Exploiting natural riboswitches for aptamer engineering and validation.

Area of Science:

• Molecular Biology
• Biotechnology
• RNA Therapeutics

Background:

• Engineered aptamers often fail to function within cellular environments despite successful in vitro selection.
• Developing aptamers with reliable in vivo functionality remains a significant challenge in molecular biology.

Purpose of the Study:

• To introduce the Graftamer approach, a novel platform for enhancing aptamer selection and in vivo validation.
• To demonstrate the efficacy of Graftamer in generating functional aptamers for small molecules like quinine, guanine, and caffeine.

Main Methods:

• Utilized combinatorial RNA pools with guanine riboswitch aptamer structural features for multiplexed in vitro selection.
• Grafted selected aptamers onto a natural guanine riboswitch expression platform for in vivo testing.
• Monitored reporter gene expression to assess aptamer functionality within cells.

Main Results:

• Successfully developed aptamers for quinine, guanine, and caffeine that retained structural integrity of the natural guanine riboswitch aptamer.
• Validated the in vivo function of quinine and caffeine aptamers through reporter gene expression assays.
• Identified RNA sequences that evade selection strategies, providing insights for future methodological improvements.

Conclusions:

• The Graftamer strategy offers a convenient and efficient method for developing and validating aptamers for in vivo applications.
• This approach accelerates the transition of aptamers from laboratory settings to functional cellular systems.
• The findings contribute to the advancement of RNA-based therapeutics and diagnostics.