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Magnet-Controlled Medicines — Andrew York & Maria Ingaramo · The New Biology

• ANDREW: This is like a once-in-a-lifetime thing. This is the coolest science I've ever been associated with. Let's take a big stinking swing at it. Scientists are professional beggars, so a means of doing something that matters so much that we might be decoupled from the begging cycle — we might become the people that get to decide what science happens. ANDREW: We had a lot of discussions about, "Is it real?" The discussions about "Is it real?" stopped when we started showing people this figure. It is a photography time-lapse of a plate of *E. coli*. Maria has taken pictures of the plate in the fluorescence channel while I'm waving a magnet around underneath the plate. There's this obvious, enormous effect. Prior to showing this picture to people, they want to debate with me whether or not magnetic effects are real. After I show them this picture, the discussion turns to practical details of how to make this into a technology. No further questions on veracity. --- Introduction HOST [00:54]: Today's podcast is with Maria Ingaramo and Andrew York, who are two pioneers in the field of magnetogenetics — which essentially has this amazing promise of, what if we could control molecules inside the body using magnets placed outside the body? Both of them are affiliated with a company here in San Francisco called Nonfiction Laboratories. The thing I really want to get at in this conversation is what it will actually take to get magnetically responsive molecules into the clinic, and what are the bottlenecks to achieving that. HOST: So, Maria and Andrew, welcome. MARIA: Thanks for having us. ANDREW: Great to be here. What magnetogenetics could mean for medicine HOST: My first question is: if Nonfiction Labs were to invent a way to reliably engineer proteins to be responsive to magnetic fields — such that you could take this magnetically responsive module and attach it to anything, not just fluorescent proteins but antibodies and other types of molecules — what would that actually mean for medicine? How might it change the way we treat diseases? MARIA: That's a very good question. I think the dream of being able to control the activity of a drug in space, without taking other cues, would be fantastic. The example you gave with antibodies — the idea of having cancer drugs, which bring with them lots of side effects, and being able to minimize those by activating the drug just where you want it: that's something we dream we can make possible. Also, many drugs today are failing because of side effects and toxicity. Being able to rescue a lot of those would be valuable, because drug development is expensive. It would be nice to make drugs that currently aren't working — because the patient cannot tolerate the side effects — into things that work. HOST: So it's not about just creating new types of drugs. It's about: can we take drugs that failed clinically because of toxicity, make them magnetically responsive, and then somehow make the drug only active near a tumor, for example, but not toxic elsewhere? MARIA: That is exactly the dream of Nonfiction Labs. It's what we're working towards. ANDREW: The cancer drugs are the thing that occurs to us first. It's the goal that's quickest to explain. If I'm in an elevator with somebody, I lead with cancer drugs because everyone can imagine, "I'd want more medicine on the tumor, less side effects everywhere else." The other applications might take a little longer to explain. If you get an organ transplant: would you like the immune system suppressed in your whole body, or just near the organ? If you take a drug that has therapeutic activity through the whole body but damages one organ in particular — like if there's a drug that damages your heart, or your liver — wouldn't it be cool if we could take the edge off the drug's potency just in the vulnerable organ and leave it potent everywhere else? Not that we're working on small molecules or painkillers, but wouldn't it be cool if Advil only worked in your head? I'd take more Advil if it didn't damage the rest of me. HOST: Do you have a sense of how many organ transplants fail because of — I know about xenotransplants, like there's not a great track record of long-lived xenotransplants even when we take organs from humanized pigs. But regular transplants, I guess my biased sense would be that they're quite successful. ANDREW: And this is a fun one. So, taking successful therapies and just reducing the nasty side effects — whatever level of immunosuppression you require for any given transplantation, wouldn't it be nice to have less? You see what I'm getting at. Whatever side effects you're getting from your cancer drug — what if you got the same efficacy, less side effects? But I think it's the rescue Maria was talking about that's where the real chance to actually save some lives is: drugs that really could cure the disease, they just do something else that's completely unacceptable. If we could rescue drugs out of the trash bin — We're basic scientists. A lot of where we're coming from is a capability: what can we do with it? But the dream that we might actually save some lives — I'd like to do that before I die. Magnets vs. light vs. ultrasound HOST: Of course, you're both working on magnetogenetics — I'm going to ask you about the name later, because I'm not sure it always makes sense. So you're building magnetically responsive molecules. But there's this bigger movement where people are making molecules that respond to ultrasound. Optogenetics is the classic that's been around for 20 years. So we can use light, sound, magnetism — people are using electricity to control cells and tissues. What are the advantages of magnetogenetics, or magnets, that we don't get with light or sound waves? ANDREW: Okay, I'll start with the easy one. As many people know — but I'll pretend somebody needs to hear it — there is a powerful, mature toolbox of optogenetic proteins (I think I agree with you about the name when we get to that). Many cellular proteins have some clearly defined function; a scientist has gone to the trouble of engineering a modified version with a light-sensitive domain attached. When you shine light on the protein, you can turn the function up or down, on or off. Incredible tools for research — such a powerful tool for dissecting the function of a protein. So optogenetics works in things that are transparent. Humans are opaque. If there was a disease where turning a protein on and off — and many drugs are made of proteins — turning a drug on and off in the human body in space and time was desirable, and the location in question was optically accessible (the back of your eye, or superficial in your skin), light can penetrate. Different people's skin has different levels of opacity, so you have to carefully account for that. HOST: People have built insulin sensors using light, right? For shallow applications. ANDREW: Exactly. For shallow applications, there's room for optogenetics to make powerful biotechnologies. But how light penetrates through tissue is a little unpredictable, a little out of control. Depending on whose tissue, how far it'll penetrate. That's the beautiful feature of magnets: they go through flesh like it's not there. So that's the obvious one. The ultrasound one is a little more nuanced. HOST: That's what I was going to ask next. Ultrasound has lots of approved diagnostics, people use it for all kinds of stuff, it's very safe. Does ultrasound have higher resolution than magnets? What are the trade-offs? MARIA: I think ultrasound is amazing. By all means, let's move the field forward in all directions. There are — this is not impossible to overcome — but at the moment the main problem with ultrasound, compared to our technology, is that we have a protein-based domain we can use to modulate the function of anything we want. In ultrasound, you have proteins that can respond to heat, for example, but that limits what you can couple to. There's not a direct way to connect this domain to control the function of the protein it's attached to. There's beautiful work controlling transcription through heat-sensitive proteins, but all your cells respond to heat one way or another, while magnets are completely orthogonal. The other way to couple it to the biology is by modulating ion channels, which is amazing for things like the brain, but that limits the functions you can control. With our system, we can take any protein — a structural protein, an enzyme — and change binding, change catalysis. Another advantage: with focused ultrasound, you need to focus it, so covering a large area becomes a limiting factor. With magnets, we cannot focus magnetic fields, but we have a lot of control over the areas. We can do big areas, and we can also do areas of no magnets that are small. ANDREW: This is cute. Just a little physics nerdery: Earnshaw's theorem says there is no local maximum of a static magnetic field. That's one of its consequences. The cool part is what it doesn't say — there's no local minimum. So we can't focus a static magnetic field to make a single focus point the same way you could with ultrasound. Seems like a weakness, but — both Adam Cohen and Hunter Davis taught us this — you can put two north poles, north-to-north, and by symmetry right in the middle, no field. Everything cancels. As you move away from that point in any direction, you're jacketed by a non-zero region of field. So you can have a point null surrounded by a volume of non-null. You can scan that null through a sample to 3D-define — to 3D-print — the region where you want your protein to function, if your protein functions in the absence of field. So, much like focused ultrasound, we can pick out a point, we can scan around the point to hit a region, to paint a region. Unlike focused ultrasound, Maria's technique can also just flood the area. If you want to activate a liter, a cubic inch, a cubic centimeter — you can do that with a magnet. I had dinner with Mikhail Shapiro a couple months ago — fascinating dude, really enjoyed it — and it wasn't like we were debating which technology was better. We were both nerds excited about what we could do with them. A static DC magnet does not require a battery. A static DC magnet is extremely cheap. Whereas if you needed a therapy that delivered focused ultrasound to one part of your tissue chronically for a week straight, I might prefer the magnet in that case. MARIA: I'd also like to point out that the magnetic field strengths we're using are actually quite convenient for therapeutics, because we're in the millitesla range. A big magnet can reach through me with no problem. We do not need MRI levels of magnetic field, which would be a little more complicated. HOST: What is millitesla range for context? ANDREW: A strong handheld neodymium magnet can have a surface field of hundreds of millitesla. At a standoff distance, you can hit millitesla several inches from a decent magnet. If you're trying to hit the whole torso, yeah, you'd be wearing a fairly elaborate device, but you wouldn't be sitting in an instrument. There would be no liquid helium involved, no superconductors. Maybe try to stay home while you're wearing one of the big boys. But for more localized therapies, the same magnet that holds your laptop shut — tape one of those to your chest, and that would define a region in the vicinity of the magnet. MARIA: But it is also good that it is strong enough that you cannot activate it without really bringing a magnet close. Pretty much the requirements would be: if you're wearing a pacemaker, you've seen all the signs — "Do not enter, be careful." It would be the same type of precaution. HOST: Just to build some mechanistic intuition for mechanosensitive proteins, or ultrasound- and magnetically-controlled proteins: the way ultrasound enters the body is via pressure waves passing through tissue. So the proteins you can open with that are usually mechanosensitive — ion channels that open when they're tugged with pressure waves — or heat-sensitive. Focused ultrasound also heats locally, so people are making thermogenetic systems: you can put nanoparticles into the body carrying a payload, and they release the payload when stimulated with heat. So, just so I understand your argument: that sort of mechanism of what you can build at the molecular level to take advantage of local heating and pressure waves is more limiting than having a completely orthogonal channel, like a magnetically responsive domain where we could tune anything. That's what you're saying — for the sonogenetic stuff? ANDREW: Something I want to emphasize: this is to my knowledge. We're not specialists in sonogenetics, so if I'm speaking out of turn, I trust folks like Mikhail to correct me. But to my knowledge, for example, if you're doing a heating-based sonogenetic perturbation — at 39 degrees Celsius, no effect; at 41 degrees Celsius, the transcription factor activates, your gene makes a lot of the protein you want; at 43 degrees Celsius, you damage the tissue, you can cook the cells. A plus-or-minus a few degrees range is a big range, but if your tissue — how well is blood circulating through that area? — if you get that a little wrong and you cook instead of activate (or do nothing), that is not a bio-orthogonal perturbation. That is a perturbation which is nuanced and related to