An in vitro selection for small molecule induced switching RNA molecules

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Selecting RNA aptamers with specific ligand-binding properties is established.
• Directly selecting aptamers that undergo conformational changes upon ligand binding has been a significant challenge.
• This limitation has hindered the development of small molecule-responsive strand displacement systems and synthetic riboswitches.

Purpose of the Study:

• To present a novel strand displacement-based selection protocol.
• To directly select RNA molecules exhibiting switching activity in response to ligand binding.
• To enable the design of more complex, environmentally responsive molecular circuitry.

Main Methods:

• A selection protocol based on strand displacement was developed.
• The RNA library was derived from a known thiamine pyrophosphate riboswitch.
• The entire selection process was performed in vitro.

Main Results:

• The methodology successfully identified RNA sequences with robust strand displacement activity.
• These sequences demonstrated significant activity in the presence of thiamine pyrophosphate.
• The selected RNA molecules exhibited riboswitch activity comparable to natural riboswitches.

Conclusions:

• The presented strand displacement selection protocol effectively selects for RNA aptamers with ligand-induced switching activity.
• This method facilitates the creation of synthetic riboswitches responsive to various ligands, including toxic ones.
• The approach is expected to advance the design of sophisticated, environmentally responsive molecular devices.