'DNA origami' is one step in hunt for advances in medicine, science

Host Amber Smith: Upstate Medical University in Syracuse, New York, invites you to be The Informed Patient, with the podcast that features experts from Central New York's only academic medical center. I'm your host, Amber Smith.

A professor of biochemistry and molecular biology at Upstate recently received a $1.5 million grant to combine two different fields of biomedical engineering, and Dr. Stewart Loh is here to discuss his work.

Welcome to "The Informed Patient," Dr. Loh.

Stewart Loh, PhD: Thanks, Amber. Great to be here.

Host Amber Smith: Now, I've seen proteins described as complex molecules that do most of the work in cells. Is that a good description?

Stewart Loh, PhD: That's absolutely a good description. They do everything in the cell that you could possibly think of, that scientists can think of.

They do a lot of things that we haven't even thought of. They literally do the heavy lifting. If you're picking up your coffee cup, that's protein molecules crawling along other protein molecules. So they really do everything that you would consider responsible for life.

And they're tiny little machines. They're nanomachines that do their jobs extraordinarily well. They can do it better than we as humans can. We can do some things that proteins and enzymes do, but not nearly as well as the proteins themselves.

Host Amber Smith: Your lab at Upstate for the past 20 years has been focused on engineering proteins. Can you explain what that involves?

Stewart Loh, PhD: Protein engineering: It's kind of a broad term. It generally means taking a protein and changing its sequence or changing the way it's modified to improve its properties, to make it more useful, to increase its shelf life, or to make it work under more harsh conditions, or to simply make more of it so that it can be used for biomedical or other purposes.

For example, protein biologicals, drugs that are given to say, cancer patients, are typically proteins, monoclonal antibodies, for example, that can attack and treat tumors. So that's a common type of protein engineering.

The type of protein engineering we do is a little bit different. We combine two or more proteins together and try to develop ways to do that such that those two functions can be combined and integrated, so you have kind of an added benefit, something that wasn't present initially in the natural proteins.

Host Amber Smith: Well, let me ask you, do enzymes play a role in this?

Stewart Loh, PhD: Yes. Enzymes are, I think, some of the most interesting proteins. They are the machines that convert something to something else.

The non-enzymatic proteins can be structural, but enzymes can transform things, which is a very useful property, so we mainly work with enzymes. Those are the molecules that we work on in the NIH (National Institutes of Health) grant that you mentioned.

Host Amber Smith: What do enzymes do, because I've always thought of them as breaking things down?

Stewart Loh, PhD: They break things down, but they also synthesize things, and they create light, which is one of the class of enzymes that we're pursuing. They will literally just generate light out of darkness. And that is a great, very kind of neat, property, but it can be extremely useful if you convert that into a molecular switch in which it will only shine light under conditions that you specify.

As I mentioned, we fuse that to another protein, so when the other protein encounters a molecule that it recognizes, then it tells the other protein to start shining light. There's many types of enzymes, and those are just a very few examples of what enzymes are capable of doing.

Host Amber Smith: The grant you were awarded will have you combine protein engineering with DNA engineering. So how will that work?

Stewart Loh, PhD: Protein engineering and DNA engineering have generally been done by two separate kinds of scientific groups, and they haven't really talked to each other too much in the past, so we're trying to change that.

DNA, as you know, is kind of the blueprint for how to make proteins, so it tells the body what to make.

And DNA engineering, typically, most people would think of that as genetic engineering in which you change the DNA sequence. And the goal of that is to change the protein that is encoded by the DNA, to have that protein either present or not or whatever.

The way we approach DNA engineering is a little different. DNA is also a molecule. It's much less complex than a protein, and it can't do all these heavy lifting and other crazy things that proteins can do, for the most part. But DNA is capable of adopting kind of simple structures. It's often referred to, actually, as DNA origami (after the Japanese paper-folding art).

So, if you think of DNA as a blueprint, you can make machines from the blueprint, but you can actually sort of do things with the blueprint itself. You can fold it into these very simple structures, which nevertheless are capable of doing a few limited things. So that's how we integrate DNA engineering with protein engineering, is we develop ways to make simple structures out of DNA, and then these integrate, they will physically combine with our protein switches to get that sort of diversity of enzyme function and couple it to these very simple things that DNA can do. And if you combine those two things, then you have a truly potentially large number of amazing things that you could really do.

Host Amber Smith: Can you share some examples of medically important products that have been produced through DNA or genetic engineering?

Stewart Loh, PhD: Sure. The SARS-CoV-2, or the COVID, vaccine is an example of the latest generation of products or biomedical technology that's generated from DNA. These are RNA vaccines, so the vaccines contain RNA, which was made directly from DNA, and so that has really facilitated vaccine technology tremendously. So there's examples of DNA technology all around us.

There's not many examples of how protein and DNA engineering can be combined to generate something useful, but there is one that really stands out, and that's the CRISPR-Cas9 gene editing technology.

And it's hard to overestimate the importance of this single technology in biology. So I think we're already at the point where there's sort of the pre-CRISPR era and the post-CRISPR era. It's been really transformative, and that kind of revolution has been enabled by a single protein DNA molecular switch.

And that protein is the CRISPR-associated enzymes, and the DNA is a synthetic RNA molecule.

And what happens is, when that enzyme binds that RNA, then it activates that enzyme. That enzyme does one thing, and that one thing is to cut DNA, and that results in this gene editing technology.

