An omega-like DNA nanostructure utilized for small molecule introduction to stimulate formation of DNAzyme–aptamer conjugates

2014 ◽  
Vol 50 (15) ◽  
pp. 1900-1902 ◽  
Author(s):  
Bingqian Liu ◽  
Bing Zhang ◽  
Guonan Chen ◽  
Dianping Tang
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Electronic Monitoring ◽  
Label Free ◽  
Dna Nanostructure ◽  
First Time ◽  
Target Analyte

A novel, label-free omega-like DNA nanostructure was for the first time utilized for the homogenous electronic monitoring of small molecules (ATP used in this case) accompanying the formation of DNAzyme–aptamer conjugates upon target analyte introduction.

scholarly journals A Simple Displacement Aptamer Assay on Resistive Pulse Sensor for Small Molecule Detection

2020 ◽  
Author(s):  
Rhushabh Maugi ◽  
bernadette gamble ◽  
david bunka ◽  
Mark Platt
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Label Free ◽  
Resistive Pulse ◽  
Pulse Sensor ◽  
Label Free Detection ◽  
Ssdna Aptamer ◽  
Aptamer Assay ◽  
Small Molecule Detection ◽  
Surface Changes

A universal aptamer-based sensing strategy is proposed using DNA modified nanocarriers and Resistive Pulse Sensing for the rapid and label free detection of small molecules. The surface of a magnetic nanocarrier was first modified with a ssDNA aka linker which is designed to be partially complimentary in sequence to a ssDNA aptamer. The aptamer and linker form a stable dsDNA complex on the nanocarriers surface. Upon the addition of the target molecule, a conformational change takes place where the aptamer preferentially binds to the target over the linker; causing the aptamer to be released into solution. The RPS measures the change in velocity of the nanocarrier as its surface changes from dsDNA to ssDNA, and its velocity is used as a proxy for the concentration of the target. We illustrate the versatility of the assay by demonstrating the detection of the antibiotic Moxifloxacin, and chemotherapeutics Imatinib and Irinotecan.

scholarly journals Mechanism-based sirtuin enzyme activation

2015 ◽  
Author(s):  
Xiangying Guan ◽  
Alok Upadhyay ◽  
Sudipto Munshi ◽  
Raj Chakrabarti
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
False Positives ◽  
Enzyme Activation ◽  
Label Free ◽  
Multiple Substrates ◽  
Dependent Protein ◽  
Biophysical Techniques ◽  
Catalyzed Reactions ◽  
Small Molecule Modulators

AbstractSirtuin enzymes are NAD+-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. There is intense interest in the activation of the seven mammalian sirtuins (SIRT1-7) in order to extend mammalian healthspan and lifespan. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Moreover, across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. None of these modes of activation are based on the unique catalytic reaction mechanisms of the target enzymes. Here, we report a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds. We characterize several small molecules reported in the literature to activate sirtuins besides SIRT1, using a variety of biochemical and biophysical techniques including label-free and labeled kinetic and thermodynamic assays with multiple substrates and protocols for the identification of false positives. We provide evidence indicating that several of these small molecules reported in the published literature are false positives, and identify others as hit compounds for the design of compounds that can activate sirtuins through the proposed mechanism-based mode of action.

scholarly journals Optimized methods for IL-17A refolding and anti-IL17A Fab production for co-crystallization with small molecules

BioTechniques ◽  
2020 ◽  
Vol 69 (1) ◽  
pp. 70-76
Author(s):  
Xiaoyun Meng ◽  
Lanjun Zhang ◽  
Hong Wei ◽  
Furong Li ◽  
Lihua Hu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Small Molecule ◽  
Yield Enhancement ◽  
High Affinity ◽  
Interleukin 17A ◽  
Affinity Peptide ◽  
First Time ◽  
Essential Prerequisite

Refolding of human interleukin 17A (IL-17A) has been reported; however, the key refolding protocol was not robust enough to deliver consistent results and to be easily scaled up for crystallization. Here we report an optimized refolding method for IL-17A. Although co-crystal structures of IL-17A with ligands have been obtained with a high-affinity peptide and an anti-IL-17A Fab as stabilizers, neither the production yield nor the characterization of the IL-17A/Fab complex was reported. To facilitate co-crystallization of IL-17A with small-molecule compounds derived from our DNA encoded library, we also describe the method for yield enhancement of anti-IL-17A Fab production and characterize the IL-17A/Fab complex for the first time, providing an essential prerequisite for structure-based drug discovery targeting IL-17A.

scholarly journals A Simple Displacement Aptamer Assay on Resistive Pulse Sensor for Small Molecule Detection

2020 ◽  
Author(s):  
Rhushabh Maugi ◽  
bernadette gamble ◽  
david bunka ◽  
Mark Platt
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Label Free ◽  
Resistive Pulse ◽  
Pulse Sensor ◽  
Label Free Detection ◽  
Ssdna Aptamer ◽  
Aptamer Assay ◽  
Small Molecule Detection ◽  
Surface Changes

