Think back to your last chemistry class. (This might have been some time ago.) For most of you, you were likely 16 or 17 years old. When I was 16 or 17, I was thinking about some girl, or football, or a party, or … some girl. I certainly wasn’t focused on chemistry. And, chances are pretty good that my teacher knew this about me and my classmates. And your teacher knew this about you.
Experiments during lab sections were simplistic for a reason.
Or runaway hormones.
OK, no reason to point fingers here. Our chemistry teachers were smart. They didn’t ask us to do anything more than: Take some stuff. Add some junk. Get something else. Collect it. Measure it. Report. In fact, the reaction for the experiment probably looked something like this:
In this reaction, acetic anhydride (junk – blue) and salicylic acid (stuff – black) are mixed together and ultimately form acetic acid and aspirin (something else). People who teach general chemistry courses love to assign this experiment. You add two chemicals together. Both of them start off in liquid phase (dissolved in water). And the molecule you are trying to make (aspirin) separates out from the liquid in solid form. From there it’s easy to remove the liquid (what you don’t want) from the solid (what you want). Pretty basic.
And, to be honest, it’s not just instructors who love these reactions. All chemists love these reactions. If all reactions were like this, our work lives would be so much easier. And isn’t that what we all want out of our jobs? A little predictability. An ensured, built in sense of accomplishment. The satisfaction that comes with knowing that your work can save someone else from a few headaches.
Inventing and running reactions as simple as this is something most chemists strive to do.
Cells and Chemistry
There are, however, some brave chemists who are actively eschewing the comfort of simplicity.
These scientists are developing chemical tools to craft new chemistry inside of cells. I need to amplify that statement: IN THE CELL. The cell is the polar opposite of the beaker you used when you were 16 and synthesized aspirin. The cell is teeming with chemicals. The cell is the most complex collection of chemistry found anywhere on earth all bundled up in a teeny tiny little package. And these chemists are using the cell as their own personal test tube. Amazing.
Reactions are continuously occurring inside the cell. Products from one reaction are the starting point for a completely different reaction, whose products go on to do something else. These reactions keep propagating, expanding, moving forward. When they stop, when the all of the reactions find balance and equilibrium inside of our cells, we die. The necessary diversity of chemistry pumping along in our cells keeps us alive. This diversity necessitates many different kinds of chemicals, all of which are doing different things. Evolution is the great usurper. If a reaction works well and makes something of value, life will find a way to hard-wire that chemistry into its genetic code.
The diversity of reactions in a cell requires chemicals that look different from one another.
The research I am profiling in the post represent an attempt to find and develop new types of chemistry that work inside of cells.
There are a few criteria for this research:
1) The chemistry cannot already exist in cells. That is, it must be something that evolution hasn’t discovered or that evolution has deemed worthless or unnecessary.
2) The chemistry being developed cannot affect the life that is always happening at a cellular level. In chemistry-speak, we call this “orthogonal”, meaning the chemistry moves in another direction, or life continues to happen uninterrupted while this new chemistry is going on.
3) The chemicals being used in this new chemistry should not look like any chemicals that Nature already uses.
Perhaps it would be prudent at this point to discuss what the point of all of this new chemistry is.
One reason for doing this research is that chemists are inherently masochistic. We secretly like impossible problems. And we all want to be that person who makes other chemists say, “Holy moley … they did WHAT?” There is no absence of (deserved) “muscle-flexing” going on by the researchers who have figured out how to perform new reactions inside of cells.
Importantly, however, more applicable reasons for doing this chemistry do exist.
Imaging cellular biochemistry
There is so much about what goes on inside the cell that we don’t understand. On a molecular level, we are pretty good at studying how molecules work in a test tube. But, as discussed earlier, the cell is vastly more complex than a test tube. One of the problems is that we can’t make out the one specific molecule (protein, DNA, sugar, etc.) that we want to look at from the rest of the tangled mess. Being able to “see” what’s going on with the molecule we want to watch (and only that molecule) is the necessary first step to better understanding the chemistry of life. In order to see biological molecules in action, a molecule called a label needs to be added. The label serves as a visible marker that distinguishes one molecule from another.
Adding new chemistry to a cell means that it can potentially do all sorts of new things. Clean up oil spills. Synthesize new drugs. Create new kinds of plastic. Do arithmetic. Derive the formula of Coca-Cola. Really, the list here is endless. Finding new technological applications is a matter of putting the right tools into the hands of a scientist who understands how to use them. And, the research being profiled here is really just that: the development of a new technological tool.
Because this tool is still being honed, I have chosen to focus on the work of imaging molecules, which is the more developed of the two applications.
Seeing Chemistry in Cells
The gold standard for imaging proteins inside of cells involves tricking cells to attach green fluorescent protein (GFP) to the tail of the protein being examined. (This is called a GFP fusion because GFP is being fused to the protein of interest). GFP is the most commonly used label that enables us to see biological molecules in cells. GFP fusions have produced a minor revolution in the way we understand cells because they allow us to actually see individual molecules being created, moving around, and responding to stimuli. The science behind the development of this technology was awarded the Nobel Prize in Chemistry in 2008.
Bacterial colonies on an agar plate, each expressing a different version/color of the standard bearer for imaging, GFP.
However, there are shortcomings to this technology. GFP can easily be used to label proteins, while labeling DNA and other biological chemicals is more problematic. The main issue, though, is that GFP is big. It is roughly the same size as any protein being examined. Attaching GFP can completely change what a protein does and where it lives inside of a cell.