the system in question. Whereas the cool thing about magnets is they go through you like you're not there. Predicting what perturbation you're delivering at what position in the flesh is just an easier problem. To emphasize the point Maria made earlier, the sonogenetic systems we're familiar with are a heat-shock-protein for transcription changes in response to temperature, which is really cool. But it's just transcription, and anything downstream of transcription is a slow process that has a lot of momentum to it. It's not something you turn on and off in a second. The other one we're familiar with is the channels that open in response to mechanical perturbations. That's really stinking cool — being able to open a channel to influence whether or not a nerve is going to fire — but again, it's limited to a channel. The technology Maria has, when she's saying she has this generic toolbox — there are a couple of different paths, but one of the most exciting is she has a luminescent protein. Think like a firefly — a protein that just glows from chemical energy — that turns down with a magnetic field. (At the moment, we hope eventually to have one that turns off; for now it turns down.) Take any optogenetic protein you're interested in — the entire optogenetic toolbox, which includes enzymes, signaling molecules, cytoskeletal binding proteins that affect cellular motility, weird niche things — a huge variety. All of them that are optically responsive could be hooked, this is our vision, to this one protein she's engineered. So essentially, 3D remote control of where blue light is expressed in the body. HOST: So you're using a magnet to turn on a light, which then turns on an optogenetic protein. It's kind of like two layers of logic. ANDREW: And there's other ways to do it, but that's a really promising one. HOST: So you're saying, even if you couldn't make a directly responsive, magnetically controlled protein, there are ways to use magnetic control to turn on optogenetic proteins inside the body. ANDREW: Which we can. The nanobodies that we've made are themselves responsive to magnetic fields, but they still need light. So one way or another, you put the light in, with either a luciferase that doesn't respond to magnetic fields or a luciferase that responds to magnetic fields — and you get double the effect. You can stack the gates to get sharper control. If you have a 50% modulation on one and a 50% modulation on the other, put them together. --- On the name "magnetogenetics" HOST: We alluded earlier to this idea of the name "magnetogenetics." I'm assuming the name came about historically because optogenetics is genetically encoded, optically responsive proteins. But of course, in the context of magnetogenetics, the thing that makes it so interesting is that the things you're delivering don't have to be genetically encoded. So is a better name just "force-controlled biology"? Why call it magnetogenetics? MARIA: Right now it means something on its own that has diverged from where it originated. Back in the day, the first opto- demonstrations were with channels. And it's very hard to deliver a channel and have it do its life. HOST: You can't inject a channelrhodopsin and hope it ends up in the right membrane. MARIA: Exactly. You were required to genetically modify the organism in order to express the channel, for it to respond to light. Since then, there was gorgeous work by other groups where they looked to plants and found that plants have these domains that respond to light, and they're very ubiquitous. Plants, thanks to the response of those domains, can grow towards light and control their metabolism — they're involved in a variety of processes. The idea of taking those domains and, in a sense, getting them closer or attached to other proteins to control their function — it's very analogous to what we're calling today magnetogenetics. But the name for that was also optogenetics, even though that protein doesn't necessarily have to be genetically encoded. They could be injected. HOST: And what are those plant domains? MARIA: Aha — those are, in a sense, the exact domain I'm using for engineering the magnetic-field response domain. So we started with these LOV domains — L-O-V, light-oxygen-voltage. They are used to build anything that is not a channel (although you can also make optogenetic channels by putting this domain in channels). But everything else that has been built in optogenetics has been built with LOV domains. HOST: A lot of them. MARIA: Yes. When we found that we could engineer magnetic-field response in the LOV domain — even if we saw it on other proteins — it seemed the right choice, because there's already so much work that we can build on from optogenetics. ANDREW: I was not optimistic when she started. Her initial discoveries were in jellyfish proteins, coral proteins, which are not the basis of optogenetic tools — optically responsive protein function. (Totally agree about the nomenclature, by the way.) So when she found that the LOV domain was also magnetoresponsive — which I didn't expect, I was not optimistic about that — that was incredibly exciting, because it went from something that's scientifically fascinating and maybe could be adapted into a technology, into something which is obviously central to a technology we have to pursue. --- Origins: birds, cryptochromes, and a skeptical reading of the field HOST: Before we go into the story of the discovery and the founding of Nonfiction Labs, I want to go way back in time to the origins of magnetic biology. My understanding is that the origins of the field were birds — basically zoologists or naturalists who would watch the migrations of birds and say, "How do birds know where to fly?" The Arctic tern flies thousands of miles every year. There are birds that fly over the Himalayas, where airplanes fly. People were studying this and saying, "What's going on?" I'm wondering: have you studied the history of this field? What have you discovered about the origin? ANDREW: Not as much as you might think. When it comes to really knowing anything about animal behaviors and certainly animal magneto-response, I'd say we're humble civilians. MARIA: That is true. However, I do have to say that I'm very thankful for all the work that has gone on characterizing mechanisms and the response of those proteins, especially because they are such tiny responses. Whether the cryptochromes are the protein that is thought to be — HOST: Yeah, tell me about cryptochromes. When did people start to — where did cryptochromes come from? Were people, because they're in the eyes of the birds, right — so people were dissecting bird eyes? ANDREW: Four years prior to today, neither one of us knew cryptochromes from Adam. Not our field, not our specialty. We have read a certain amount of literature — enough to affirm some opinions and some suspicions, both about the technical realities of the field and about the social factors that led to those technical realities. But we can tell you, people have been reading about cryptochromes to some degree for a couple of years — that's our level of expertise on cryptochromes. So with those error bars, hit it. MARIA: Thank you. I do appreciate all the work that has gone in, because it kind of provides a framework through which to look at our own protein. There are a lot of studies on electron transfer and effects of magnetic fields on that that come from that literature. I sympathize with them because effects are very, very small. Whether it is the protein that is responsible for birds sensing magnetic fields, I don't know, given the very small effect. HOST: When you say small effect, what does that mean? How are people studying cryptochrome proteins, and what is the response being measured? MARIA: Usually in terms of biochemistry — I'm a biochemist, I like to see responses that we can repeat in a tube — one of the best and most-used assays is transient-state absorption. It's not necessarily just a change in absorption. I wish it was just a change in absorption. It's an absorption that happens for a very short period of time after excitation. So you're looking for a small change. You shoot with a pulse of light, you wait a certain amount of time — could be femtoseconds, could be milliseconds, could be minutes — you shoot with a second pulse of light that might be a different color, you check how much of that second pulse of light got absorbed and how did it change if I turned the first pulse of light on and off. ANDREW: Transient absorption. I would call it a somewhat exotic measurement that does not necessarily measure a physiologically relevant quantity. It's a great measurement. It'll tell you a lot about a protein. It will not establish that a protein necessarily has a given biological function. MARIA: When you see a 40% change, that's not even a 40% change on the bulk of the protein. It's a 40% change of that population that you had excited for a very short period of time. HOST: But what does that have to do with magnetic fields? ANDREW: It's the only signal in many systems that didn't totally ignore magnetic fields. So there was the hope — with all of the error bars I gave about my expertise — there was the hope, the observation, the belief that behavioral assays on birds suggested, how can the bird sense magnetic fields? It would be nice if we could connect that to a root mechanism: "This protein changes its behavior in this way." A really beautiful assay would be like, "Ah, when I turn the field on and off, the phosphorylation of this protein changes by 50% in a super quantifiable way." I am not familiar with work that makes claims like that. We are familiar with work that says: when you turn the magnetic field on and off in a pretty big way, for a cryptochrome which is very cold, very acidic, in a weird reducing chemical environment that is not obviously physiologically relevant, you will get a tiny change in a tiny effect in an exotic measurement. That is the work I'm familiar with that shows cryptochrome does not entirely ignore magnetic fields. HOST: Why do they study it in strange buffers? ANDREW: When you read a paper, the paper doesn't come right out and say it, but my suspicion — slash opinion — if you read between the lines: if it had worked at body temperature, if it had worked at physiological pH, if it had worked in physiological chemical conditions, that's what they would have published. They don't say they tried it and they failed, but you don't do that stuff if it worked on easy mode. So my personal suspicion about a lot of the magneto-response field is that, whether the effects are real or not real, it's really hard to study. And the people doing this work are heroic and working really hard. HOST: It feels like there's so many people who have studied this in such a rich history. Why would so many people get sucked into that if the effect is so minor — and if it doesn't even seem like that's the real mechanism? ANDREW: So, before we knew anything about this, before we touched this in any way, I was asked, at a company we both worked at, to give an entertaining talk for everybody at the company — not just the scientists, the purchasing department, the security staff. Just tell a story that anybody can appreciate. I was handed an article from *Scientific American* that said, "This is how birds navigate." I was expected to basically just read the article for a five-minute talk. But I think one of our superpowers is — for better or worse — we don't have a lot of respect for authority. So rather than simply recite the article as gospel truth, I figured I should at least read the source article. The source article was, I think, a *Nature* paper or a *Science* paper — in a reputable journal — that I was utterly unconvinced by. I thought, "Surely this isn't the basis for this belief. Let me go look at their foundations." So I go to their list of citations and I go through looking for the smoking gun. It was like those dreams where you're trying to throw a punch and your arms don't work. You get to this layer of citations, I was like, "Well, none of these is obviously true. Let's go to their layer of citations." And you go all the way down to the bottom and find a single solid thing. Now, perhaps because I'm a bad reader, perhaps because I'm not very perceptive, or perhaps because I didn't find the right papers — we found one paper that was absolutely, definitely true. It had nothing to do with biology, nothing to do with biochemistry. It was a synthetic organic fluorophore. It was striking — seeing the strongest magneto-response that's ever been published is 80%, clearly visible by eye, when a human hand just enters the frame of the video and holds a magnet up to a tube with a synthetic organic fluorophore, and it gets obviously about twice as bright. The second strongest effect is like 0.01% in a super exotic measurement with no obvious biological relevance. Just the staggering gap between the two claims was striking. HOST: So the cryptochrome stuff wasn't convincing, but you found stuff in organic chemistry that *was* convincing — which made you believe, presumably, "Oh, molecules in some contexts can be magnetically responsive." ANDREW: And luckily that context was fluorescence. And luckily Maria is a biochemist protein engineer who engineers biologically fluorescent molecules. --- Adam Cohen's molecule HOST: Before we go to that — in your leap to say, "Oh, I wonder if this shows up in proteins" — what