Analysis of aptamer-target binding and molecular mechanisms by thermofluorimetric analysis and molecular dynamics simulation

Abstract

Introduction: Aptamers are valuable for bioassays, but aptamer-target binding is susceptible to reaction conditions. In this study, we combined thermofluorimetric analysis (TFA) and molecular dynamics (MD) simulations to optimize aptamer-target binding, explore underlying mechanisms and select preferred aptamer.Methods: Alpha-fetoprotein (AFP) aptamer AP273 (as the model) was incubated with AFP under various experimental conditions, and melting curves were measured in a real-time PCR system to select the optimal binding conditions. The intermolecular interactions of AP273-AFP were analysed by MD simulations with these conditions to reveal the underlying mechanisms. A comparative study between AP273 and control aptamer AP-L3-4 was performed to validate the value of combined TFA and MD simulation in selecting preferred aptamers.Results: The optimal aptamer concentration and buffer system were easily determined from the dF/dT peak characteristics and the melting temperature (Tm) values on the melting curves of related TFA experiments, respectively. A high Tm value was found in TFA experiments performed in buffer systems with low metal ion strength. The molecular docking and MD simulation analyses revealed the underlying mechanisms of the TFA results, i.e., the binding force and stability of AP273 to AFP were affected by the number of binding sites, frequency and distance of hydrogen bonds, and binding free energies; these factors varied in different buffer and metal ion conditions. The comparative study showed that AP273 was superior to the homologous aptamer AP-L3-4.Conclusion: Combining TFA and MD simulation is efficient for optimizing the reaction conditions, exploring underlying mechanisms, and selecting aptamers in aptamer-target bioassays.

Stability and affinity evaluation of aptamers as a biorecognition system for testosterone and its analogs

“…Another point to evaluate sodium and magnesium ions is that the relation between the effects and the amount/concentration of these ions, cannot be considered a basic or direct effect, because only a specific range of concentration can hold the conformation while a low concentration conducts an unstable aptamer structure, and the counterpart, high concentration, cause electrostatic repulsions that interference on the aptamer binding [30,31]. In vitro methodologies allow us to determine a working concentration of both ions and also evaluate for each aptamer the ability to maintain its structure through the changed conditions.…”

“…When looking at chemical stability, we evaluate ions commonly present in buffers, sodium ion starts from a worked concentration of 50 mM and modify it into a higher and lower value, and magnesium ion performs in general concentrations, according to the literature, since it is not an obligate required ion for our future worked solution on the binding event [30,31]. For sodium ion concentration, [Na + ], the ΔG value (kcal/mol) decreases as the sodium concentration increases in response to the capacity of sodium as a stabilizing cation for nucleic acids, reducing the solubility that surrounds the sequence, allowing it to generate a greater number of intra-interactions.…”

Strategies for targeted gene delivery using lipid nanoparticles and cell-derived nanovesicles

“…163,164 Aptamers can effectively bind to the target protein only when both of them maintain their tertiary structure, and the isolated CDNs can easily be seperated by adjusting the ionic strength and metal ion concentration to cleave the bonding between aptamers and CDNs. 165,166 Zhang et al utilized a DNA aptamer immobilized matrix for isolating CDNs having the MUC1 protein. 166 Despite the several advantages of obtaining a highly selective class of CDNs, this method comes with a high cost, requires proper storage of reagents and cannot be utilized for large volume samples because of the cost, and certain studies do not require a specific sub-class of CDNs.…”

“…Also, CDNs are isolated in various ways including ultracentrifugation, filtration, immunoaffinity, polymer precipitation, and stimuli techniques. 134–181 According to the references we mentioned, various combinations of the isolation method and targeting strategy of CDNs exist. Not only is it important to decide whether to use LNPs or CDNs to target specific cells, but it is also important to carefully determine what each individual strategy is.…”