Think of GFP fusions in terms of the following scenario: You and I have been coworkers for several years. We have a good relationship. Our company relies on our teamwork to properly function. One day, out of the blue, I show up to work with a Siamese twin. You are completely taken off guard. Your expectations of who I am and what I do are drastically altered. Suddenly, we don’t work so well together. The company starts to fall into disarray and can’t quite recover. Alternatively, if instead of showing up with a Siamese twin, I come to work wearing glasses, you may notice and comment on it, but it’s not going to change our working relationship.
Chemists who are developing these new imaging techniques are basically trying to shrink the size of their add-ons by switching from Siamese twins to spectacles. Unfortunately, it is impossible to drop a thousand pairs of glasses over a company headquarters and have them land perfectly on a single individual. Throwing a dash of labels at a cell and expecting one to perfectly attach to the protein you would like to image isn’t feasible. Likewise, you can’t just exploit the everyday chemistry that goes on inside of cells to attach new imaging labels to a protein or sugar or DNA. If you try to do that, you’ll end up labeling (and seeing) lots of things when you only want to see one.
A cell is like a bowl of vegetable soup. When you or I add salt to our soup it flavors the entire dish. The goal of bio-orthogonal chemists would be to salt only the peas in the soup (i.e. add a new chemical to one and only one protein). Furthermore, they would only want to flavor the peas on one specific wrinkle. In this simile, the bio-orthoganal chemists are an extreme version of avant garde chefs like Jose Andres, Ferran Adrià, Heston Blumenthal, and Grant Ashatz, striving for perfection in complexity at a molecular level.
In order to label the molecule where you want to, there are two things that you have to do. First, you have to be able to alter that protein/DNA/sugar with a unique chemical group (not found anywhere else in the soup/cell – i.e. adding more chemical diversity to the cell). Second, you have to add a label that will only react with the chemical group that you’ve just put on the protein/DNA/sugar.
Bio-orthogonal Techniques in Practice
Likely, the chemist most responsible for popularizing the use of these bio-orthogonal techniques is Carolyn Bertozzi who is a professor at the University of California, Berkeley. Her research mainly focuses on understanding the biological role of glycans (groups of sugar molecules strung together) play. Recently her group has been using bio-orthogonal approaches to observe glycans in zebrafish. The image below, which shows labeled glycans (green) in a zebrafish embryo, is the result of some of this research. To produce this image Bertozzi’s group had to go through a few steps. First, they added azide-modified sugars to the container holding the zebrafish. (An azide is a chemical with three nitrogens in a row, and, conveniently, azides are not found anywhere else in zebrafish.) The zebrafish incorporated these modified sugars into their glycans. Second, they added a fluorescent probe (green star) attached to a chemical (an alkyne) that reacts only with the azides in the cell.
M.G. Finn at the Scripps Research Institute uses a similar reaction scheme to attach labels to the outside of viruses. His label, in this instance, isn’t a luminescent molecule like the one Bertozzi uses. In the image below, Finn has labeled the virus with a protein that surrounds a core of iron and oxygen (basically rust). The rust (dark circles in the image) that surrounds the virus (white blobs in the circle) lets us view the virus in sharper detail.
Studying RNA synthesis in cultures of mice cells and tissues, Adrian Salic of the Harvard Medical School has prepared an alkyne-modified uridine compound. (Uridine is used in RNA but not in DNA.) The image shown below shows a green DNA stain alongside their red RNA stain. Importantly, the technique that they described in this study enabled the observation of RNA synthesis in cells and tissues from mice.
These bio-orthogonal techniques have really produced some stunning results and captivating images. (Terrific reviews covering these techniques in scientific journals can be found here and here). But, there are several shortfalls that still need to be overcome. First, the labeling reactions are slow. They are slower than the time it takes for biochemical reactions to happen in the cell. Ideally, the reactions would be just as fast if not faster. Doing so would allow us to observe not only where molecules are in a cell, but accurately know how these molecules react and are shuttled around. Second, some types of bio-orthogonal reactions require copper ions with a charge of +1. These ions are required to make the reactions go quickly, but they are also toxic to cells.
Future of Bio-orthogonal Chemistry
As I mentioned earlier, these new chemical tools really give scientists entirely new landscapes upon which they can work. And, it’s fun to speculate? What would you do if you had these tools at hand? I would try to figure out a way to watch what the cell does with its junk proteins (proteins that have become deformed like, for instance, the proteins involved with Alzheimer’s and Parkinson’s diseases). How do normal cells treat junk proteins? Where do most unfolded proteins form inside of the cell? Where are they taken after the cell figures out that these proteins are no good? How long does this process take? How are these processes different in unhealthy cells?
My brain is spinning a little bit trying to figure out all of the molecules I would need to control to be able to do this study.
I think I need to sit down.
Maybe I should go have a bowl of vegetable soup … with salt.
Image Credits: Image 1: Matthew Hartings, Image 2: Matthew Hartings, Image 3: Roger Tsien, Image 4: Matthew Hartings, Image 5: Image of embryo was taken from this reference cited in post and modified by Matthew Hartings, Image 6: Image of viruses from this reference cited in the post and modified by Matthew Hartings, Image 7: Image of mouse tissue taken from this reference cited in the post and modified by Matthew Hartings.
Author Bio: Matthew Hartings is an assistant professor of chemistry at American University where specializes in studying how metals and biological molecules interact. He writes about how chemistry and everyday life mix and mingle (most notably in cooking) for his blog ScienceGeist. Importantly, though, he’s a very happy husband and a father of three wonderful children. You can find Matt on Twitter at @sciencegeist.
The views expressed are those of the author and are not necessarily those of Scientific American.