was the actual experiment that Adam Cohen did at Harvard that had this big effect? What was the experimental setup? MARIA: So Adam made a molecule where he engineered, as well as he could, a molecule that would do electron transfer. He assumed that you could potentially control that with magnetic fields, and it worked. The only problem with that molecule is that it only works in a very specific solvent, and it's a little finicky to maintain the magnetic-field effect, but it is very, very large. He was actually the first one to use that molecule and to make that magnetic null and be able to image through — like, a piece of glass that has the molecule in it. HOST: What do you mean by this null imaging through glass? MARIA: If we could have a molecule — let's say it's fluorescent — if you added a magnetic field, it would turn off. We know that if you put two magnets that are north-to-north, or south-to-south, then in the middle there's going to be no magnetic field. And that determines where your signal can come out from. So if you have, let's say, this fluorophore inside of a mouse, when the light tries to come out of the mouse, it gets distorted because of scattering. ANDREW: I'll show this demo. You've probably all seen this — light is quite good at passing through tissue. It is quite bad at passing through tissue in straight lines. So you know if light is generated inside the tissue, it'll come out, but it'll come out in a random direction at a random position. So the innovation in Adam's case was: if you can also further define where it must have come from — because it will only glow at the magnetic null — here's this point where, if any light came out, it must have come from here. So you just measure how much light comes out, and then you scan that null around. You can build up a map of where your sources were, where your emitters were, without any light ever traveling ballistically, without any light traveling in straight lines, which is normally what we require to see through a transparent object. Light has to go in straight lines. MARIA: Exactly. So, in a sense, a lot of the fluorophores we were developing — you could think of them as a way to improve imaging deep in human tissue. HOST: The chemicals would not go in the body, would they? MARIA: The ones that he synthesized, no. But the ones that we could make without protein could. ANDREW: I remember very vividly — whenever Maria's about to embark in lab work, I try to spend a little time on the phone first. Maybe somebody knows something that could save us a little time. So we called up Adam. We love Adam. And we told him, "Hey, your work's incredible. Why aren't you still working on this?" And he described the buffer to us, he described the conditions, he said how difficult it was to get the effect robust and strong. And Maria says, "Oh, do you think it could work in fluorescent proteins?" He says, "If you can find anything where the fluorescence response to magnetic fields is in liquid water at body temperature, you call me." And that's what happened. She found it. We called him and he immediately jumped right back in. He says, "Awesome." And it's just really beautiful work, obviously inspired by him. Then we got to play our part and pass the ball back. I love that guy. It's nice working with people that make you glad you built on their work and glad they built on your work. --- The Markus Meister critique HOST: So you were giving a talk at Calico. Did you also work there, Maria? MARIA: Yes. So we've been working together for a very long time. HOST: What's interesting is you had arrived at this thing of, like, "These papers seem fake." ANDREW: That was my opinion. Yeah. HOST: Well, what's interesting, of course, is that there's a famous 2016 paper from Markus Meister at Caltech where he quantifies the same thing — he says most of the magnetic effects reported in papers cannot be true. For example, there was a paper where they attached ferritin to a channel and then used a magnet to open the channel by pulling on the ferritin. And Markus Meister was like, "You would need 100,000 times more iron atoms to exert enough force to open that channel." Thermodynamically impossible. So people were already kind of criticizing this field when you came into it. It's just kind of surprising to me that you were like, "Oh, let's try to find this effect in biology" when Markus Meister had already said the effects in biology are kind of garbage. ANDREW: This is a good one. So, dead on — my default position was, I would not expect to find a magnetic response in a biological system, based on both what I know by physics and what I saw in the literature. And I know you meant this in a nuanced way, but the word "fake" — I do think a lot of magnetogenetic effects were not real. I didn't get the impression the paper was written in bad faith. I got the impression they're attempting a difficult task and failing in a very human, reasonable way. I know that's what you meant. The work from Adam — the effect was so huge, and it was a system where, sure, the chemistry is different, the context is different, but the photophysics of fluorescence is fairly universal. Proteins from jellyfish and corals are fluorescent; the synthetic organic fluorophore is fluorescent. So this clear demonstration that it was not physically impossible — it might not be biologically compatible, but it was definitely not a violation of thermodynamics, not a violation of energy conservation — gave us the confidence that we're not doing something which is fundamentally insane. We're just doing something I'm not super optimistic about. One of Maria's secret weapons here is the way she does science. She is incredibly good at making many bets in parallel, and she's an obligate gambler. She's a degenerate gambler. She likes to wager. When you're doing protein engineering, there's a lot of "mutate and screen" — you do a bunch of things that, yeah, no one of them should work, but just make it cheap and fast enough, and very reliably some of them will. I don't know anybody that more reliably produces value every month, despite any single thing she does being borderline crazy. So just throwing one more crazy thing in the hopper was a pretty light lift for her. She's engineering fluorescent proteins — might as well wave a magnet past them while she's doing all the other work she's doing. In hindsight, the real work wasn't protein work. The real work wasn't the biochemistry. The real work was getting really good at measuring very small effects. But the cool thing is, a lot of her existing skills — the work she was already doing — involves, in engineering, you always start with going from zero to one. So you measure some small effect. She'd done that with biosensors — make something which is sensitive to calcium, make something which is sensitive to metabolites like NAD. They start off insensitive, and your very first hit, where it goes from insensitive to sensitive, could be garbage, could be noise. The garbage costs you very little the way she does her science. The hits are priceless. So taking a tiny effect and engineering it to make a big effect is a skill she had been practicing for years. The additional labor to add a magnet to it, as crazy as it sounds, was pretty marginal. --- The actual experiment HOST: So tell me about the experiment. MARIA: I remember this story slightly differently — because it actually took me probably at least three months to get good at measuring magnetic-field effects. The first thing I did was to wave magnets at proteins and I didn't see anything. But I was pretty obsessed with finding it. So I had to master the art of measuring fluorescence changes induced with magnetic fields in the systems I had access to, which were flavins with tryptophan, or — ANDREW: Oh, that's a good point. It was well established prior to anything we did that, in addition to the system from Adam, if you just take flavin and mix it with hen egg white — MARIA: Lysozyme. ANDREW: Not a biochemist. A fairly boring protein mixed with a vitamin that's in most foods we eat. It was known that the fluorescence from that system was weakly magneto-responsive, which gave her a reference to get good at, to practice with. MARIA: We needed a positive control, and on top of it to rig something up to control the magnetic field. So it was a little bit of an investment, but I'm glad we pursued it. Once I got into the universe of being able to remotely see things that responded to magnets, then waving the magnet worked. HOST: So tell me about the experiment. You saw it in flavins with lysozyme, and you said, "I want to find a single protein that might have the same effect." Walk me through that. What was the experiment that you ran? MARIA: Oh, so I had been working with lots of fluorescent proteins before — I was doing a lot of engineering in that area. So I had lots of very interesting and unusual photophysics proteins, and I expressed them all in *E. coli* and put them on the microscope. I had a little servo motor with a magnet on a popsicle stick that was moving on top of it. So I could see the *E. coli* and the lysozyme — HOST: Wait — so you're growing cells in a dish and they're expressing a protein? MARIA: Yes. So I'm growing *E. coli* like normally, and then I just scoop some *E. coli*, put it on a slide to look in the microscope, and then on the microscope I had a little servo motor that would move the magnet in and out. HOST: And then you're just recording this to see if there's any fluctuations? MARIA: Exactly. HOST: And you have some image segmentation? MARIA: Yes, usually I draw a little square and I'm like, "Do I see any changes?" And out of that, we got — GFP, the most common fluorescent protein ever, the one that is most widely used — was wiggling a little bit. And that became super, super interesting. The problem is, I'm a biochemist, so the first thing I did was, "I want to purify this protein and I want to look at this protein without all of the extra *E. coli* junk." And the moment I do that, there's no more magnetic-field effect. And the effect that I was seeing in *E. coli* was really small, but it was not noise. It was definitely something I could — HOST: I want to ask about that, because — I'll put the chart up on the screen — there's a famous, well, niche, cult-classic chart, and the fluorescence is, let's say, 44,000 units, whatever it is, and with the magnet it's a change of like 100, which is a fraction of a percent. So most people would look at that and be like, "I don't know — it's so tiny." ANDREW: Here's the nice part. One: totally agree, everything you said is fact. The cool part about the signal — we place a sharp distinction between signals that look like *this* and signals that look like *this*. Even if it is a one-part-in-400 change, which I think is about how big it was, it was a one-part-in-400 change that was massively bigger and cleaner-looking than the noise. So we look at it and we say, "We know damn well it isn't noise." That does not mean it's real. Holy cow, did we spend a while establishing that it was something we could actually trust enough to speak aloud in public. The major anxieties were: okay, it's not noise, but it's probably heating, because we use an electromagnet. Oh — well, hence the popsicle stick. No heating from the popsicle stick. Oh, well, it's probably vibrations from the popsicle stick. Well, the electromagnet doesn't vibrate, and the signal looks the exact same if it's a permanent magnet or an electromagnet. Oh, maybe the magnet's pulling on the optics. We swap in a sample — for example, Maria said the purified protein is still fluorescent but is not magnetoresponsive. We don't see the effect. The effect follows the chemistry, not the mechanics of the setup, not the thermodynamics, not the vibration. The effect follows the chemistry of the sample. That left us feeling a lot more comfortable, a lot more confident, taking a tiny-ass signal and attributing it to the thing we wanted — which is dangerous — rather than the 10 other fake things it could easily be. HOST: True. But then you took it out of the cell and it doesn't work. And then did you start to question yourself again? MARIA: Yes. Yes. The beginnings of this project were a lot of questioning. At that time I was working with Rebecca, who was staying with me for the summer. ANDREW: From Adam Cohen's lab. She was interning with us. Lucky us. MARIA: Yes, we were very lucky, and she's a very calm, very methodical person. We saw the effect together, and she's like, "It's there. We just have to figure out how to bring it back." ANDREW: She's used to signals that are there one day, gone the next. "Why didn't I go work on a more robust signal?" — maybe consistent with the point you're making. And Rebecca said, "It was there yesterday. It'll be there tomorrow. Keep looking." MARIA: So what we did was screen a library of metabolites, because we were like, "There's something inside of *E. coli*." I knew that, because if I mushed *E. coli* and added it and mixed it with my pure protein, I would get the effect. Like a cell-free extract. And pure *E. coli* was not giving me the magnetic-field effect — which, by the way, is not what I learned now, looking back. I wasn't in the right universe to — the imaging conditions need to be very good, like your power levels, where you're looking in your spectra. They need to be right in order for you to see the effect in fluorescence. So it was pretty lucky that