mRNA Structural Modifications

RNA is generally considered a more sensitive and unstable molecule compared with DNA. A major liability factor is the ubiquitous presence of RNases, which can rapidly hydrolyse mRNA molecules, dictating the necessity of a RNase-free environment from the research and development laboratory upwards to the final formulation facility of the finished product. The saga of mRNA instability continues in vivo where it is exposed to a physiological

temperature of 37°C, increased concentrations of magnesium ions as well as the enzymes

of the host cell. 

Considering the above, Seattle Genova has put efforts in modifying the basic elements of mRNA and formulating it in ways that can putatively increase its stability.

Five integral elements of mRNA are of pivotal importance for its therapeutical potential and have been the target of modifications: 5’ cap, 5’ and 3’ UTRs, ORF and the poly(A) tail. 

Our Solutions for mRNA Structural Modifications

5’ cap

During mRNA manufacture, the 5′ capping performed at the stage of IVT carries the inherent danger of yielding a population of mixed mRNA species, some incorporating the 5′ cap in the correct orientation and some in reverse which leads to untranslatable mRNA. In Seattle Genova, we developed novel caping technologies for 5’cap modification to surpass this hurdle, which ensures incorporation in the correct orientation. 

5’ UTR

5’ UTRs are mRNA regulatory sequence elements implicated in recognition of the transcript by the ribosomes, facilitators of the interactions with the translational complex and targets of many mRNA binding proteins such as the ones regulation nuclear export of mature transcripts and subcellular localizations. 

Efficient mRNA design should take into account that Start, Stop and non-canonical Start codons present in uORF can hamper the main ORF translation. Lengthwise, it has been found that increased length of 5’ UTR can be detrimental for mRNA eIF4F-depended translation so shorter sequences are generally preferred.

ORF

ORFs are the main coding sequences that are translated to peptides which reside between 5’ and 3’ UTRs. Widely employed modifications of the ORF regions include codon optimization and the use of base analogues. 

Codon optimization entails the swapping of codons for synonymous ones, abiding to the codon bias of the organism in which the mRNA is to be introduced. A complete optimization effort intergrades much more than simple selection of tRNA favored codons: it takes into account the GC content of the final transcript, as well as the strategic introduction of sub-optimality.

Use of base analogues can confer advantages both by ensuring proper protein folding

through modulating, as above, production rate and by rendering mRNA invulnerable

towards the cell’s innate immune response to foreign mRNAs. 

As in codon optimization, prudency is advised when considering the use of base analogues in mRNA as means to evade immunogenicity and increase translator efficiency. 

3’ UTR

3’ UTRs are located immediately after the Stop codon and is known to serve a multifarious role in mRNA localization, stability, and translation. When choosing a 3’ UTR for a therapeutic mRNA, length, sequence and secondary structure have to be taken into account.

poly(A) tail

The presence of poly(A) tail on functional mRNA is essential and its length is crucial. The poly(A) tail can act as a facilitator in the formation of a circular closed loop state with the 5’ cap that inhibits deadenylation and promotes polysome formation. 

Optimal poly(A) tail length: Certain length of poly(A) tail is thought to be ideal, increasing translation efficiency and at the same time contributing to uridine depletion by reducing the relative uridine content of mRNA. 

Modifications of the poly(A) tail: Employment of analogues, specifically for the tail, is a terrain with at least two peculiarities. First of all, the general consensus is that the tail is intolerant of other bases than adenine, so the base used must strictly be an adenosine analogue. Moreover, the modified base must be incorporated at the very end of the tail and fluorophore incorporation might be inappropriate for therapeutical applications. 

Our mRNA engineering technologies offer you a streamlined solution for mRNA stability improvement, simplifying your process and saving time. Seattle Genova can take you all the way from the very beginning of mRNA design to the efficacy studies with animals.