Single-round isolation of diverse RNA aptamers from a random sequence pool

Area of Science:

• Molecular Biology
• Biotechnology
• Biochemistry

Background:

• Aptamers are oligonucleotide ligands with specific binding affinity to target molecules.
• Traditional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for RNA aptamer generation involves iterative amplification and isolation, which can yield uncertain results.
• Existing methods face challenges with experimental noise and efficiency during the amplification of rare target-binding RNA molecules.

Purpose of the Study:

• To develop a novel, robust method for generating RNA aptamers.
• To overcome the limitations of traditional SELEX, including its iterative nature and dependence on target immobilization.
• To create a primer-less, non-repeated isolation technique for aptamer discovery.

Main Methods:

• A new RNA aptamer isolation method was developed, characterized by being non-repeated, primer-less, and target immobilization-free.
• The method employs RNase digestion to remove non-aptamer sequences, leaving only target-bound aptamer molecules.
• Remaining undigested sequences are amplified with a known sequence, followed by restriction digestion and high-throughput sequencing for aptamer identification.

Main Results:

• The developed method successfully generated multiple RNA aptamers targeting α-thrombin and TGFβ1 proteins independently.
• This approach is robust to experimental noise and independent of specific aptamer types.
• The method has the potential to generate thousands of aptamer candidates, enabling prediction of common sequence or structural properties.

Conclusions:

• A novel and efficient method for RNA aptamer generation has been established, overcoming key limitations of SELEX.
• This technique offers a robust and versatile approach for aptamer discovery against various protein targets.
• The method's ability to yield a large number of candidates may facilitate deeper understanding of aptamer characteristics.