Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Summary

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Abstract

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

Introduction

Interaction between molecules is the basis of nature. Hence, scientists in many fields of basic and applied research try to understand the fundamental principles of molecular interactions of different kinds. MicroScale Thermophoresis (MST) enables scientists to perform the fast, precise, cost-efficient, and quality-controlled characterization of molecular interactions in solution, with a free choice of buffers. There are already more than 1,000 publications using MST, from 2016 alone, describing different kinds of analyses, including library screenings, binding event validations, competition assays, and experiments with multiple binding partners^1-8. In gene....

Protocol

1. Preparation of the Aptamer Working Stock

1. Follow the manufacturer's instructions and dissolve the oligonucleotide (5-Cy5-CCTG GGGGAGTATTGCGGAGGAAGG-3, sequence from reference¹⁸) in water, reaching a 100-µM final concentration.
2. Prepare the aptamer working solution by diluting the oligonucleotide stock to 200 nM with binding buffer (20 mM Tris, pH 7.6; 300 mM NaCl; 5 mM MgCl₂; 0.01% Tween20).
3. Incubate the mixture for 2 min at 90 °C, let the sample immediately cool down on ice, and use the sample at room temperature.

2. Preparation of the Ligand Dilution Series

....

Results

In this study, MST was applied to characterize the binding site of the DH25.42 DNA aptamer¹⁸ on ATP. In contrast to other studies characterizing the interaction of ATP or ATP-mimicking small molecules with proteins randomly labeled with one or more fluorophores^38-40, this study includes a labeled version of the 7.9 kDa ssDNA aptamer with one Cy5 molecule on the 5´ end. Different ATP derivatives and related molecules, all differing from ATP in various positions, were .......

Discussion

Quality controls:

Unspecific sticking/adsorption of sample material to surfaces, as well as aggregation effects, have a dramatic influence on the quality of the affinity data. However, only a few state-of-the-art technologies offer accurate and rapid options to monitor and avoid these effects. MST offers integrated quality controls that detect and help to overcome these issues, allowing for the stepwise optimization of the technical setup. Important information on sticking and fluorescence effect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aptamer binding buffer			20 mM Tris pH 7.6; 300 mM NaCl; 5 mM MgCl2; 0.01% Tween-20
Fluorescently labeled ATP aptamer	IDT, Leuven, Belgium		sequence: DH25.42 50-Cy5-CCTGGGGGAGT- ATTGCGGAGGAAGG-3
ATP	Sigma Aldrich, Germany	A2383	10 mM stock solutions stored at - 20 °C
ADP	Sigma Aldrich, Germany	A2754	10 mM stock solutions stored at - 20 °C
AMP	Sigma Aldrich, Germany	A2252	10 mM stock solutions stored at - 20 °C
Adenine	Sigma Aldrich, Germany	A8626	10 mM stock solutions stored at - 20 °C
SAM	Sigma Aldrich, Germany	A7007	10 mM stock solutions stored at - 20 °C
dATP	Sigma Aldrich, Germany	11934511001	10 mM stock solutions stored at - 20 °C
CTP	Sigma Aldrich, Germany	C1506	10 mM stock solutions stored at - 20 °C
GTP	Sigma Aldrich, Germany	G8877	10 mM stock solutions stored at - 20 °C
Monolith NT.115	NanoTemper Technologies, Munich, Germany	MO-G008	Blue/Red Channel MST device with standard detector, Monolith NT115 pico is MST device with high sensitivity detector
Monolith NT.115 capillaries Standard	NanoTemper Technologies, Munich, Germany	MO-K002
Eppendorf PCR tubes	Eppendorf, Germany	30124537
Monolith control software. 2.1.33, pre-installed on the device	NanoTemper Technologies, Munich, Germany
MO.affinity analysis v2.1.1	NanoTemper Technologies, Munich, Germany
Kaleidagraph 4.5.2	Synergy Software