During my first and second year at Stanford, I worked on a project in chemical biology to study zebrafish. This research was performed in James Chen's group in Chemical and Systems Biology.
For those who are not particularly familiar with biotech industry, Moderna is a (rather high-profile) company founded in 2010 by several Harvard faculty. Its focus is drugs based on RNA (DNA's less boring counterpart), rather than the conventional small-molecule approach. In particular, the biotech industry has long relied on the protein-target approach, in which one first identifies a faulty protein of interest (e.g. a checkpoint gone haywire in cancer cells) and then designs a molecular substance to block or activate that protein. However, this often doesn't work as expected, causing unwanted side effects or even horrible toxicity. Hence, the biotech industry has turned to other technologies, such as biologics, and, in Moderna's case, mRNA. Biologics are the kinds of proteins-turned-drugs that have basically included all the major breakthrough cancer therapies of the last few decades, including Herceptin (breast cancer), Rituxan (lymphoma) and Keytruda (melanoma).
The other big thing that might turn out to revolutionize our current therapeutics are oligonucleotides, such as RNA. Because many diseases are caused by the nuclear material of their vector (most prominently viruses), RNA provides a unique strategy towards attacking new targets or even circumventing existing problems. Despite their great promise, it's still far too soon to determine whether this new strategy will pan out.
Now, it turns out that long before people began thinking about using synthetic oligonucleotides for therapeutic purposes, researchers have employed them for probing biological systems. In develomental biology, scientists often want to ask questions along the lines of when and where to specific anatomies develop? More specifically, we often are trying to figure out how specific genes, molecules, and environmental cues interact with both spatial and temporal control to generate the body plans of animals we see around us. For example, how do we get left-right symmetry and asymmetry? How does anterior-posterior organization arise? How do limbs develop? Since all these processes are controlled at heart by the action of genes and proteins, oligonucleotides present a powerful tool for probing them. In essence they allow us to selectively turn off (and on) genes at different points during development and at different locations in the body. With this knowledge, we can begin to untangle the complex biological networks that orchestrate the creation of a living organism from a single cell.
Morpholinos are a kind of oligonucleotide that chemical biologists have created to control such developmental processes. Recall from biology that each RNA nucleotide is composed of a sugar backbone (ribose), a nitrogenous base (adenine, guanine, cytosine, or uracil), and a phosphate group. In morpholinos, however, there are two major changes with perturb the chemical stability: first, the ribose is converted to a morpholine ring, a six-membered oxygen and nitrogen heterocycle; and, second, one of the oxygens on the phosphate group is converted to a nitrogen, making a phosphoramidate group. The details are not so important, but the effect of these changes is that morpholinos are much more stable in vivo than RNA, which disintegrates rather quickly (and thus renders it difficult to use for biological experiments).
As a result, a synthetic morpholino, armed with the correct complementary gene sequence to a target mRNA, can hybridize (bind) to that mRNA in vivo and knockdown (i.e., shutoff) the gene of interest. Although morpholinos are a formidable tool for investigating effects of specific genes, they suffer from precision both spatially and temporally. When working with zebrafish, the typical protocol involves injecting the embryos at the single-cell (zygote) stage with the morpholino of choice, but this inevitably causes the morpholino to spread throughout the fish as it grows, preventing one from, say, turnoff a gene in only the fish's tail. At the same time, it is challenging to control the time at which the morpholino acts. After injection, the morpholino simply diffuses throughout the cells, encountering mRNAs and turning off genes in a stochastic fashion.
As I mentioned at the beginning of this section, these two aspects—spatial and temporal control—are essential to a detailed understanding of development; but the native approach of injecting morpholinos seems to preclude this. A key development then was the idea of caged morpholinos (cMOs). What if we could turn on and turn off the morpholino activity while it was already in the zebrafish, perhaps by activating it through an external cue? The idea then is as follows: through a chemical modification of the morpholino, we attach the two ends together (caging) so that it is unable to bind to the target mRNA. Then, we use light (which can be tuned very selectively) to break apart the chemical cage and allow the morpholino to hybridize.
Why, then, does this give us spatial and temporal control? It is because the experimentalist can decide exactly when and where to shine light onto our zebrafish, thus enabling precise and localized activation of morpholino activity. In practice, one usually employs a confocal laser to ensure a small area of focus and illuminates the embryo at varying stages of development. With this method, people have been able to probe several essential genes for zebrafish function, including ntla (no-tail) and Tbox genes, which control front-back patterning in the embryo (see here or here for more details).
Under construction...!