Thermodynamic analysis of cooperative ligand binding by the ATP-binding DNA aptamer indicates a population-shift binding mechanism

Zachary R Churcher,Aron A Shoara,Sladjana Slavkovic,Philip E Johnson,Anne E Johnson,Yanrui Zhu

doi:10.1038/s41598-020-76002-8

Zachary R Churcher, Aron A Shoara + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-76002-8

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Abstract

The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods. This aptamer follows a structure-switching binding mechanism and is unusual in that it binds two copies of its ligand. We have used isothermal titration calorimetry methods to study the binding of ATP, ADP, AMP and adenosine to the ATP-binding aptamer. Using both individual and global fitting methods, we show that this aptamer follows a positive cooperative binding mechanism. We have determined the binding affinity and thermodynamics for both ligand-binding sites. By separating the ligand-binding sites by an additional four base pairs, we engineered a variant of this aptamer that binds two adenosine ligands in an independent manner. Together with NMR and thermal stability experiments, these data indicate that the ATP-binding DNA aptamer follows a population-shift binding mechanism that is the source of the positive binding cooperativity by the aptamer.

Highlights

The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods
We used Isothermal titration calorimetry (ITC) methods to determine that the ATP-binding aptamer binds two copies of its ligand in a positive cooperative manner
The ITC data for all ligands studied best fit a cooperative model as opposed to a two-site independent model (Table 1)

Summary

Introduction

The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods This aptamer follows a structure-switching binding mechanism and is unusual in that it binds two copies of its ligand. We have used isothermal titration calorimetry methods to study the binding of ATP, ADP, AMP and adenosine to the ATP-binding aptamer Using both individual and global fitting methods, we show that this aptamer follows a positive cooperative binding mechanism. The ITC data were fit individually and using global fitting methods to show that the ATP-binding aptamer follows a cooperative binding mechanism and to determine the binding thermodynamics for both ligand-binding sites

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Results

Conclusion