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Published on: February 28, 2015
Stepping Library-Based Post-SELEX Strategy Approaching to the Minimized Aptamer in SPR
Xiaoqin He1, Lei Guo1, Junlin He1
1State Key Laboratory of Toxicology and Medical Countermeasures, and Laboratory of Toxicant Analysis, Academy of Military Medical Sciences and ‡State Key Laboratory of Toxicology and Medical Countermeasures, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences , Beijing 100850, China.
A new post-SELEX optimization strategy using a stepping library successfully minimized aptamer 807-39nt to In27. This optimized aptamer retains binding affinity and offers potential for enhanced diagnosis and therapy.
Area of Science:
- Biotechnology
- Molecular Biology
- Biochemistry
Background:
- Aptamers evolved via SELEX are often lengthy (70-130 nucleotides).
- Truncation and optimization are crucial for aptamer diagnostic and therapeutic applications.
- Post-SELEX optimization aims to simplify sequences and enhance binding affinity.
Purpose of the Study:
- To introduce a novel post-SELEX optimization strategy utilizing a stepping library.
- To identify a minimized aptamer with retained or improved binding affinity.
- To define key nucleotide contributions and explore binding behaviors of the optimized aptamer.
Main Methods:
- A stepping library was designed to explore all probable nucleotide truncation directions.
- Surface Plasmon Resonance (SPR) was used for screening and evaluating label-free aptamer sequences.
- Site mutagenesis and competition/binding assays were employed to analyze nucleotide contributions and binding behaviors.
Main Results:
- A minimized aptamer, In27, was successfully identified from a small library (35 sequences).
- In27 features a single loop structure, lacks the original stem, and maintains full binding affinity.
- Key nucleotide contributions were identified, leading to a degenerated sequence with comparable or enhanced affinity.
Conclusions:
- The developed stepping library strategy is an efficient method for post-SELEX aptamer optimization.
- The minimized aptamer In27, with its defined sequence and binding characteristics, shows significant potential for diagnostic and therapeutic applications.
- This work provides a valuable approach for aptamer engineering and development.
