As soon as the researchers started to learn that the genetic machine was far more complicated than the first models designed back in the fifties or sixties, when DNA was discovered, they realized that we could create our own molecules made of genetic code either of DNA or RNA. Recently, following the discovery of the ncRNAs, it was indeed faster to introduce the first artificial RNA molecules into cells in the lab and learn that this could be an useful tool to understand how the genes work. The next step was a simple conclusion: if we can interfere on protein production (or gene functioning) in these cell cultures, can we do the same in a living tissue? The answer is again yes. More than this, some of these molecules are already registered as medicines or in clinical trials.
Artificial RNA molecules are made of blocks of nucleotides and the combination of nucleotides in certain specific sequences can be easily performed in the lab. In theory, if someone wants to block a given mRNA, let´s say the mRNA which encodes the fibroblast growth factor receptor 3 (FGFR3) protein, it is just the work of combining the right sequence of nucleotides that would match, or complement, the sequence present in the FGFR3 mRNA.
But how could this combination stop the protein production?
Let´s take a look in a rule all genetic machinery follows: the 5’ (we say five prime) to 3’ (three prime) reading direction. These numbers refer to the carbon atom position in the sugar ring (a ribose) which is part of the nucleotide. You can learn more about this here. The entire transcription and translation machinery follows this rule. We have already seen a little about this in the last article. In the DNA (this is valid for RNA, too), we have two strands combined in a double helix. One of the strands, which we call sense strand, starts at 5’ and ends in 3’. The other, or complementary strand, will combine with the sense strand in the opposite direction, from 3’ to 5’ (being called anti-sense strand). What does this have with RNAi? The ncRNAs work by binding to a region close to the 3’ end of the sequence of the mRNA and block the mRNA reading by the ribosome. I invite you to watch again the animation about RNA interference sponsored by the NATURE Journal. This time, pay attention to the position the miRNA will bind the messenger RNA. There is more than one kind of RNA interference and this animation shows the two main kinds: the first one that appears in it is the one which leads directly to RNA degradation; the second shows the interference happening after the ribosome binding to the mRNA.
When researchers build a molecule of nucleotides they look for the same region the natural ncRNAs target and as it is located in the end of the mRNA sequence, they were called anti-sense oligonucleotides. Oligo is a root for few, so the word means literally few nucleotides. We need only a short sequence of nucleotides to make protein translation coming to a stop. Nonetheless, research on RNA interference has been very creative and is not limited to this natural mechanism of action. In fact, there are some antisense therapies already in clinical trials where the molecule binds to a particular section of the mRNA to produce the desired effect. For instance, there is an antisense olgonucleotide being developed to treat Duchenne Muscular Dystrophy, a very severe kind of genetic muscle malformation, caused by a defect in a very important protein called dystrophin (free text). The molecule works by binding a specific part of the dystrophin immature mRNA causing a change in the way it is prepared for translation, a strategy called exon skipping.
If you have been following these articles, you probably will remember the aptamers. Aptamers are very adaptable molecules made of nucleotides. We showed that a specific aptamer could be used to bind the extracellular domain of FGFR3 and block the receptor signaling activation. Well, the ability of these oligonucleotides is not limited to bind proteins. Of course, they can bind also other nucleotide sequences, which includes, potentially, the FGFR3 mRNA, through the antisense strategy. These two potential applications of aptamers in achondroplasia are still waiting for an interested investigator.
Finally, if making oligonucleotides is that easy, then what we are waiting for? Let's just make an oligonucleotide to bind the FGFR3 mRNA and make it happens: no FGFR3, no bone growth arrest. We already have examples that this can be made here.
The great challenge
A lot of potential approaches to treat a vast number of conditions, including achondroplasia is becoming available. What is preventing us to use an antisense oligonucleotide to stop a single protein, the FGFR3, production inside a living body? The main problem is not about creating the molecule, it is about delivering it to the target cell, the chondrocyte.
We will work on the delivery challenge in the next article.
You can learn more about RNA interference reading the papers I mentioned in the end of the last article.
Post a Comment