way. I have seen now that the autofluorescence of *E. coli* is magnetic-field dependent. You just need a lot more power than what I was using at that time. So there are actually flavins that are interacting with random proteins inside the *E. coli* cell, and they're modulating the fluorescence a little bit. ANDREW: It might be worth emphasizing — when we say you turn the field on and off and you see the effect go up and down, what we don't see is a square wave. We're not, like, "The brightness just follows the magnetic field," because the magnetic field is basically following a square wave. What we see is this curving, sawtooth shape. So to us, that usually means: we're not directly changing the brightness with the field. The light is photoswitching the sample. The light is changing the sample from one state to another, and that rate is what the magnetic field is affecting. So earlier when Maria said power has to be right — a lot of the time when we send samples to other people and they don't see a signal, the thing that goes wrong most frequently in someone else's hands is their light wasn't bright enough. Because the light has to be bright enough in the first place to drive switching between these two states to a substantial, measurable degree, and then the rate of that switching is what the magnet will turn on and off. So the exponential curve is what the magnet will modulate. When she says she had to be in the right universe — lucky for us, she happened to be using a bright enough light to drive the effect she observed. --- Directed evolution and MagLOV HOST: So after you found this protein and you said, "Okay, I'm convinced that it's real," the next thing you did was directed-evolution experiments where you said, "Can we make the effect much larger?" Tell me about that. MARIA: At that time, we had identified — the result from the screening of the metabolites was, you could restore the effect on GFP by adding a few metabolites. Flavin was one of them. We like flavin because it's cheap, easy, and there are more enzymes and genetic tools that make it nicer to work with. If you wanted to, let's say, attach a flavin on a protein forever, there are tools for that. ANDREW: Super biocompatible. MARIA: There are other metabolites — probably the ones inside *E. coli* are not necessarily flavin, they're some of the other ones — that, when brought in close contact with GFP, would make you see that magnetic-field response. But I also identified another fluorescent protein called the LOV domain, which is what we use today. And that protein, when I purified it, was still magnetoresponsive. HOST: Without flavin? MARIA: Without flavin. But the LOV domain has a cofactor, in a sense. The heart of the LOV domain is a flavin. So the LOV domain is just really good at sequestering its own flavin and having it be part of it. HOST: But to clarify — the LOV domain grabs flavin from the cell? MARIA: Yes. HOST: And are there human cell types that do not produce flavin? MARIA: We're bad at making flavin, but we're good at eating it. So it's in you, but not because you synthesized it. HOST: So if you were to put a LOV-domain therapy into a human, it would find flavin somehow? MARIA: It works in mammalian cells. It definitely would find — most likely it would find its own flavin. It's a ubiquitous vitamin used for transfer of electrons inside the mitochondria. HOST: So you did this directed-evolution experiment on the LOV domain, and you found a protein called MagLOV that has very strong magnetoresponsive effects. Do you have a sense of what those mutations actually did that makes the protein so responsive to magnetic fields? I love this — because I literally don't understand that. Does anyone understand the mechanism of what's happening here for very strong effects? ANDREW: Oh, let's lead off with: we have a lot of information about the mechanism. A lot of data, a lot of constraints, a lot of thoughts, a lot of opinions. I will also state: we know we don't really know. We would love to know. But it's the hilarious thing about directed evolution — you need to be really good at measurement. HOST: Before we go into this — how did you actually do the evolution experiment? You're mutating the gene encoding LOV, and then you're screening in your same assay with microscopy, and then you're picking cells that seem to have a strong effect? MARIA: Yes, that is pretty much it. HOST: Wow, so tedious. MARIA: I am a gambler. So every time I do it, it's like, "Yes, I got a good one!" HOST: How long did that take? MARIA: The way I have to do this is, because I want to maintain identity between a phenotype and the DNA that encodes for that protein — because I can't just screen for brightness and take whoever is brightest — I have to switch the magnet on and off and ask the same *E. coli*, "Are you responding?" So I do it in a photography system. You take pictures of a plate of *E. coli* with colonies, and they're all clonal colonies, so each one represents one variant of the protein. I make libraries that are relatively small. It just means I have every single substitution at every position, but not more than one mutation per LOV domain. HOST: So it wasn't random mutagenesis? MARIA: No. I don't usually do random mutagenesis because it tends to lead to very large libraries, which is amazing if you can screen millions of them. But because I have to take a picture and move the magnet and see what happens, it becomes a little bit of a slower process. But the data is very, very rich. It moves forward. In my experience, it's been very productive. ANDREW: She can do 40 of these in parallel. One iteration takes between one and two weeks. And it's something she has taught undergrads how to do, and it works well in the hands of undergrads. So on the one hand, it's tedious. On the other hand, taking the *Avena sativa* LOV domain straight out of the oat plant and turning it into this staggeringly magnetically responsive engineered protein — three months. Once she was good at the measurement, which took a long time, the actual engineering — snap. That's kind of a beautiful thing about directed evolution. As tedious as it sounds, I think there's a reason more people don't do it, and one of the reasons I got animated about this: Maria is one of the very few — possibly the only — people on the planet who is currently actually engineering proteins to be more immediately magnetoresponsive. It's not that hard. It's not trivial — there's an activation energy, there's a skill, there's a craft. But why aren't more people doing this? Tons of academics are downstream of her work, taking her proteins and studying them, asking cool questions. It sounds intimidating, but science is hard. Everything in science is a slog. Any wet lab — mouse work is soul-crushing. As tedious as this is, one to two weeks to make a substantial improvement that hundreds of thousands of people are going to build on and cite and be excited about? Could be worse. MARIA: They're relatively cheap experiments. I like cheap experiments because it means I can try more things at the same time. HOST: So you found a protein that had five amino-acid substitutions from the wild type, and the effect went up to like 75% responsiveness. Where were those mutations happening? Let's get at the mechanism. MARIA: Evidence that we've gathered since we started working with these domains tells us that what we're modifying is electron transfer. So we are, in a sense, controlling how long a molecule remains charged by adding the magnet to it. Based on a huge, very long history of studies on the LOV domain, you can trace some of those mutations and generally attribute them: "Yes, now I see why this appeared, and I see why I need this mutation." The first mutation you always get breaks the photoswitching cycle, right? ANDREW: Yes. MARIA: So the LOV domain has kind of a complicated photo cycle. The LOV domain takes a photon, it gets excited. Then you can either fluoresce, or you can go into a triplet state — a lower but super-long-lived energy level. And those triplet states — HOST: Now very reactive. MARIA: And in the wild-type LOV domain, that can react with a cysteine, and it reacts relatively quickly. And that initiates a huge conformational change. HOST: But to clarify — so there's this charged — it doesn't emit fluorescence, it enters a triplet state, and that makes it reactive. And then it dumps that energy onto a cysteine, which is just an electron transfer. When you say electron transfer, it's just the movement of an electron. Why would the movement of one electron completely change the shape of the protein? MARIA: That's actually quite interesting. And I don't necessarily have a pretty good explanation for it. ANDREW: Yeah, we're not specialists. We were not specialists in the LOV domain three years ago. MARIA: On how to simulate those changes and how that translates into a changing structure. ANDREW: But that's like the native protein, before she did any of the engineering. And the first mutation she finds every time she does directed evolution is: destroy that. HOST: Destroy what? ANDREW: The cysteine. The cysteine. MARIA: Yeah, it's the first mutation every time. HOST: So it sits in this charged triplet state? ANDREW: Exactly. HOST: And it can't dump its electron? ANDREW: Yeah. It probably does charge transfer to somewhere, but we suspect the charge transfer does not result in that classic change of the bond and change of the shape of the protein. We spent a while actually trying to establish whether or not the protein still changed shape in her magnetosensitive engineered versions, because it's believed to do so in the default wild-type version. HOST: But in the wild-type version, you're listing these two states — saying it can either release fluorescence or enter a triplet state. Is the triplet state exceptionally rare? Is it usually — it just gives off light? MARIA: Yes — except in LOV domains. ANDREW: Yeah, that is weirdo. LOV domains are weirdos. They have a — HOST: So this is just stochastic? MARIA: Yes. The protein is like, "Depending on local fluctuations, it might do this or it might do this." ANDREW: In GFP, the branching ratio is 100 singlets — 100 normal fluorescence events for every one triplet. In the LOV domain — MARIA: It's like 60% quantum yield. ANDREW: According to the literature. We didn't personally verify this. MARIA: So in the LOV domain, most of them — half of them — are going into the triplet state, and they're available to do this type of electron transfer that we're controlling with the magnet. ANDREW: Of all of those steps, the only one we believe is magnetically sensitive is whether or not that charge transfer occurs. That's the thing we think goes up or down as we turn the magnet on or off. HOST: Wait, so just so I understand — so you're biasing — you can engineer the proteins to bias which state it enters, so that instead of fluorescing it will mostly drive electrons to this triplet state? ANDREW: We hoped that would be magnetosensitive. We don't have evidence of that, and we don't currently believe that to be true. What seems to be the case is: if you end up in the triplet state, what you do next is magnetic-field dependent. And the cool part there is, the lifetime of the singlet is like nanoseconds; the lifetime of the triplet for the LOV domain is tens of microseconds. So way longer than fluorescence, but still pretty transient. Whether or not this charge-transfer event happens — which we believe is often an internal event, the electron goes from this spot in the protein to that spot in the protein, that's our cartoon model — whether or not that occurs depends on the magnetic field. If it occurs, the resulting charged form is super long-lived. Like instead of nanoseconds or microseconds, it's seconds to minutes, depending on which mutant of the protein. HOST: What is the magnet doing? We have a protein. It's in this triplet state. You've caught it in that time where it's like the few microseconds. You put a magnet on it. What is actually happening? MARIA: This is where we get into quantum. I can tell you, and I can repeat my understanding. When you're in the triplet state, you have different energy levels where your electrons can be, depending on their spin — which is the thing that magnetic fields modify. They perturb the spin a little bit. So when you add a magnet, you actually separate those energy levels a little bit more, so you can't go back to your singlet very easily. So you're stuck in that state for a little bit longer. So we're pretty much capturing things. ANDREW: It might be worth emphasizing: up until now, everything we're telling you, if you dig a little deeper, I can tell you, "Here's the experiments we did that convinced me this is true. Here are the supporting facts in the literature I have a lot of faith in." What Maria's describing now is the very root question: of all the processes that we think are not magnetosensitive, the one we think is magnetosensitive — whether or not this charge transfer occurs — the internal details of "How is it the charge transfer can be magnetosensitive?" There is exactly one known theory for how a charge transfer could be magnetosensitive. It may or may not be correct, but there's no second theory. It's called the radical-pair hypothesis. Magnetosensitivity of charge transfer always gets attributed to the radical-pair hypothesis because we don't have a second hypothesis. What I rarely see in the literature, or