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A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
RNA and DNA aptamers in cytomics analysis
Henning Ulrich1, Antonio Henrique B Martins, João Bosco Pesquero
1Universidade de São Paulo, São Paulo, Brazil.
Current Protocols in Cytometry
|September 5, 2008
Summary
Systematic evolution of ligands by exponential enrichment (SELEX) generates aptamers, which are high-affinity binders. These molecules offer an animal-free alternative to antibodies for targeting diverse biological molecules.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Antibodies are widely used for biological target identification but cannot be raised against all molecules.
- Systematic evolution of ligands by exponential enrichment (SELEX) offers an alternative method for generating high-affinity binders.
- Aptamers, selected via SELEX, can be developed against a broad range of targets, including toxins and non-immunogenic molecules.
Purpose of the Study:
- To describe the SELEX process for generating aptamers.
- To highlight the advantages of aptamers over antibodies, including in vitro generation and applicability to diverse targets.
- To present protocols for developing fluorescent-tagged RNA and DNA aptamers for cytomics analysis.
Main Methods:
- SELEX involves selecting RNA or DNA molecules from a combinatorial library based on target-binding affinity and specificity.
- Modified pyrimidines can be incorporated to create nuclease-resistant RNA aptamers.
- Protocols are provided for developing fluorescent-tagged aptamers.
Main Results:
- SELEX generates high-affinity aptamers with antibody-like specificity and affinity.
- Aptamers can be generated against virtually any biological molecule, overcoming limitations of antibody production.
- Nuclease-resistant RNA aptamers are suitable for in vitro and in vivo protein interaction studies.
- DNA aptamers generally do not require modifications for most applications.
Conclusions:
- Aptamers represent a versatile class of molecules for biological research and diagnostics.
- The SELEX methodology provides a powerful platform for developing novel aptamers against diverse targets.
- Fluorescently tagged aptamers are valuable tools for cell surface protein analysis in cytomics.
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