PhD Scientific Days 2024

Budapest, 9-10 July 2024

Poster Session E - Molecular Medicine 2.

Creation of eIF4G-selective Aptamer Containing Circular RNAs

Author(s)

Alexandra Tar1, Anna Tory1, Tamás Mészáros1
1: Semmelweis University, Institute of Biochemistry and Molecular Biology, Department of Molecular Biology, Budapest

Text of the abstract

Introduction
Circular RNAs (circRNAs) are covalently closed RNA molecules that are of increasing interest in the field of protein synthesis and vaccine development primarily due to their nuclease resistance. Given the absence of the 5' end of these RNAs, the translation must be initiated a cap-independent manner, which requirement is generally fulfilled by utilizing various IRES sequences. Previously published data indicated that the translation efficiency of circRNAs can be enhanced by using circRNAs containing an eIF4G-specific aptamer.
Aims
Our objective is to insert eIF4G-selective aptamers that were isolated in our group into a circRNA producing vector. Next, the applicability of these aptamers will be evaluated in a human cell line and in a human cell extract-based in vitro translation system.
Method
Aptamer candidates were isolated by systematic evolution of ligands by exponential enrichment (SELEX). The selection steps were carried out by alternating the target molecules, i.e. MIF4G domain of the human eIF4G protein and the eIF4E binding peptide motif of eIF4G. Following SELEX, the enriched library was cloned in an appropriate vector and analyzed by nucleic acid electrophoresis of colony-PCR products and sequencing. The interaction analysis of aptamer with the target protein was performed using AlphaScreen. CircRNAs containing eIF4G selective aptamers are produced using the NEBuilder® HiFi DNA Assembly Cloning Kit and the permuted intron and exon method. The success of circRNA production is evaluated by gel electrophoresis. The translation efficiency of produced circRNAs is studied by a cell- and a cell extract-based system.
Results
Computational analysis by MEME Suite motif finder revealed the enrichment of distinct nucleic acid sequence motifs during SELEX. AlphaScreen interaction analysis showed that several aptamer candidates interact with the MIF4G domain. The positive control circRNA was successfully produced and detected by agarose gel electrophoresis.
Conclusion
Our aptamer selection strategy led to the enrichment of the original oligonucleotide library and numerous aptamer candidates interact specifically with the MIF4G domain. Currently, we are employing the A549 cell line and HeLa cell extract-based in vitro translation system to investigate the applicability of aptamers in circRNAs.
Funding
OTKA-ANN-139564