in our discussion with colleagues, is quantitative predictions that follow directly from the radical-pair hypothesis that are falsifiable. What everyone seems to say is, "Ah, the charge transfer seems to depend on the magnetic field — must be the radical-pair hypothesis." More than one group, I believe, is trying to not just attribute this to a model, but falsify the model if it's false, or support it if it's true. I think one of the predictions is: how will the probability of charge transfer depend on the magnetic field? Because what we see is — not enough field, you don't see anything; in a field, you see an effect; too much field doesn't improve on it. The effect saturates. It's not "I add more field, I get more effect." It's like, 10 millitesla, good effect; 0.1 millitesla, no effect; 50 millitesla, similar to 10. Diminishing returns. One of the predictions of the radical-pair hypothesis is that below some threshold, the sign of the effect ought to reverse. I'm not personally familiar with this, but my colleagues have told me these are the predictions they've made. So if someone wanted to falsify the radical-pair hypothesis — quantitatively, how that probability of charge transfer depends on the magnetic field strength — that's how you'd falsify that hypothesis. For that reason, I tend not to say the words "radical-pair hypothesis," because I know I don't know what I'm talking about. I strongly believe, with lots of evidence, that charge transfer depends on the magnetic field. And the literature will predict the thing that the physics Maria is telling you right now. HOST: But why is this actually changing the fluorescence of the protein? You're telling me it's not fluorescing, it's entering another state — and then you shine a magnet and then the fluorescence goes down. What is happening there? MARIA: Because you're in a sense — if you're controlling how much is getting stuck in this other state, the long-lived charge form — ANDREW: Super long-lived compared to any of the other states in question. MARIA: Yes, which we believe at that point is not fluorescent. Then you're stealing from the population that would normally be doing fluorescence. HOST: Oh, I see. So it's just that a subset of the proteins in your sample are not fluorescing. That's it? And that's why you see a dimming effect. ANDREW: Yep. They end up charged, they end up dimmed. HOST: So if you see a 75% effect, it just means you're coaxing more proteins into a charged long-lived triplet state? ANDREW: Yeah, exactly. Or a long-lived charged state — the triplet state is super short-lived. Sorry for the pedantry. HOST: But doesn't that mean, then, that you're biasing the protein away from the singlet? Is that the magnetic effect? ANDREW: This is the one that might be worth — these are all the main characters of our story. Basically: ground state, default form of the protein. Shoot the ground state with — in the case of the LOV domain — blue light. It gets excited. Default excitation is singlet. In the case of the LOV domain, there's a hell of a lot of singlets that turn into triplets. (This is our cartoon model.) Lifetime of the singlet, few nanoseconds — goes back to the ground state, gives you a photon. Lifetime of the triplet, tens of microseconds — one way or another, goes back to the ground state. Both of these have a lot of energy in them. One of the things the triplet can do is participate in charge transfer. If it participates in charge transfer, then it ends up in this super-long-lived state — the seconds-to-minutes state. That seconds-to-minutes state, in many contexts, we believe is essentially just a non-fluorescent state that doesn't participate in the photo cycle anymore. And just like you said, that shelved population just doesn't participate anymore. So you go dark. We don't know as many of the properties of that state as we'd like to. So it's a good question — when she's mutating her protein, is she changing rates of triplet generation? Is she changing rates at which the triplet turns into the charge form? Is she changing rates at which the charge form returns to the ground state? We don't always stop to — This is a cool thing about directed evolution: you don't need to understand a goddamn thing. You can just keep engineering without looking at what you're doing at all. We love mechanism. We love understanding. We don't *need* understanding. So a really common workflow for Maria is, she's making a ton of progress — she and her colleagues at Nonfiction are making a ton of progress engineering proteins, making them do incredible new things, not necessarily understanding any of how they work. And in parallel with that, when we're drinking a beer, we talk about what the data has taught us. MARIA: And we love to work with people, because these are very specialized measurements that, even if we wanted, it would take a lot of years of expertise to develop. So, in a sense, working with people that are doing this kind of transient spectroscopy — ANDREW: She tends to pass the things she's engineered — that are wild and exotic and huge-effect-size — to academics. And there's a beautiful synergy there: these academics that just kind of by identity would not have done the mindless engineering — it's not mindless, but maybe feels to them mindless — the engineering work of just optimizing the protein, they wouldn't do it. But now we've handed them a really big effect size that they can study in a very precise, relaxed way. We've got lots of prototypes like this. Another nice thing — this is something I didn't realize, but it was kind of forced on us. Good luck engineering the hell out of a system without coming to understand it by accident, just in passing. So I think it's often regarded that a side effect of curiosity-driven basic science is invention and technology. And sometimes, yeah. A side effect of *just* engineering the damn thing is you learn a lot about the basic science, whether you want to or not. MARIA: It is true, because before looking into this, we had no idea — we never heard about the radical-pair mechanism. We had never read any magnetic-field-related literature. From scratch, we did this whole go-around-the-table, and before we heard about the radical pairs we were like, "This is all consistent with electron transfer from a long-lived triplet state." It's just funny, either way. It's nice to have the literature to refer to and put more of a framework on things. The fact that we came back all the way to flavins — it's funny, because I never went — and like most people would start with flavins, and they just came out from a screen. ANDREW: And a beautiful thing is, so many of these people that are doing the hard work of studying these tiny effects in endogenous systems — to win arguments with other academics — she just handed them easy mode. And okay, they didn't want to understand this engineered system; they wanted to understand native biology. But it turns out it's really productive in many cases for them to start by getting really good at and understanding the engineered system. After you're good at easy mode, now do medium mode, now do hard mode. Just like she couldn't see the signals when she first looked for them — now it's second nature. --- The conformational-change red herring ANDREW: At one point, when she'd engineered the LOV domain — MagLOV — to be extremely magnetically responsive: we know *Avena sativa* LOV, the thing straight out of the oat plant, is believed to change shape when you shine light on it. And it's commonly stated in the literature that that shape change is how you make other optogenetic systems. You take this light-responsive protein that changes shape when you shine light on it, you attach it to a cellular protein, and perhaps the change in shape of this protein disrupts the function of that protein. We wanted to control the function of proteins. So we thought — we've changed our thinking since then, but we thought — "Oh, we need to show that the magnetic field controls the shape of this protein." I don't believe that anymore. We need to show that when you attach this protein to that protein, we turn the function on and off. The cool part about that approach is, just measure it. You don't need to say anything about the mechanism. But for quite a while, several months, we were hung up on the mechanism. We wanted to show the shape change. HOST: Because the idea was you could attach MagLOV to anything, put a magnet on it, and turn off that thing inside the body, basically? ANDREW: That's it. It would be changed — because at that point we thought it had to be a conformational change. So Maria designs this beautiful, elegant assay to establish once and for all that the shape of this protein is changing not just when she shines light on it (which is already a literature-based belief) but also when you turn on a magnet. There's this family of calcium sensors where a fluorescent protein from a jellyfish is attached to a calcium-binding domain — a fusion. Proteins from two different creatures, but you fuse them together. The idea being: this calcium-binding domain changes shape depending on the calcium, and it warps the shape, presumably, of the fluorescent protein. By looking at the changes in the fluorescence, you can infer changes in the calcium. So she thought, "I'll make a similar system. I want to establish a shape change. Okay, it's not a calcium-binding domain — it's this light-sensitive, magnetosensitive, hopefully-shape-changing domain. I'll attach it to another fluorescent protein." This one already fluoresces green, so she used a red fluorescent protein. She uses mScarlet. So she's got her hopefully-shape-changing magnetosensitive domain, and her red fluorescent protein. And she attaches the two parts that are supposed to change shape to two different parts of this protein — but she doesn't know where to attach it a priori, because we're not protein designers. So she just tries a bunch of things — she does an insertion library, makes a bunch of different versions of the protein where they're attached in different ways. She screens the library to check for the red fluorescence. How is a magnetic field going to affect the red fluorescence if it doesn't change the shape of this one? So she's looking for hits where you shoot this guy with his color, you turn the magnet on and off, presumably change his shape — a hit would mean, "Oh, this shape change affected the red fluorescence," which the magnet shouldn't do anything to. This one alone is not magnetosensitive, especially if there's no flavin. But she puts them together. She gets a hit! Super excited. I think it was right before Christmas. Leaves for Christmas. Comes back after Christmas. "Okay, let's sequence the hit." It's a terminal goddamn fusion. HOST: Why is that so disappointing? ANDREW: It's a one-point connection. There's no way — pardon my language — there's no way for the shape change here that we can imagine to change the shape of this guy. So a hit doesn't make any sense. It's boiled nonsense. And we realize, "Oh, goddammit — it's probably charge transfer." That's how we got onto the charge-transfer kick. Good luck explaining that with a shape change, which was our dogma — our guiding light — for a while. It really emphasized for us: focus on what you can measure. Hypotheses about intermediate states are awesome. Focus on the observable, focus on the quantifiable. Since then, Maria's screening philosophy has shifted from reasoning about an intermediate quantity — shape change — to focusing more on a terminal quality. For example: she wants to make a drug that only works near a magnet. Just check if the drug works or not. Measure that, and then mutate the protein to turn the thing you wanted up and down. Don't screen for an intermediate proxy that you think will lead to what you want. Screen directly for what you want. HOST: Have you found that when you're trying to engineer other proteins, do you always need a flavin-like thing? Or are there proteins that you've now found that have no cofactors that are just magnetically responsive? MARIA: What we've been doing since then has been working with this LOV domain, which has the flavin cofactor. Flavin — there are other molecules that could potentially replace flavin. This is just what we're doing because of convenience, and because we've engineered based on this particular protein. It's actually turning out to be easier than I expected, in the sense that we've been able to control lots of protein-function activities. I assumed that each one of those activities would be an enormous feat of engineering and time, and they're not necessarily — any of them — done amazingly, amazingly well. But the fact that we've had hits relatively easily, and we can control enzymatic activity, control transcription, control binding — it's a lot of activities that very easily we get a small effect, but it's there. So that has made me pretty excited. And then there's the engineering to make it bigger, which takes a little bit of effort and time. ANDREW: The hallmarks for magnetosensitivity, for everything that she's seen — everything shares at least two features. She got the system into a very energetically excited state — the triplet state. And the triplet state participated in some type of charge transfer. That's a magnetosensitive step. And then that charge transfer had some measurable consequence. In the case of fluorescent proteins, it's been known for a while that when you change