the significant reductions we observe for the binding probability are likely the result of more binding events whose breaking force is close to the force sensitivity threshold set by the unzipping force of naked DNA, and are therefore missed. Surprisingly, two additional force peaks were detected in the positions previously unidentified inside the Lhb gene body sites 1 and 2, Supplementary Figure S7. Unzipping Egr-1 from the ZF3 direction requires less force A Schematic representation of forward and reverse unzipping. The data for binding probability B and breaking force C are analyzed and presented as in Figure 1. Next, to further check whether perturbation of Egr-1 from the ZF3 direction is a site-specific property, or a more general property of the structure of the Egr-1—DNA complex, we reverse-unzipped the Egr-1—consensus complex. These results suggest that the reduction in forces required for disruption of the protein through ZF3 is a general property that reflects Egr-1 binding to DNA. Finally, we wanted to understand whether Egr-1 dissociation is faster upon a local disruption of ZF3, as compared with with a similar disruption of ZF1. Accordingly, we mimicked a local disruption of each ZF by partially unzipping DNA from the forward direction for disruption of ZF1 or reverse direction for disruption of ZF3. Next, we exploited our laminar flow cell to move the fluctuating DNA to the channel in which Egr-1 was present. Exposure of the DNA to the Egr-1 channel led to a quick transition of the DNA into a closed form, and a complete repression of the fluctuations, indicating Egr-1 binding. Reappearance of the fast fluctuations indicated dissociation of the protein. Multiple events of binding and dissociation could be observed for a single molecule with continuous exposure to Egr-1 Supplementary Figure S8c. To observe a single event of dissociation, we moved the construct back to the channel in which Egr-1 was absent, to prevent re-binding. The time lapsed until the reappearance of fluctuations Figure 6B allowed us to measure the characteristic dissociation time of a single Egr-1 molecule under a tension applied on the ZF1 or ZF3 side. Altogether, our results suggest that a local perturbation of ZF3, either by force or by modulation of the flanking sequences proximal to it, leads to more rapid dissociation of Egr-1 from its binding site. Force disruption of ZF3 increases Egr-1 dissociation. A A single-molecule containing a consensus motif in the C1 context is unzipped until the fork reached the binding site. Binding of Egr-1 to the DNA stabilizes the closed conformation, leading to a sudden repression of the fluctuations, which lasts until Egr-1 dissociation. The time difference between binding and unbinding dissociation time is measured for forward disruption of ZF1 and reverse disruption of ZF3 unzipping. C Mean dissociation time, calculated for forward and reverse unzipping. Despite the universal requirement for gene-specific TFs to bind a highly specific recognition element in their target gene promoters in order to induce transcription, the identification and binding process is still surprisingly poorly understood. In this study, we have characterized the interactions of Egr-1 with its binding sites at the promoter of the Lhb gene. Measuring both the protein binding probability to the different sites and the mean force required to disrupt the bound proteins, allowed us to characterize the binding of Egr-1 at the single-molecule level. Our experiments show that the differences in sequence between these sites, and the specific genomic context where they are located, dictate different modes of interaction with the DNA. Interestingly, the breaking forces measured for the three conserved sites in the Lhb promoter did not correlate with their binding probability, suggesting that it is not only a modulation of the affinity of a site which is functionally important, but the specific structure of the complex, as dictated by the binding sequence and its context, is important too. Our results show that the protein—DNA complex is particularly sensitive to the properties of the DNA flanking the binding site at the side corresponding to interactions made by ZF3. Unzipping the DNA in the reverse direction, thus approaching the complex from the ZF3 side, resulted in lower breaking forces, indicating that the complex is more easily displaced if ZF3 is disrupted first. A novel method monitoring binding of Egr-1 via the reversible repression of local fluctuations in the DNA allowed us to determine that the dissociation time of Egr-1 is much shorter under a perturbation on the ZF3 side than it is under a similar perturbation on the ZF1 side, further supporting a functional role for ZF3. This is the conformation originally described in ref. Notably, our findings have implications for the interaction of Egr-1 with other TFs. Binding of a second TF to DNA in close proximity to the ZF3 side of a bound Egr-1 can facilitate its dissociation, thus effectively providing a mechanism of cooperativity between the TFs. Interestingly, site -2 in the Lhb gene is located immediately upstream of the binding site for Pitx1, and downstream from the binding site of SF These three TFs have been found to act in a highly cooperatively manner in the GnRH-induced expression of Lhb 39 , 66 , 67 , and the mechanisms outlined here may provide a molecular mechanism by which this cooperativity is achieved. Our results can also shed light on the interaction of Egr-1 with other cellular molecular motors translocating on DNA. Some of these sites are bona fide regulatory elements, but most will likely be located at the body of genes. Previous studies have highlighted the importance of these quasi-specific sites for the kinetics of the Egr-1 finding its regulatory targets, as they can slow down the search process However, binding of Egr-1 at high density on the DNA will likely also affect the function of molecular motors, e. Interestingly, while previous works have shown that many helicases are capable of displacing proteins bound to DNA see for example refs. Our results suggest a mechanism that can assist in the displacement of the quasi-specifically bound Egr-1 proteins, provided that they are in the right orientation, such that the polymerase encounters the ZF3 side first. Interestingly, the binding sites we detected on the gene body of Lhb 1,2 in Supplementary Figure S7a are positioned in an orientation such as an RNA polymerase transcribing the Lhb gene will first encounter Egr-1 from the ZF3 direction. Finally, our findings on Egr-1 shed light on novel elements likely affecting also the interaction of other TFs with the DNA. Previous studies analyzing genomic variation in humans 71 have shown that most significant genetic variants are located in non-coding regions, and that complex organismal phenotypes are the result of altered binding of TFs. However, only a small fraction of these variations in TF binding is caused by variants directly disrupting the TF recognition motif Our current study indicates the potential role of even small changes in DNA sequence in regions flanking the previously recognized binding sites, and suggests that these could well form a broader mechanism involved in altering transcriptional regulation by DNA binding factors in general. Elucidation of these molecular mechanisms will lead to a clearer understanding on the organization of regulatory elements at gene promoters, will help understand inter-individual phenotypic variations and may improve our ability to dissect the molecular basis for disease susceptibility. We are thankful to Drs. Author Contributions : S. K supervised the research. Frankford Research Fund. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Nucleic Acids Res. Find articles by Sergei Rudnizky. Find articles by Hadeel Khamis. Find articles by Omri Malik. Find articles by Allison H Squires. Find articles by Amit Meller. Find articles by Philippa Melamed. Find articles by Ariel Kaplan. These authors contributed equally to the paper as first authors. Open in a new tab. Click here for additional data file. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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