A universal aptamer-based sensing strategy is proposed using DNA modified nanocarriers and Resistive Pulse Sensing for the rapid and label free detection of small molecules. The surface of a magnetic nanocarrier was first modified with a ssDNA aka linker which is designed to be partially complimentary in sequence to a ssDNA aptamer. The aptamer and linker form a stable dsDNA complex on the nanocarriers surface. Upon the addition of the target molecule, a conformational change takes place where the aptamer preferentially binds to the target over the linker; causing the aptamer to be released into solution. The RPS measures the change in velocity of the nanocarrier as its surface changes from dsDNA to ssDNA, and its velocity is used as a proxy for the concentration of the target. We illustrate the versatility of the assay by demonstrating the detection of the antibiotic Moxifloxacin, and chemotherapeutics Imatinib and Irinotecan.

scholarly journals Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

2021 ◽  
Author(s):  
Clément Sester
Keyword(s):  
Signal Transduction ◽  
Structural Change ◽  
Small Molecules ◽  
Small Molecule ◽  
Electrostatic Interactions ◽  
Label Free ◽  
Ligand Interaction ◽  
Wide Range ◽  
Spurious Signals ◽  
Entropically Driven

<p><b>Aptamers are synthetic nucleic acid single-stranded oligonucleotides that bind to a wide range of ligands, including cells, proteins, DNA strands, metal ions, and small molecules, with high specificity and affinity. Aptamers have also proven to be highly stable, readily adaptable to chemical modifications, and exhibit reversible binding. As a result, aptamer-based biosensors (aptasensors) are promising replacements for antibody-based biosensors in many applications, particularly for small molecule ligands. This thesis explores an aptamer that binds the drug methamphetamine, and its prospects when incorporated in an electrochemical (e-chem) signal transduction platform. Specifically, we examine the range of interactions between the aptamer and ligand, and with electrodes, and identify a number of challenges in generating robust e-chem aptasensors.</b></p> <p>Due to their size and limited number of functional groups, further understanding of the aptamer-small molecule ligand interactions is required for the design of future aptasensors – particularly the thermodynamics and structural information about the aptamer-ligand interaction. In fact, detecting small molecules with aptasensors can become challenging because target addition may induce little structural change, and therefore numerous nonspecific interactions may emerge as transduced signals from the biosensor. In this thesis, the combination of spectroscopic and calorimetric analytical techniques reveals a conformational selection binding model, in which binding is entropically driven, and the meth binds via hydrophobic and electrostatic interactions and only induces a modest structural change. This first-of-its-kind study is important for the selection and the design of the aptasensor transduction system.</p> <p>Electrochemical (e-chem) aptasensors offer high inherent sensitivity and practicality as a signal transduction platform. Indeed, different e-chem aptasensor formats have been published before, including labelled and label-free sensors. In screening the viability of three commonly used methods – including labelled and label-free, as well voltammetric and impedance-based methods – we find that each of them suffers from instability of the aptamer-functionalised electrode. This instability compromises our ability to resolve real signals and prompted us to develop ways to understand – and suppress – this baseline drift.</p> <p>The functionalization of the electrode is the critical step in terms of self-assembled monolayer (SAM) stability and SAM aptamer density. Consequently, different protocols of SAMformation were explored and evaluated with respect to stability. We find that instability arises from the uncontrolled arrangement of thiolated aptamers on gold electrodes (including aptamers lying down on the electrode), which is in turn affected by the density of aptamers that can be coupled to the surface. As a consequence, a new protocol is developed using disulfide aptamer pairs to increase the density of correctly tethered aptamers, and generate a stable SAM.</p> <p>Because of the high sensitivity of electrochemical platforms, numerous spurious electrochemical signals may be produced, and controlled for in order to confirm a positive ligand-binding signal. The specificity of the aptamer-target interaction can be checked by testing the response with an interferent molecule, or by substituting the aptamer with a non-binding nucleotide sequence. In this work, these control experiments reveal that target and interferent molecules interact directly (and in different ways) with the bare gold surface, as well as perturbing signals from the aptamer in ways that cannot be linked to a specific aptamer-ligand complex formation. Ultimately, these spurious signals compromise our ability to confirm a real binding signal.</p> <p>The results from this work provide the first clear picture of how an aptamer binds to its small molecule target – which we find is entropically driven, and with only minor structural change induced in the aptamer stem. In addition, the label-free EIS measurements on aptamer SAM electrodes reveal the nature of instabilities, and reveal spurious signals that cannot be sufficiently suppressed at this stage. This knowledge highlights the difficulty in fabricating e-chem aptasensors, and will assist in overcoming challenges faced during research and commercialization of aptasensors area, as well as contributing new insights into troubleshooting, data acquisition, and data validation.</p>

scholarly journals Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

2021 ◽  
Author(s):  
Clément Sester
Keyword(s):  
Signal Transduction ◽  
Structural Change ◽  
Small Molecules ◽  
Small Molecule ◽  
Electrostatic Interactions ◽  
Label Free ◽  
Ligand Interaction ◽  
Wide Range ◽  
Spurious Signals ◽  
Entropically Driven

