Site-Specific Evolution of DNA Aptamers to Detect ADAR1p150 in Complex Biological Samples

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Adenosine deaminase acting on RNA1 (ADAR1), especially the ADAR1p150 isoform, is crucial in cancer immunity and serves as a prognostic biomarker.
• Detecting ADAR1p150 specifically presents significant challenges in research and clinical settings.

Purpose of the Study:

• To develop a sensitive and specific detection platform for the ADAR1p150 isoform.
• To create a quantitative tool for assessing ADAR1p150 levels in biological samples.

Main Methods:

• Utilized Blocker-SELEX, structure-guided virtual screening, and affinity-based optimization to select a high-affinity aptamer (Zα_LS_11) targeting the ADAR1 Zα domain.
• Employed NMR titration and mutagenesis to confirm aptamer-target interaction and epitope binding.
• Constructed an aptamer-antibody sandwich assay coupled with qPCR for sensitive ADAR1p150 detection.

Main Results:

• The aptamer Zα_LS_11 demonstrated high affinity (K_D = 4.76 ± 2.91 nM) for the ADAR1 Zα domain, with specificity for ADAR1p150.
• The developed aptamer-antibody sandwich assay showed a linear detection range from 62.5 to 4000 ng/mL.
• The platform successfully distinguished ADAR1p150 expression levels in cell lysates under different stimulation conditions.

Conclusions:

• A robust and quantitative aptamer-based detection platform for ADAR1p150 has been successfully developed.
• This tool offers significant potential for evaluating tumor immune status and patient prognosis.
• The methodology provides a valuable approach for monitoring dynamic changes in ADAR1p150 expression.