Evolution and protein packaging of small-molecule RNA aptamers

Area of Science:

• Biotechnology
• Molecular Biology
• Chemical Biology

Background:

• RNA aptamers are valuable tools for molecular recognition.
• Encapsulating functional RNAs can enhance their stability and utility.
• Developing efficient methods for producing and analyzing functional RNA systems is crucial.

Purpose of the Study:

• To develop a generalizable method for producing functional RNA aptamers within virus-like particles.
• To demonstrate that encapsidated aptamers retain their small-molecule binding affinity.
• To establish a convenient, label-free analytical technique for these systems.

Main Methods:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was used to identify a high-affinity RNA aptamer against a drug-like molecule.
• Aptamers were embedded in longer RNA sequences and expressed within virus-like particles using a novel technique.
• Backscattering interferometry was employed to measure the binding affinity of encapsidated aptamers to small-molecule ligands.

Main Results:

• A high-affinity RNA aptamer (K(d) = 50 nM) was identified against a representative drug-like molecule.
• Encapsidated aptamers, including variants and a theophylline-binding aptamer, exhibited binding affinities comparable to their free counterparts.
• The virus-like particle system successfully produced and sequestered functional RNA molecules.

Conclusions:

• The developed system provides a general approach for producing and sequestering functional RNA molecules within virus-like particles.
• Encapsidation does not significantly impair the binding affinity of RNA aptamers to their target ligands.
• The method offers a convenient, label-free analytical technique for evaluating functional RNA systems.