RNA stability is a critical factor in the success of RNA transfection. RNAs are inherently unstable molecules, and their degradation can limit the duration and effectiveness of RNA transfection. Here’s an overview of RNA stability and degradation in transfected cells:

RNA Stability

The stability of RNA molecules in cells can vary greatly, depending on their type and sequence. Some mRNAs are very stable and can persist in cells for hours to days, while others are rapidly degraded. Factors that can influence RNA stability include:

• Sequence elements: Certain sequences can stabilize or destabilize RNA. For example, AU-rich elements (AREs) in the 3′ untranslated region (UTR) of many mRNAs can lead to rapid degradation.
• RNA modifications: Certain modifications can increase the stability of RNA. For example, the 5′ cap and 3′ poly(A) tail of mRNA protect it from degradation. Another modification, pseudouridylation, has been shown to increase the stability of mRNA in the context of mRNA vaccines.
• Binding of RNA-binding proteins: Certain proteins can bind to RNA and protect it from degradation. The binding of these proteins can be influenced by sequence elements and RNA modifications.

RNA Degradation

RNA degradation in cells is a regulated process that involves several pathways:

• Exonucleolytic degradation: This is the process where nucleases degrade RNA from its ends. In eukaryotic cells, the exosome complex degrades RNA from the 3′ end, while Xrn1 degrades RNA from the 5′ end.
• Endonucleolytic cleavage: Some RNases can cleave RNA in the middle of the molecule. An example is the RNase III enzyme Dicer, which cleaves double-stranded RNA (such as pre-miRNA or long dsRNA) into short fragments.
• Decapping and deadenylation: For mRNAs, the removal of the 5′ cap or the 3′ poly(A) tail (a process called deadenylation) is often the first step in degradation.
• RNA interference: In the RNA interference pathway, siRNA or miRNA incorporated into the RISC can lead to the degradation of target mRNAs.

Strategies to increase RNA stability, such as sequence optimization and RNA modifications, can be employed to enhance the efficiency and duration of RNA transfection. However, the inherent instability of RNA remains one of the challenges of this technique.