the charge state of a fluorescent protein, you do modify the fluorescence — usually you just turn it off. We believe — I won't say we have the same evidence for this — but the antibodies that she reported recently, we strongly suspect the mechanism there is: she's generating triplets in a LOV domain which is attached to these antibodies, and whether or not they participate in charge transfer at the antibody, we suspect that is the magnetosensitive step. We suspect that a charged antibody and an uncharged antibody have different binding affinities for their target. I don't want to say we have evidence for that, but it's not the most insane speculation I've ever made. HOST: And you trust the results? ANDREW: It definitely — and also with enzymes. HOST: Sorry — did you say you always have some kind of cofactor in the LOV domain? MARIA: Yes, there's always flavin. Always has flavin. ANDREW: Okay. So those two hallmarks: a triplet state, some type of charge transfer, and then some consequence of charge transfer. Everything she's doing with a LOV domain is a flavin, no matter what. In her original discovery in proteins from jellyfish, proteins from corals, flavins were certainly capable of being the cofactor. But as she mentioned briefly earlier, there were other non-flavin cofactors that we suspect they're all implicated in charge transfer. We suspect anything that's good at exchanging charge with a triplet in a GFP could be a cofactor. The reason I got super-excited when you asked the question is something I don't think she necessarily has the luxury of pursuing — the company is trying to cure diseases, so that's something of a focus. But there are quite likely endogenous biological systems that produce energetically excited states as a side effect of metabolism, let's say. Perhaps some of those are triplet states. Perhaps some of those triplet states participate in charge transfer. Perhaps those charge transfers have some measurable impact. There may well be strongly magnetoresponsive systems in the body if there are excited triplets that participate in charge transfer. It just may be that what's downstream of them is kind of hard to measure. You see what I'm getting at? Quite possibly this particular form of magnetosensitivity is present in the body. It's just not upstream of something that we're good at seeing a one-part-in-400 change in. MARIA: I was going to correct the word "strong" — I think what you mean is there are some responsive systems, but not strong. --- The Quantum Biology Institute HOST: Well, it's also such a beautiful segue, because my next question is: there is this growing literature from the Quantum Biology Institute and elsewhere that show very weak magnetic fields — weaker than the Earth's natural magnetic field — are enough to do things like change the lag phase of *E. coli* growth, or these other physiological effects that they've seen with development. What is sort of the plausible mechanism, or reason, that evolution would create such a thing if these types of weak magnetic fields don't even exist? Is it just that this is a feature of biology that has never been selected against? What is actually going on here? ANDREW: Maria's results are consistent with what you just said — meaning, I think, consistent with what you said: biology did not have access to 10-millitesla fields to evolve in the presence of. And in her hands, some systems seem to try to evolve them. It doesn't work very well. If you try to make GFP brighter, you're almost bumping into energy conservation — that's tough. But maybe another reason it's tough is, evolution already, for whatever reason, tried to make GFP pretty good at being bright. So making it a lot brighter, you're already at the peak of a fitness landscape. Everything she's engineered for magnetosensitivity has all the hallmarks of something which is nowhere near the peak of a fitness landscape — because why would it be? Why would it ever be able to be selected for that? When there's something which depends on the protein sequence, isn't forbidden by physics, and hasn't previously been optimized by evolution — in her hands, it usually responds like crazy to mutate-and-screen, whereas things which have been optimized don't. Actually, shout out to the Quantum Biology Institute. One of the things I really love about them: whenever you say "magnetosensitivity," a lot of folks hear "tinfoil hat." They hear crazy stuff that you shouldn't trust. And you heard me express some skepticism about some other work earlier in the conversation. For magnetosensitivity and biological systems in particular, I think peer review is great, but reproducibility is the only thing we can really trust. Anybody that makes a claim — if it works in somebody else's hands, that's when you should start taking it seriously. And what I love about Maria's work is, she makes the effects so big that it just trivially works in everybody's hands. What I love about the Quantum Biology Institute is, these guys are taking reproducibility very seriously in a way that I think a lot of academics don't have the luxury of. Felice knows: if she wants to be taken seriously, she can't just share some data and make a claim. She has to reproduce claims that have been made; she has to make claims that are reproducible. It's the only way I'm ever going to trust that stuff. It has to work in more than one lab. HOST: Have people reproduced that — like the lag phase of *E. coli*? It's clear though that nobody knows the mechanism, right? It's like we're measuring effects, and they're like, "Well..." ANDREW: I don't know shit about that. I guess what I love is, unlike a lot of groups I'm familiar with, I think they've done something really public. "Hey, let's announce what we're doing. We're trying to reproduce this claim. We're going to share everything we can." I think it's the only way to take the field out of the dark ages and into the "I actually believe it" phase. Sorry, maybe that was off-topic. MARIA: I would take different approaches, because they're very, very small effects. I don't know — even selecting for *E. coli* that — pick a metric and engineer. My heart comes from an engineer. Why would we want *E. coli* that responds to magnetic fields? I don't know — we can think of something. ANDREW: Don't win an argument about a small effect. Make a big effect, and then there's no argument. HOST: Oh, you're saying that the effect is small, and so there could be sort of unplanned selection effects that are changing the lag phase? MARIA: That's fun. I didn't think of that. Yeah. But if they are, can we make them respond to — like, be very slow, very, very — HOST: I see. So you're saying engineer *E. coli* to have super-strong magnetic-field effect. No one's ever done that, right? Has anyone done organism-level selection for — MARIA: No. But *E. coli* is perfect for that. HOST: Yeah, you could totally put *E. coli* into a continuous bioreactor with some kind of magnetic-field setup. But I don't even know what to expect would happen, I guess. MARIA: I think it would probably be very interesting. Let's assume that flavins and some other metabolites have an effect, and this is the source of the difference in lag. Then you start seeing all of these metabolic enzymes — and it will correlate. Potentially they are more magnetic-field-responsive enzymes to start with, which would be, I think, a neat result. ANDREW: I'll make a slightly spicy point. Take this with a grain of salt and know that I'm saying it with a smile. But a lot of folks that are interested in endogenous magnetoresponse are building on her work in an engineered system that they're not necessarily interested in. We're not really building on the existing literature for arguments about endogenous response. It's more a philosophical point: it's kind of funny how the engineering helped us with the science, and possibly just because I don't read as much as I should, the science didn't so much help us with engineering. HOST: But to clarify — have you built any proteins, enzymes, antibodies completely by rational design? Or is it all still directed evolution? Or it's like, you're going to look for hits, because that's not really engineering. It's not engineering until it's rational. ANDREW: Well, let's call it — pick a name and we'll call it that. But it sure ain't rational. MARIA: I think what I like about what I do is that you do enough of them, and you start seeing rules. The rules don't necessarily — like, you can start putting, you can look in the literature, "Oh, if we remove the cysteine, now the electron is hanging out here for longer. That kind of makes sense in terms of this model." Even for the engineering itself — you do engineering, engineering, and you start getting rules of "We want it sturdier. Here we're changing charges. We're moving — we think we're going from here to here on the electrons, and we're making a path." --- Founding Nonfiction Labs HOST: Tell me about founding Nonfiction Labs. You're both at Calico, right, at the time. And you decide to leave that to do this for-profit company. Why not just stay and do the R&D and then license out the technology, or something like that? MARIA: We were having a very, very good time at Calico. Calico was an amazing place. We had a lot of freedom, we had our time, in a sense. After we discovered the magnetic-field effect, it's not something that is necessarily aligned to the rest of the company, but we thought it was really neat and worth doing — because we could, and to take it the next step further from just fluorescence and just microscopy. HOST: And they would have kept supporting it? ANDREW: We had our freedom. What we didn't have was mission alignment. This is, like, a once-in-a-lifetime thing. This is the coolest science I've ever been associated with. Let's take a big stinking swing at it. I did what we could to explore the possibility of taking a big swing at it internally. It just really didn't line up with the mission. So the idea of splitting our time between the most important thing I could imagine working on and the thing I'm supposed to do seemed like a bad idea for everyone involved. Let's go all in. Let's roll the dice. MARIA: I had met Richard Fooss, my co-founder, and he seemed perfect for this. He actually was with us while all of this was being born in terms of the science. So he knew it from the beginning, and he was excited about it too. We started brainstorming and thinking what could be the most impactful application. He was amazing. He set it all up, and here we are trying our best. ANDREW: As you might imagine, our motivations are primarily nerd-based. This is the coolest science we know how to be part of. We believe it can create just magical-seeming technologies, capabilities. And let's not forget — it's not that we're motivated by money, but money is how you get cool shit done. Sometimes begging is a really effective strategy to get some beautiful science done, and I have been a professional beggar my entire life, quite happily so. Yes, I definitely want more money to do more science. But given a chance to do even better — give it a shot. Don't get me wrong, you know, I think Maria's company as a nonprofit would do just fine. I think it would make just as much sense. I think it'd be just as much fun. And I think at this point it wouldn't even make much difference. But in the event that our dreams come true, in the event that what we see is almost a technological inevitability — well, somebody's going to get rich off it. Why not the people who invented it and discovered it? Again, not that we really care about being rich, but goddamn, would we like complete intellectual freedom and the ability to bring beautiful ideas into existence with the help of as many of our brilliant friends as we can. HOST: We're filming this at the Biohub, and Andrew, you work at the Biohub, and Maria, you work at Nonfiction Labs. What was the story of leaving Calico, starting Nonfiction Labs — but now you're at the Biohub? ANDREW: I love the people here. I've been collaborating with the folks here for a decade or so. Well, not — okay, as long as they've been here, I've been collaborating with them. I love the spirit of this organization. I love the mission. I love the values. There are a lot of ways to make traditional academic progress on this incredible scientific opportunity. And I think being at a nonprofit in the past has functioned as a funding agency to bring cool science into existence, fund people to do cool science. I thought I could potentially — basically I just want to drive the field forward. It's the coolest thing I've ever been associated with. How can we capitalize on this as much as possible? How can we bring as much human opportunity into existence as possible? One way to do it is to come to a place that has incredible values, incredible resources, incredible people. And yeah, I'm not in control of those people. I'm not in control of those resources, but I have a voice. If I have good ideas, maybe they'll listen to me. Whereas starting a company — Maria's in 100% control, her and Richard. If they got an idea, they can try it. There's nobody older than them, bigger than them, that's going to stop them. MARIA: It's not relaxing, but I do enjoy the pace. I do enjoy the flexibility, and the fact that if you think of something that needs to be done — the only question is, why didn't you do it yesterday? ANDREW: At Calico we had access to incredible resources, and