Partial base pairing between 5' and 3' UTRs artificially regulates IVT mRNA expression levels

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Eukaryotic mRNAs feature a 5' cap and 3' poly(A) tail crucial for expression regulation.
• mRNA circularization, mediated by proteins like eIF4E, PABPC1, and eIF4G, underlies this regulation.
• In vitro-transcribed (IVT) mRNA expression is a key area in biotechnology.

Purpose of the Study:

• To investigate the impact of 5'-3' untranslated region (UTR) base pairing on IVT mRNA expression.
• To explore the relationship between UTR pairing patterns and mRNA expression levels.
• To establish a thermodynamic framework for designing and predicting IVT mRNA expression.

Main Methods:

• Designing and transcribing IVT mRNAs with varying 5' and 3' UTR base-pairing potentials.
• Quantifying the expression levels of these IVT mRNAs.
• Analyzing the correlation between UTR pairing thermodynamics (Gibbs free energy) and expression levels.

Main Results:

• Partial base pairing between 5' and 3' UTRs of IVT mRNAs promotes mRNA circularization.
• This induced circularization artificially enhances mRNA expression levels.
• Expression levels are influenced by the lengths of complementary and non-complementary regions within UTRs.
• A sigmoidal relationship exists between expression levels and the Gibbs free energy of UTR pairing.

Conclusions:

• Engineered 5'-3' UTR base pairing is a viable strategy to enhance IVT mRNA expression.
• The study provides a novel framework for rational design of IVT mRNA UTRs.
• Thermodynamic parameters, specifically Gibbs free energy of UTR pairing, can predict IVT mRNA expression levels.