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Engineering base-excised aptamers for highly specific recognition of adenosine
Yuqing Li1, Biwu Liu1, Zhicheng Huang1
1Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo Waterloo Ontario N2L 3G1 Canada liujw@uwaterloo.ca.
Chemical Science
|June 4, 2021
Summary
Researchers engineered an adenine-excised DNA aptamer for highly specific adenosine detection. This novel biosensor distinguishes adenosine from ATP, overcoming limitations of previous aptamer-based analytical methods.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Molecular Biology
Background:
- DNA aptamers are crucial for analytical biosensors, particularly for detecting adenosine and adenosine triphosphate (ATP).
- Distinguishing adenosine from ATP is essential for practical biosensor applications but remains a significant challenge.
- Existing aptamers often lack the specificity required to differentiate between adenosine and similar molecules like ATP.
Purpose of the Study:
- To develop a novel DNA aptamer strategy for highly specific adenosine detection.
- To engineer an existing adenosine aptamer to differentiate adenosine from ATP and other related molecules.
- To create a cost-effective and facile method for enhancing aptamer specificity without using SELEX (Systematic Evolution of Ligands by Exponential Enrichment).
Main Methods:
- A one-site adenosine aptamer was modified by excising an adenine nucleotide from its DNA backbone.
- Adenine-excised aptamer specificity was analyzed using SYBR Green I (SGI) fluorescence spectroscopy.
- Binding affinities and thermodynamics were characterized using isothermal titration calorimetry (ITC).
Main Results:
- The engineered adenine-excised aptamer demonstrated highly specific binding to adenosine, with other analytes (AMP, ATP, guanosine, cytidine, uridine, theophylline) showing no binding.
- ITC confirmed two cooperative binding sites for adenosine on the engineered aptamer, with a dissociation constant (Kd) of 14.8 ± 2.1 μM at 10 °C.
- The modified aptamer achieved a limit of detection of 46.7 μM for adenosine in diluted fetal bovine serum.
Conclusions:
- Excising an adenine nucleotide from an adenosine aptamer backbone effectively creates a highly specific biosensor for adenosine.
- This facile, non-SELEX engineering approach enhances aptamer functionality for improved analytical applications.
- The developed adenine-excised aptamer offers a promising tool for distinguishing adenosine from ATP in complex biological samples.