we had a mission that did not overlap with this mission. And it turns out that having access to far fewer resources but focusing clearly on a mission where everybody is pulling in the same direction — goddamn, has she been productive since she started the company. Whereas if we'd been in an organization that was better resourced — a wonderful organization, but an organization that fundamentally does not prioritize this work — it turns out you're just staggeringly more impactful, even if you have tenfold less money. --- Investors and market HOST: I'm curious — when you go to investors for the company, what do they think of this? You've mentioned it's a $100 billion market. We can make so many drugs better. But is there a poisoned-well effect, because there is so much fake stuff in the field? What do investors say when you approach them? ANDREW: I don't think there's a poisoned-well effect based on previous attempts at magnetogenetics. Prior to Maria's engineering work that made the effect enormous, we had a lot of discussions about "Is it real?" The discussions about "Is it real?" stopped when we started showing people this figure. I'll describe it verbally: it is a photography time-lapse of a plate of *E. coli*. Maria's taking pictures of the plate in the fluorescence channel while I'm waving a magnet around underneath the plate. There's this obvious, enormous effect. Prior to showing this picture to people, they want to debate with me whether or not magnetic effects are real. After I show them this picture, the discussion turns to practical details of how to make this into a technology. No further questions on veracity. MARIA: But I think it is true that there's a lot to learn on the technology, and a lot to still engineer. We're still very early. We are still at the "get the technology very, very well" stage. The beauty is that we can move fast, and we're moving fast. I suspect in one year we'd be in a mouse. But right now we are at the biochemistry level, and it's a little harder to have conversations about future drugs — especially because people are not used to this kind of super-unusual way of completely changing — it's not an antibody. ANDREW: If you want to make safe money, invest in real estate. HOST: Is most of your time at the company just basic R&D right now? Like, "Let's try to expand this to new types of proteins. Let's figure out what's happening." You've said that you've now translated this effect to enzymes like luciferase, antibody binding. One thing I've noticed is in the antibody work, for example, in the presence of the magnet, the antibody stops binding. Do you have any sense of whether or not you can flip these effects? Or is it only one or the other? MARIA: So we have data from maybe a few months ago where we got mutants where the effect is reversed. And it's very interesting. We're doing some experiments — porting certain parts of one antibody to the other. HOST: This is for antibodies? MARIA: Mm-hmm. HOST: Oh, okay. So it's like the antibody binds tighter in the presence of the magnet. MARIA: They're very fresh results, so — ANDREW: We didn't tweet those results, because we don't — we like to have things pretty solid before we announce them. We don't like to retract our claims. MARIA: But we're working hard on understanding why that happens, and how to engineer that on purpose. Even if I'm not very good at actual rational design, I like learning rules — and usually the behaviors tend to more or less boil down to specific aspects that you can try to engineer into a protein. ANDREW: There's two factors here, and one of them is: you get what you select for. So why do her antibodies go a particular way? Well, the first hit happened to go that way, and we'll steer into that. Although it is certainly true that some things, the selection is a slog, and some things, the selection is a breeze. When she made the magnetically responsive fluorophore, that was a few months and it was incredible. The magnetically responsive luciferase, I think far more time has been put into it. The modulation gets bigger, the progress is beautiful — we have a nice animation of, you know, zero effect, tiny effect, medium effect, bigger effect. But had that same effort been put into a fluorescent protein, the return on investment would have been much, much higher. So we don't know why one thing's easy and one thing's hard. In a similar spirit — can things flip both ways? When I showed you the result from Adam Cohen in the synthetic organic molecule that inspired Maria to look in fluorescent proteins, that got brighter when you put a magnet near it. When she found this effect in GFP, when she found this effect in the LOV domain, you put the magnet near it, it gets dimmer. So if I were a smarter man, maybe I could tell you why. But just the fact that some are brighter, some are dimmer — perfect. We'll get what we select for. --- What would a medicine actually look like? HOST: What would the end state for these be? If you are able to make a medicine, what would it actually look like? Let's say you take an existing antibody used to treat cancer. What are the actual modifications you have to make? How do you know it's going to persist as long in the body as just the natural antibody? What are the sorts of things that you have to think about in terms of translating this into medicines? MARIA: The way it would look — the parts you would need is the fuel. So you would need to have some sort of injectable fuel. You need the luciferase that is chewing that fuel and activating the LOV domain. So that would be all your antibody. In a sense, you're injecting the antibody, you're injecting the fuel. You could probably take the fuel by a pill — the fuel would be a small molecule, not a protein, so you wouldn't have to inject it necessarily. Maybe in the very future. Right now we're using something that's not very easy to deliver. HOST: But you think you'll always need the light component? MARIA: You need a way to get into the triplet state, other than external illumination — an enzyme burning of fuel is the other — HOST: Magnetic effect. MARIA: That's right. It only stems after stimulation with a photon, or a photon's worth of energy. HOST: But doesn't that complicate things? Are there safe fuels? Will the fuel distribute through the body in the way that you need? MARIA: I'll give you a lame answer. It works in mice. So, whatever that's worth, it's a very common imaging method in mice, and you can do it repeatedly. However, yes, there are concerns. There's also lots of concerns regarding how immunogenic these proteins may be. ANDREW: Just to expand on that for folks listening: when we say "it works in mice," it is a common assay in drug development to put a luminescent protein inside of a mouse. The luciferase that burns the fuel, and the luciferin (the fuel), are both totally compatible with putting in a mouse and produce glow that you can measure from outside the mouse. HOST: For example, you would do that if you wanted to figure out where an AAV capsid goes in the body — you can package it with a promoter driving luciferase, you inject it into the mice, it infects all the tissues, and then you actually record where photons are coming out of the mouse to figure out where that AAV capsid went. ANDREW: Yeah, exactly. But you're saying — has anybody ever done this on humans? No. MARIA: Yeah, why would they? That would be hilariously unethical. But now that there's an actual reason to do it, I am cautiously optimistic that there's no reason in principle why it should fail in a human. There's just the practical reality that going from a mouse to a human is a lot of work. In a sense, going back to that original experiment where we were looking and asking the question of "Can we evolve *E. coli* and come out with metabolic enzymes?" — there's no reason, like, probably FAD will be involved, but those are not the only molecules involved. The idea of having a molecule that can chew up sugar and make excited high-energy triplet states — it's not insane. It's something that happens all the time in metabolic reactions. There's high energy. ANDREW: If she had infinite money, she would probably be chasing other magnetoresponsive chemical systems. She has one that's incredible. It's like, "Wow, that's as good as we need and more." But her nature, if she had infinite funding, would be: let's go find something that's even better. Let's go find something like an endogenous human system that happens to be magnetoresponsive that we can stack our engineering on top of. The dream would be a protein that *does* change conformation with a magnetic field, so that you can tether it to things and then you don't need to worry about light. HOST: But you're saying, because you have to worry about the light, you need to inject fuel. Walk me through what the molecule actually looks like. You have an antibody. It's fused to a MagLOV domain, which is fused to luciferase? MARIA: Yeah. The current design has a nanobody with a LOV domain, and that gets controlled by light from a luciferase that is not magnetic-field dependent. But you can put both of them — you can use one to drive the other. This luciferase can have a LOV domain on its own. So you have two LOV domains on the design. It's a nanobody, LOV domain, luciferase, LOV domain. And they're all fused together into a single molecule. HOST: And the idea is that this would only work for therapies that are just intravenously injected? ANDREW: Anywhere where you can deliver a protein. Intravenous injection is a great way to deliver a protein. There are other ways you could imagine to deliver a protein. Like, what's an RNA therapy? It's a means of getting yourself to make the protein. HOST: But you have to package all this. ANDREW: Oh, sure. It's too big to fit in an AAV, for example. MARIA: It's actually not that big, because all the parts here are very tiny. So yeah, it's small, actually. HOST: And this wouldn't get to the brain, would it? MARIA: If you can get a protein to the brain — there are tricks to getting proteins into the brain, potentially. Like people have used these transferrin antibodies where you can get peptides through. ANDREW: Maybe let's put this in the category of — I don't know, we are not specialists in delivery, and that is not, this year, the primary bottleneck. That said, whatever the challenges or opportunities are for delivering protein therapies to the brain, this would have the exact same difficulty/achievability profile. MARIA: That is true. ANDREW: Maybe that's something worth emphasizing. The fact that this is just a protein — it's not a big chunk of metal, it's not a nanoparticle. HOST: Why and the fuel? Which is a small molecule. ANDREW: So, totally agree. But the fuel is easy mode. Getting a small molecule everywhere in the body — hell yeah. Getting a protein wherever you want it in the body — I respect that. But it's way easier than getting a nanodiamond, a quantum dot, a microbubble. HOST: But couldn't you find — instead of injecting a molecule, why not just try to find some kind of light source that burns a fuel that the body already has? MARIA: I think what could fall out of the experiments of the *E. coli* and selecting — and what could fall out of looking for endogenous triplets and molecules that change shape based on those reactions — is the fact that you'll be using things that are already inside your cell. ANDREW: We don't currently — you're making a great point — we looked for this, we talked about this, we looked for a luciferase which could burn an endogenous fuel rather than an exogenous fuel. On the one hand, we would have been hyped if we'd found it. On the other hand, there's something kind of cool about — for example, some antibody drugs, as I understand it, have a super-long half-life in the body. You inject them, but they live in you for like a month. On the one hand, amazing. On the other hand, do you want to be magnetosensitive for a month? Maybe not. But if you need both — if you need the protein and the fuel — the protein might be annoying to deliver but stay in you for a while. If the fuel, in the fantasy now, if the fuel was a pill you took with a half-life of 30 minutes, you get the — we would love to have an endogenous fuel that we burned, but an exogenous switch with a separate half-life from the protein therapy? It's also useful. MARIA: Referring to something that you said on what you would need — one of the advantages of what we're doing is that it's control of biology. So the antibody is one way of doing it. But you could imagine, let's say, activating CAR-Ts at the tumor, where all your engineering happens in a cell. So the cell is engineered to express luciferase and to make luciferin. Then you could do the CAR-T activation once you only on the parts where you have the magnet. I think potentially there are other paths forward. ANDREW: And as much as what she described represents an enormous amount of work to do, optogenetic CAR T-cells — basically existing tools we don't have to reinvent. I believe there is an optogenetic CAR T-cell. So the fantasy she was painting largely already exists. It's less of a fantasy and more of, like, composing two existing things in a difficult, laborious, but intellectually straightforward way. HOST: If you were to deliver these as a protein therapeutic, you also have to engineer them to be long-lived. So are you now in the domain of, like, semaglutide-style engineering — of fusing fatty-acid