Antisense Oligo Matters

I shared it here first. Disclaimer: I am a newbie in the RNA therapeutics. I learned about RNase H(I), an antisense oligo-based interference, during this month (That much unbelievable newbie!

Hey, no worries, I am eager to learn more, and that’s the changing the expertise topic joy!). It got my attention a lot about how to make use of such a system. I am happy to share my newbie insights here with you. If you are a beginner in the antisense oligo design (or just want to refresh some molecular biology concepts), I hope you will enjoy it, too.

Nucleases briefly

Nucleases are a type of cell scissors. They can cleave the phosphodiester bonds between the sugar-phosphate backbone of nucleic acids. They act through hydrolysis since they use water to break these bonds. It is vital for many processes in the cell, from DNA replication to repair.

Phosphodiester bond depiction simplified. Ribose sugar (pentagon) + phosphates (circle)

Endo- vs exo-nucleases

Endo means into, and exo means outwards. This makes sense if you know endonucleases act inside of (kinda middle) the strands, whereas the catalytic activity of exonucleases is the removal of the nucleotide at the end of (kinda outside) the strands. Fun fact: restriction enzymes used in cloning and synthetic biology in general are a type of endonucleases.

What is RNase H

RNase Hs are a type of endonucleases. Although RNase H cleavage might be non-specific, it can be directed for targeted cleavage in some cases. This is where antisense oligos come in!

What is antisense oligo and how it works

Let’s say you have a single-stranded messenger RNA (mRNA) molecule that you want to be degraded before being translated into protein or other processes. One way of removal is cleaving it and leaving it for degradation (so that it can no longer be a message carrier).

RNase H cleavage of RNA-DNA heteroduplex. For more info about commercial RNases, NEB

As RNase H needs and recognizes the double-stranded complex (e.g., RNA-DNA), you can control where the RNase H enzyme binds and cleaves your target RNA. If you know where to target, you design a complementary strand oligo of that location, usually 15–25nt, and (kinda) guide RNase where to bind and act. This short complementary strand is called “antisense oligo (ASO)”.

“Sounds great, but how precise is this cut, and what happens to the oligo inside of the cell?” If you are wondering, let’s dig into this mechanism.

How to design ASO?

Suppose you are working in a cell environment. In that case, you need to consider many factors while designing therapeutics (e.g., the delivery, nanoparticle toxicity, cellular uptake, guiding the delivery location, cargo release dynamics, stability, and functionality of RNA or ASO). I will not cover all these here.

There are six main areas for fine-tuning the ASO design for RNase H-based therapeutics.

What to consider how to design ASO (simplified categories from my perspective)

Cells usually react to foreign molecules (in this case, your nucleic acid cargo), which decreases the stability of your ASO. These ASOs might trigger a strong immune response. It is important to decrease immunogenicity and increase the stability. Meanwhile, you need to ensure the modifications you applied to ASO are not toxic either.

Affinity strength is how strong your ASO and RNase H bind to the target. The precise location of the cleavage is associated with the affinity of the ASO to the target.

Enzyme binding is not enough! Although you can guide for certain locations and target and eliminate the immunogenicity, the catalytic activation (doing what the enzyme is supposed to do, cleavage) is also crucial.

All the parameters above can be controlled by base/sugar/backbone modifications of ASOs.

What matters? 3', 5' terminus, both or none?

Before discussing how to modify ASO to control the activity, precision, toxicity, and immunogenicity, I will explain the binding and activity mechanism of RNase H more here.

We learned that it is an endonuclease, and we can use ASO to guide it to a certain location. If you want to control the enzyme, you need to know how it binds and catalyzes the reaction.

The good news is that we know whether it has a site preference. The studies showed that the 3' terminus DNA ASO binding site (to the 5' terminus RNA target to be cleaved) is more important for RNase H. The bad news is that if it binds too strongly to particular 3'DNA/5'RNA, then it might increase the possibility of off-targets. You do not want your ASO to be too strong or too weak in terms of affinity to the target. There are strategies available to fine-tune (please check out further readings)!

Gapmer Strategy

Different base modifications are used to modify ASOs. However, where to apply these is required to have sufficient activity.

Summary of the most commonly used base/sugar/backbone modifications. For more details, IDT

Previously, researchers used mixmers: mixing the modified and unmodified bases. However, this was not enough to control the location and catalytic activity. Later, the gapmer strategy is developed. ASOs are designed as gapmers such that the bases with higher affinity binding modifications (e.g., LNA +) are used for both ends (5' and 3') and lower/regular affinity bases in between.

Example: +N+N+NN+N*N*N*N*N*N*N*N*N+NN+N+N+N

I will keep some of my literature notes open, so stay tuned if you are interested in “how-to-RNA therapeutics”.

Further Reading/References:

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Ortaya Karışık (Fatma Betul Dincaslan)
Ortaya Karışık (Fatma Betul Dincaslan)

Written by Ortaya Karışık (Fatma Betul Dincaslan)

FeBe/ Molecular Biologist and Geneticist / Bioinformatician/ Single Cell Assayist / Socially developed nerd

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