Thermodynamic Analysis of Aptamer-Ligand Binding by Isothermal Titration Calorimetry

Aptamers are short, single stranded nucleic acid molecules typically 15 to 60 nucleotides in length with the capacity of binding diverse molecular targets, ranging from small molecules to whole cells primarily due to their specific three-dimensional structure. The cocaine binding aptamer is a DNA aptamer that contains 3 stems built around a 3-way junction. This aptamer was selected by Stojanovic in 2000 using classic SELEX to bind cocaine, but not for its common metabolites, benzoylecgonine and ecgonine methyl ester. It is widely used as a model system in the development of a variety of biosensor applications. The aim of this research was to gain an insight into understanding how aptamers interact with their ligands by measuring thermodynamics. Since very little work has been done in this field using isothermal titration calorimetry (ITC) studies, the thermodynamics of small molecule binding to the cocaine-binding aptamer was investigated in detail. The study included both quinine-based and non-quinine based antimalarial compounds. Some of the results that this study yielded are the importance of a quinoline ring in the ligand, the second binding site on the aptamer, the new tightest binding ligand for the cocaine binding aptamer amodiaquine, and a ligand (artemisinin) that does not contain quinoline ring but binds tightly to the cocaine-binding aptamer. In order to determine the selectivity of the antimalarial compounds (amodiaquine, mefloquine, chloroquine and quinine) for the cocaine-binding aptamer, the investigation was further expanded to other DNA structures such as three-way junctions and duplex DNA of varying length. Results showed that quinine and chloroquine are specific for the cocaine binding aptamer, while amodiaquine binds DNA in general. Artemisinin, a non-quinine based antimalarial compound is a generic DNA binder, a previously unknown property of this antimalarial agent. Similarly to the cocaine-binding aptamer, the ATP-binding aptamer binds two copies of its ligand. But unlike the cocaine-binding aptamer, the ATP aptamer binds its ligand in a cooperative two-site binding manner. In addition, this aptamer must have both sites functional; otherwise, the ligand will bind very weakly. Studies also showed, that if two binding sites are separated, the aptamer becomes more structured and stable, and binding model switches from cooperative to independent for adenosine but not ATP

Isothermal titration calorimetry studies of aptamer-small molecule interactions: practicalities and pitfalls

Isothermal titration calorimetry (ITC) is a powerful technique for studying binding interactions. From a single ITC experiment it is possible to quantify the affinity and thermodynamics of a binding event. Here, we outline an experimental approach for performing ITC experiments with a focus on aptamer-small molecule interactions. We also discuss some common problems that can be encountered and how to resolve these issues

Designed Alteration of Binding Affinity in Structure-Switching Aptamers through the Use of Dangling Nucleotides

The ability to change binding affinity in a controlled fashion is a key step in the rational design of biomolecules in general and functional nucleic acids in particular. Here, we use dangling nucleotides to alter the binding affinity of structure-switching aptamers. Dangling nucleotides can stabilize or destabilize a nucleic acid structure with a known ΔG°37. When the dangling nucleotide stabilizes the structure, less free energy from ligand binding is needed to fold the molecule and hence the ligand is observed to bind tighter than in the absence of the unpaired nucleotide. For a destabilizing dangling nucleotide, the opposite occurs, and the observed binding is weaker. We demonstrate this concept using both the cocaine-binding aptamer and the ATP-binding aptamer systems. We find that for both aptamers there is a direct, but different, relationship between the predicted stabilization and the change in the observed binding free energy

Nanomolar binding affinity of quinine-based antimalarial compounds by the cocaine-binding aptamer

An unusual feature of the cocaine-binding aptamer is that it binds quinine much tighter than the ligand it was selected for, cocaine. Here we expand the repertoire of ligands that this aptamer binds to include the quinine- based antimalarial compounds amodiaquine, mefloquine, chloroquine and primaquine. Using isothermal titra- tion calorimetry (ITC) we show that amodiaquine is bound by the cocaine-binding aptamer with an affinity of (7 ± 4) nM, one of the tightest aptamer-small molecule affinities currently known. Amodiaquine, mefloquine and chloroquine binding are driven by both a favorable entropy and enthalpy of binding, while primaquine, quinine and cocaine binding are enthalpy driven with unfavorable binding entropy. Using nuclear magnetic resonance (NMR) and ITC methods we show that these ligands compete for the same binding sites in the ap- tamer. Our identification of such a tight binding ligand for this aptamer should prove useful in developing new biosensor techniques and applications using the cocaine-binding aptamer as a model system