<p><b>Aptamers are synthetic nucleic acid single-stranded oligonucleotides that bind to a wide range of ligands, including cells, proteins, DNA strands, metal ions, and small molecules, with high specificity and affinity. Aptamers have also proven to be highly stable, readily adaptable to chemical modifications, and exhibit reversible binding. As a result, aptamer-based biosensors (aptasensors) are promising replacements for antibody-based biosensors in many applications, particularly for small molecule ligands. This thesis explores an aptamer that binds the drug methamphetamine, and its prospects when incorporated in an electrochemical (e-chem) signal transduction platform. Specifically, we examine the range of interactions between the aptamer and ligand, and with electrodes, and identify a number of challenges in generating robust e-chem aptasensors.</b></p> <p>Due to their size and limited number of functional groups, further understanding of the aptamer-small molecule ligand interactions is required for the design of future aptasensors – particularly the thermodynamics and structural information about the aptamer-ligand interaction. In fact, detecting small molecules with aptasensors can become challenging because target addition may induce little structural change, and therefore numerous nonspecific interactions may emerge as transduced signals from the biosensor. In this thesis, the combination of spectroscopic and calorimetric analytical techniques reveals a conformational selection binding model, in which binding is entropically driven, and the meth binds via hydrophobic and electrostatic interactions and only induces a modest structural change. This first-of-its-kind study is important for the selection and the design of the aptasensor transduction system.</p> <p>Electrochemical (e-chem) aptasensors offer high inherent sensitivity and practicality as a signal transduction platform. Indeed, different e-chem aptasensor formats have been published before, including labelled and label-free sensors. In screening the viability of three commonly used methods – including labelled and label-free, as well voltammetric and impedance-based methods – we find that each of them suffers from instability of the aptamer-functionalised electrode. This instability compromises our ability to resolve real signals and prompted us to develop ways to understand – and suppress – this baseline drift.</p> <p>The functionalization of the electrode is the critical step in terms of self-assembled monolayer (SAM) stability and SAM aptamer density. Consequently, different protocols of SAMformation were explored and evaluated with respect to stability. We find that instability arises from the uncontrolled arrangement of thiolated aptamers on gold electrodes (including aptamers lying down on the electrode), which is in turn affected by the density of aptamers that can be coupled to the surface. As a consequence, a new protocol is developed using disulfide aptamer pairs to increase the density of correctly tethered aptamers, and generate a stable SAM.</p> <p>Because of the high sensitivity of electrochemical platforms, numerous spurious electrochemical signals may be produced, and controlled for in order to confirm a positive ligand-binding signal. The specificity of the aptamer-target interaction can be checked by testing the response with an interferent molecule, or by substituting the aptamer with a non-binding nucleotide sequence. In this work, these control experiments reveal that target and interferent molecules interact directly (and in different ways) with the bare gold surface, as well as perturbing signals from the aptamer in ways that cannot be linked to a specific aptamer-ligand complex formation. Ultimately, these spurious signals compromise our ability to confirm a real binding signal.</p> <p>The results from this work provide the first clear picture of how an aptamer binds to its small molecule target – which we find is entropically driven, and with only minor structural change induced in the aptamer stem. In addition, the label-free EIS measurements on aptamer SAM electrodes reveal the nature of instabilities, and reveal spurious signals that cannot be sufficiently suppressed at this stage. This knowledge highlights the difficulty in fabricating e-chem aptasensors, and will assist in overcoming challenges faced during research and commercialization of aptasensors area, as well as contributing new insights into troubleshooting, data acquisition, and data validation.</p>

scholarly journals Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese

2020 ◽  
Vol 295 (12) ◽  
pp. 3875-3890 ◽  
Author(s):  
Kyle J. Horning ◽  
Piyush Joshi ◽  
Rachana Nitin ◽  
Rekha C. Balachandran ◽  
Frank M. Yanko ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Inductively Coupled Plasma ◽  
Mammalian Cells ◽  
Longitudinal Assessment ◽  
Inductively Coupled ◽  
First Time ◽  
Homeostatic Response ◽  
Cell Lethality ◽  
Number Of Cells

Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma–MS, we validated that this assay, termed here “manganese-extracting small molecule estimation route” (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.

Graphene–hemin hybrid nanosheets as a label-free colorimetric platform for DNA and small molecule assays

RSC Advances ◽  
2014 ◽  
Vol 4 (109) ◽  
pp. 64252-64257 ◽  
Author(s):  
Cui Hu ◽  
Qiang Xi ◽  
Jia Ge ◽  
Feng-Yan Luo ◽  
Li-Juan Tang ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Label Free

A novel colorimetric platform has been developed for detecting DNA and small molecules based on a graphene–hemin hybrid nanosheet in a homogenous solution.

scholarly journals Structure of the master regulator Rns reveals an inhibitor of enterotoxigenic Escherichia coli virulence regulons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Charles R. Midgett ◽  
Kacey Marie Talbot ◽  
Jessica L. Day ◽  
George P. Munson ◽  
F. Jon Kull
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Family Member ◽  
Shigella Flexneri ◽  
Decanoic Acid ◽  
Enterotoxigenic Escherichia Coli ◽  
Gram Negative Bacteria ◽  
Enteric Infections ◽  
First Time ◽  
Arac Family

AbstractEnteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

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