So, what we envisioned as part of this project is, what if we could do that sort of thing to other enzymes? If this single switch can result in this sort of technology, imagine what could be done if we can activate other enzymes also with a similar event.

Host Amber Smith: This is Upstate's "The Informed Patient" podcast. I'm your host, Amber Smith, talking with Dr. Stewart Loh.

He's a professor of biochemistry and molecular biology at Upstate, and we're talking about the grant he was awarded to combine protein engineering and DNA engineering in a unique way.

What is your goal in combining protein engineering and DNA engineering? Do you have something that you hope to create or learn?

Stewart Loh, PhD: Yeah. We sort of have a grand goal, then we have practical goals. And the practical goal is, we want to be able to develop biosensors for detecting specific things in cells. For viral infections, for example, we want to be able to manipulate cells. For example, we want to be able to kill cells that have a certain type of DNA in them, like a pathogen (disease-causing micro-organism) DNA, a viral DNA, a cancer DNA.

So those are what we want. Those are the kind of the short-term goals that we think we can achieve within the time scale of this grant.

The bigger perspective is how to kind of open up the entire proteome of cells (all the cells' proteins), and not only animals, but plants, bacteria. Make those proteins controllable, and who knows what can be done after that?

We're looking at basic technology, at basic science, and we're also looking at those specific, medically related applications.

Host Amber Smith: Is there a name when you combine protein and DNA engineering? Does that have a name of its own?

Stewart Loh, PhD: Not really. As I mentioned, there's not too many people that are doing this, so there hasn't been kind of a nickname developed for it yet.

Host Amber Smith: Well, have other scientists successfully combined protein and DNA engineering?

Stewart Loh, PhD: Some, yeah. There's not a lot. I think we're one of the relatively few labs in the country that actively try to develop mechanisms for how to couple these two different technologies. Not just sort of conceptually, but physically: how to take a DNA molecule and have it interact with a protein switch and have that interaction turn the enzyme or protein on and off again. There's not too many labs doing that.

Host Amber Smith: Can we fast-forward years or decades? I don't know how long this will take, but how would some of what you're doing ultimately be able to help, let's say, transplant patients?

Stewart Loh, PhD: Right. We hope to have some results on that not in the 10-year time scale, but more like the several-year time scale. And what's really needed in not only transplant patients, but sort of a general medical goal, is to be able to rid the body of infected cells. And the particular target we're going for in this grant is cytomegalovirus (called CMV for short).

But you can also think of this as HIV or hepatitis or any sort of virus that persists in the body that you want to get rid of. Five years ago, I would say there's no technology for doing this, but now we're starting to see some, but it's still a huge need.

What's difficult about that, with CMV virus and others, as well, is the virus kind of goes latent in your body. And all the drugs that are being developed to treat viruses will kill actively reproducing viruses. But these generally don't, so what we hope to achieve with this grant with CMV, in transplant patients, is that many of us are infected with CMV already. We just don't know it. That's generally OK, but in the case of a transplant, then transplant patients are typically immunocompromised or given immunosuppressants, and in that case, the CMV disease flares up, and that can be a fatal thing. This is a very serious problem.

I mentioned before that we've already made a protein switch that turns on. It will generate light in response to something, and that response to something is, if that molecule senses there is CMV viral DNA present. So, we already have a sensor that's going to say we can get samples from a patient, and we hope to show this very soon, and we'll be able to say, is this person infected with CMV or not?

And we can then look at transplant tissue and say, is this infected with CMV or not? And that's important.

More important than that is, we want to be able to rid the body of these disease cells in the first place. So, we're applying our technology not only to proteins that generate light, but to proteins that will actively kill a cell if it's turned on.

So the challenge then is to make that molecule only come on when it sees CMV virus. So that's what we hope to be able to test in the coming years as this grant goes on.

Host Amber Smith: So what you learn by studying cytomegalovirus, you might be able to apply to other viruses, too, in the future?

Stewart Loh, PhD: Absolutely. That is a very straightforward thing because our molecules are very modular, so we just have the same protein. And I have to tell you that engineering proteins is hard. It's very difficult. It takes a long time. It takes many graduate students doing this for years to be able to get a successfully working protein switch.

The DNA part, on the other hand, is pretty easy. That can be done just by a single student on their laptop computer. You can design a DNA to do what, basically, what we want to do. So that part's easy. The whole strategy is, we already have in our hands these proteins that work as very nice switches, and then all you have to do is just plug in different DNAs that interact with different viruses, with different viral DNAs, and you have the same effect.

So I will say that it is a very straightforward thing to translate this from CMV to HIV to SARS-CoV-2.

We already have a working SARS-CoV-2 biosensor, and it is simply a matter of going through the DNA toolbox, which is a very simple thing to do, and making just different simple DNA origami-type structures, which can be done by one person on their laptop. just designing these, on their computer. And then the result that we get with SARS-CoV-2 and CMV, we think we can get that same effect for any one of a number of different viruses or bacteria or fungi or whatever pathogen is out there that you want to either detect or treat.

Host Amber Smith: Well, Dr. Loh, your work sounds so exciting. I appreciate you making time to tell us about it.

Stewart Loh, PhD: Thanks very much. I enjoyed it.

Host Amber Smith: My guest has been Upstate professor of biochemistry and molecular biology Dr. Stewart Loh.

"The Informed Patient" is a podcast covering health, science and medicine, brought to you by Upstate Medical University in Syracuse, New York, and produced by Jim Howe.

Find our archive of previous episodes at upstate.edu/i nformed.

This is your host, Amber Smith, thanking you for listening.