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Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis
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Self-Calibrating Ratiometric DNA Aptamer Probe for Quantitative ATP Imaging in Living Cells.

Yunsong Xu1, Kunihiko Morihiro1, Rui Cong1

  • 1Department of Chemistry and Biotechnology, The University of Tokyo, Tokyo, Japan.

Chemistry (Weinheim an Der Bergstrasse, Germany)
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Summary
This summary is machine-generated.

We developed a new DNA sensor to accurately measure adenosine triphosphate (ATP), the cell's energy currency. This reliable sensor distinguishes cancer cells from normal cells, aiding in disease diagnosis.

Keywords:
ATPDNAaptamersfluorescenceratiometric sensors

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Medical Diagnostics

Background:

  • Accurate monitoring of intracellular adenosine triphosphate (ATP) is crucial for understanding cellular metabolism and diseases.
  • Existing fluorescent sensors face challenges due to high ATP concentrations and variable probe uptake.
  • Current DNA aptamer probes offer selectivity but often lack internal calibration or show limited signal changes in ratiometric designs.

Purpose of the Study:

  • To develop a ratiometric DNA duplex sensor for reliable intracellular ATP monitoring.
  • To overcome limitations of existing sensors, including poor signal change and lack of internal calibration.
  • To enable accurate imaging of ATP levels in live cells and differentiate cancer cells from normal cells.

Main Methods:

  • Designed a ratiometric DNA duplex sensor using a Cy5-labeled ATP aptamer and a thiazole orange (TO)-labeled ECHO probe.
  • Engineered the sensor to release a reporter strand upon ATP binding, causing a decrease in TO fluorescence while Cy5 signal remains constant.
  • Incorporated a polythymidine spacer to prevent Förster resonance energy transfer (FRET) between TO and Cy5, enabling a stable Cy5/ECHO ratio.

Main Results:

  • The sensor demonstrated robust performance across physiological ATP concentrations with high nucleotide selectivity and serum stability.
  • Live-cell flow cytometry and confocal imaging revealed significantly higher Cy5/ECHO ratios in cancer cells compared to normal fibroblasts.
  • The sensor exhibited minimal cytotoxicity, confirming its suitability for live-cell applications.

Conclusions:

  • The developed internally self-calibrating aptamer sensor provides a reliable platform for intracellular ATP imaging.
  • This sensor effectively differentiates cancer cells from normal cells based on their ATP levels.
  • The technology holds promise for advancing cancer diagnostics and studying cellular energy metabolism.