Structure–affinity relationship of the cocaine-binding aptamer with quinine derivatives

In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy

Analysis of the Interaction between the Cocaine-Binding Aptamer and its Ligands using Fluorescence Spectroscopy

We used fluorescence spectroscopy to measure the binding affinity and provide new insights into the binding mechanism of cocaine and quinine with the cocaine-binding DNA aptamer. Using the intrinsic fluorescence of quinine and cocaine, we have observed quenching of ligand fluorescence upon binding of aptamer. Quantification of this quenching provides an easy method to measure the binding constant using small amounts of sample. The observed quenching coupled with a red shift of the Stokes shift in the emission spectrum indicates that quinine and cocaine interact with the aptamer through stacking interactions.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Rapid characterization of folding and binding interactions with thermolabile ligands by DSC

Differential scanning calorimetry (DSC) is a powerful technique for measuring tight biomolecular interactions. However, many pharma- ceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding inter- actions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Using experimental data for two DNA aptamers that bind to the thermolabile ligand cocaine, we present a new global fitting analysis that yields the complete set of folding and binding parameters for the initial and final forms of the ligand from a pair of DSC experiments, while accounting for the thermal conversion. Furthermore, we show that the rate constant for thermolabile ligand conversion may be obtained with only one additional DSC dataset

A proof of concept application of aptachain: ligand-induced self-assembly of a DNA aptamer

A challenge for the use of aptamers as biosensors is how to signal the occurrence of their ligand binding event into a signal that can be exploited in a detection scheme. Here, we present the concept of “aptachain” formation, where an aptamer is split into two overlapping or staggered strands and assembles into an extended oligomer upon ligand binding. This assembly of aptamers can then be used as a way to detect ligand binding by the aptamer. As an example of this concept, we employed the cocaine-binding aptamer as a model system, used its ability to tightly bind quinine and demonstrated its capability in a gold nanoparticle-based biosensing application. We used isothermal titration calorimetry to demonstrate that, when split into two overlapping DNA strands, the aptamer remains functional. Size-exclusion chromatography showed that the quinine-bound oligos form a larger assembly of aptamer units than in the absence of ligand. Finally, we used the oligomer forming ability of the aptachain oligos in a biosensor application for quinine that brings gold nanoparticles closer together resulting in a shift in their plasmonic resonance to a longer wavelength and an observed colour shift. We propose that splitting aptamers into overlapping strands that form oligomers in the presence of a ligand, aptachain formation, will be generally applicable to aptamers and prove useful in a variety of biotechnology applications.York University Librarie

Doxorubicin-Induced Platelet Activation and Clearance Relieved by Salvianolic Acid Compound: Novel Mechanism and Potential Therapy for Chemotherapy-Associated Thrombosis and Thrombocytopenia

Doxorubicin (Dox) is a widely utilized chemotherapeutic; however, it carries side effects, including drug-induced immune thrombocytopenia (DITP) and increased risk of venous thromboembolism (VTE). Currently, the mechanisms for Dox-associated DITP and VTE are poorly understood, and an effective inhibitor to relieve these complications remains to be developed. In this study, we found that Dox significantly induced platelet activation and enhanced platelet phagocytosis by macrophages and accelerated platelet clearance. Importantly, we determined that salvianolic acid C (SAC), a water-soluble compound derived from Danshen root traditionally used to treat cardiovascular diseases, inhibited Dox-induced platelet activation more effectively than current standard-of-care anti-platelet drugs aspirin and ticagrelor. Mechanism studies with tyrosine kinase inhibitors indicate contributions of phospholipase C, spleen tyrosine kinase, and protein kinase C signaling pathways in Dox-induced platelet activation. We further demonstrated that Dox enhanced platelet-cancer cell interaction, which was ameliorated by SAC. Taken together, these findings suggest SAC may be a promising therapy to reduce the risk of Dox-induced DITP, VTE, and the repercussions of amplified platelet-cancer interaction in the tumor microenvironment