PhD Scientific Days 2023

Molecular Sciences - Posters K

Efficiency Improvement of In Vitro Translation Systems with an Aptamer-based Approach

Text of the abstract

Introduction
Availability of high-purity and ample amount of protein of interest is a fundamental requirement of research, diagnostics, and therapeutics. Eukaryotic cell-free in vitro translation is a popular protein producing system, however, its efficient use still has limitations. One of the main pitfalls is 5’-methylguanosine capping of the mRNA, for it is a complex and inefficient process under in vitro conditions. According to our hypothesis, this shortcoming can be evaded by replacing the cap with the insertion of eIF4G specific RNA aptamer into the 5' untranslated region of the mRNA.
Aims
The long-term objective of our research is to develop an efficient, human cell extract-based in vitro translation system that exploits the high binding affinity and selectivity of RNA aptamers. We aim at selecting human eIF4G binding RNA aptamers and creating aptamer holding vectors, which increase the efficiency of the HeLa cell lysate-based in vitro translation system.
Method
Aptamer candidates were produced by systematic evolution of ligands by exponential enrichment (SELEX). 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 into a previously assembled vector construct containing a green fluorescent protein (GFP) coding sequence downstream from the insertion site of the aptamers. Several clones were analyzed by PCR, lab-on-a-chip nucleic acid electrophoresis and sequencing. The functional screening of aptamer candidates was implemented in a HeLa cell lysate-based system with the quantification of produced GFP through measuring the fluorescent intensity of translation mixtures.
Results
Nucleic acid sequence of aptamer candidates was determined by Sanger sequencing of 96 of SELEX obtained colonies. Computational analysis of these sequences by MEME Suite motif finder revealed several longer and shorter motifs, furthermore, some of the oligonucleotides have been found in duplicated copies.
Conclusion
Our aptamer selection strategy resulted in enrichment of the original oligonucleotide library hinting the success of SELEX. However, these results do not prove applicability of the obtained aptamer candidates. Currently, we are in the process of demonstrating their applicability by applying a HeLa cell lysate-based commercial in vitro translation system.
Funding
OTKA-ANN-139564

E-mail: taralexandra95@gmail.com
Institute: Semmelweis University, Doctoral School of Molecular Medicine
Supervisor: Dr. Tamás Mészáros