chains, putting in unnatural amino acids so they don't get degraded by peptidases? So it's not actually just this little chain. MARIA: It's very possible. I agree. It's very possible. I am eager to get to mouse experiments to try to get a sense of where we are in terms of aspects like the pharmacokinetics and efficacy. Even very preliminary experiments will be very, very informative. ANDREW: The two paths — one where you try to get the systems in the body that make proteins to make this protein. We love that it's an entirely protein system. There's no big chunk of metal. There's nothing you have to inject — you need the small-molecule fuel, of course. But one of the cool advantages of "Oh, if you're going to make the protein outside of the body" — well, that opens up a whole other toolbox. Like we were talking about — isotope substitution, adding heavy atoms — things that you couldn't get the body to add to a protein natively in ways that we know about. Oh yeah, if you throw an iodine in there, you can probably get the triplet yield through the roof — things like that. I'm not saying that's Plan A or a major focus right now, but if it's exogenously prepared and injected, it makes one set of things easy. If endogenously produced, proteins your own body makes might have an easier time getting the post-translational modifications. MARIA: And it is true that other luciferases do have substrates that you can ingest — that you can feed to mice in the water, and then they will luminesce for a whole week. There are consumable luminescence substrates. It's just a different luciferase from the one we're using, but we made a few so we could switch. --- First indications and regulatory path HOST: Realistically, what is the first indication? I know you're very much in R&D phase, but if you were just to hazard a guess, what is the application that seems most like, "Oh, we can make a really big impact here"? MARIA: The targets that we have now that we're making these antibodies for — they're not necessarily the ones that we will move forward with, but they're all cancer targets. The reason for that is, it seemed like the most straightforward biology to implement, and there's just so much cancer research and good models for it that we picked those. It doesn't mean that they're the ones that, maybe two years from now, we switch. ANDREW: Among the reasons they retargeted the antibody was: let's reassure ourselves that we're not making a hyper-specific tool for only one problem. Let's make sure this is a general tool that can be retargeted to the target of choice, and that seemed to work quite well. HOST: What do you mean by retargeting? MARIA: The idea is that most of the nanobodies look very similar, and it's only the regions that contact the antigen — the ones that vary. So these CDRs, as they're referred to, you can switch them from one antibody to the other. HOST: Sorry, you mean one target to another? MARIA: Mm-hmm. So if you have two antibodies and this binds target A and this one binds target B, you can take the surface part of this one and graft it on a second one, and then it will bind that other target. We've actually been — I wasn't expecting this to work so well, but it works. ANDREW: You have an antibody against protein A. Can we make it instead bind protein B? And the beauty of that is that we only need to make one backbone that is good in the magnetic-field control. And then we hope — HOST: So you have a universal tool for any protein target? MARIA: Exactly. But we haven't done it enough times. We've only switched around like three times. We're like, "Super generalizable." ANDREW: You know the way a physicist counts? Zero, one, infinity. MARIA: Yeah. So we're at infinity. HOST: But three is — have you failed yet? MARIA: No, we haven't. HOST: So every time you tried it, it worked. ANDREW: Perhaps. MARIA: I made three different nanobodies that bind three different targets, all with exactly the same architecture, except the binding domain is for different targets. HOST: Exactly. And to convince yourself that this is a thing that might be clinically plausible — what sorts of preclinical experiments would you do? How do you even do them? Do you need to do preclinical experiments that nobody has ever done before, because you're adding in a magnetic field? What sorts of things are you thinking about? MARIA: Definitely the experiment that I really want to get to is to have a little mouse — one tumor on the right that expresses an antigen, one tumor on the left. The tumor on the right has a magnet, and the other one doesn't. We can look at one bound more than the other one. And then compare that to the antibody without the magnetic-field effect, and hopefully we have more where we want it. That's the experiment in my head that we are working towards. HOST: And you would also measure the toxicity. Nobody has delivered this fuel, luciferin — MARIA: Although in this case, in mice, we have. But I totally agree with your point. HOST: No, but I agree. MARIA: It's like, we need way more strict toxicity studies on the mouse, for sure. HOST: Yeah. Has anybody ever measured the toxicity of luciferin? Or have they not even really thought to do that because, "That's mice. We're not using it to make therapies"? MARIA: I feel like there is — if you look in the literature and you start — there's data. And depending on how — I'm not sure that there's a very, very specific "we looked at this," but there's evidence for some level of toxicity, and then evidence for "we can do it many times and nothing happens, they seem okay." HOST: Are there specific forms of cancer that have, you know, that are treated with nanobodies, where the nanobodies have particularly potent off-target effects — where you're like, "This seems like a plausible sort of starting point that we would target"? ANDREW: Before, when we were just dreaming — when Maria just had the discovery and we weren't thinking about the company yet — we spent a full week just sitting in the office, like, "What's the best thing we can do?" Some of our colleagues at Calico were part of the creation of this drug Herceptin — that is a pretty good drug for curing certain types of cancer. And among other things, it is believed to damage the heart. The trade-off of, like, "Yeah, good treatment for cancer; take a certain amount of heart damage; worth it in many cases." That was our first like, "Oh, obviously — wouldn't it be great if you didn't damage the heart? Wouldn't it be great if there was more drug on the tumor, less damage on the heart?" I'm not saying Herceptin is the first thing she ought to do, but it's definitely the one that left us with, "Okay, we're onto something. That's a problem we might be able to solve." HOST: And that's just an antibody, Herceptin? ANDREW: Antibody, yes. HOST: Yeah. At least it's an antibody. But you think you could target the same thing with a nanobody. MARIA: Yes, actually. HOST: Which kind of segues nicely into the regulatory question, which is: is Nonfiction Labs planning to develop its own drugs and bring them through trials? Or are you planning to do Phase 1 and then sell the IP? Because nobody has ever done the fuel thing. Even if you took an existing target, you're using a new architecture for the molecule. There are so many changing variables. How do you think about that? MARIA: Our idea is to definitely do partnerships. I personally do not want to develop myself the skill of navigating regulatory — ANDREW: You covered a lot of ground between the quantum mechanics and cancer therapy. You don't also need to know — MARIA: So we might change our mind, when we talk to Richard, my co-founder. ANDREW: I think Richard has more stomach for that work than Maria does. So I think the company has stomach for that type of work. She personally perhaps was not made to navigate a regulatory process. MARIA: That doesn't mean I'm not interested. It just means, like, I need someone — I think we need somebody to join the team that cares deeply about this. ANDREW: To the point you made earlier about, of all the things you could do first, what's the one you think you ought to do first? We've got some thoughts, we've got some opinions. But this might be a good time to emphasize — if anybody listening is rich and really wants us to prioritize a particular disease, you could get our full attention and have a tremendous influence on which thing we attack first. Just saying. And then to the current topic: Genentech, the classic — the grandmother of all biotechs — navigated the question of, "All right, we can get bugs to poop insulin. How shall we turn this into money?" And I think the answer is, I don't think they did. I think — who was it? — Eli Lilly got — HOST: Eli Lilly bought it, yeah. ANDREW: So the first — I think insulin, human growth hormone — they passed the ball to someone else. They got famous off the first couple. And then the next couple, they were in a position to do it themselves rather than pass the ball and watch somebody else run it. HOST: Yeah, like if you have a lucrative partnership that leads to high royalties or something, it makes you rich enough that you can start doing your own drugs. MARIA: We can start doing our thing. Yeah, that would be the dream, right. --- What does the field need? HOST: What's needed to build the field? MARIA: I think the field is getting started. It is a perfect intersection of quantum mechanics, biology, physics, engineering. So multidisciplinary labs, or a lot of people that want to work together. What I'm hoping is that there will be a very nice community around this that will be very open, with representations from many fields. ANDREW: Something we didn't see coming, but we have absolutely found delightful: nobody in these conversations knows everything. Nobody gets to be gatekeeping. Nobody gets to be smug and superior. Do you know quantum mechanics? Do you know immunology? Tell me what cancer target we should pick first. Tell me about the radical-pair mechanism. Oh — that's right, there are limits to your knowledge. Perhaps all of us are worthy of respect and a voice and a place in the discussion. See, I'm a physicist — I know a thing or two. Not about immunology, which has maybe been made clear over this time together. Maria is a biochemist — quantum is not central to her training. She's not incapable of it, but — MARIA: And that's not to say the field has been going on for a long time, but I think you will get a big push with big signals now. ANDREW: Most of the folks in the field so far — because virtually everyone in this field, we're watching it be born, and we were lucky enough that, because of the beautiful things Maria's engineered, we're kind of at the center of it — so everybody wants to join the field basically asks her first, "Hey, send me some protein." She sends them protein, so we get to know them all. They're all really cool, all well-behaved, bright, hardworking, motivated. So far — some fields are cursed with an established old person that believes a false thing, that if you contradict them, you're not getting funded. We don't have that yet. HOST: That will be you in 20 years. ANDREW: Yeah, yeah, yeah. Looking forward to it. HOST: You should work on your beard. ANDREW: I will be the gray-bearded gatekeeper. "I've heard that radical-pair hypothesis before." We watched this in the aging field. There were certain reasonable scientific questions we'd ask that we'd watch our senior colleagues — who were veterans of the aging field — kind of their hackles would go up, and we'd realize, "Ah, it doesn't matter if I'm right or not. I need to shut up." We don't have that yet. So what does a field need? It needs visionaries — delusional, motivated lunatics. They're going to try their asses off. It needs funders. Right now the funders are the typical funding sources. There's some funding sources in the Bay that like to fund crazy new visionary ideas that could change the world. There are branches of the government that like to fund — you talk to DARPA — they like to pump a lot of money into a crazy thing that could be huge but might not work. Thank gosh that exists. Getting, for example, NIH funding for this — I think it's quite possible, but maybe after a couple more *Nature* papers. What does the field need to come into existence? MARIA: Honestly, like — Andrew will give protein to anybody, give advice, input to anybody. I feel like the big contribution we did to the field was to come in with protein engineering. So let's see where that goes. More engineering — we would love to have more colleagues who are interested in doing protein engineering, because it's not just you, but it's not five other people. Whereas there's, I want to say, probably hundreds of other labs that are interested in the science, the mechanism, the questions raised by magnetosensation. That's awesome. Let's also do some more engineering. HOST: And it is kind of surprising that the total amount of funding, as far as I understand, is like a few million dollars for all of magnetogenetics, right? Whereas Sonogenetics now has a $40 million ARPA-H program. Mikhail just raised $250 million, I believe — Merge Labs, right? People are using focused ultrasound to control biology at huge scales now. But magnetogenetics is still like $5 million total, maybe? ANDREW: Maria's been printing literal miracles. Literal miracles. Back-to-back-to-back *Nature* papers, and things that were confidently predicted to be thermodynamically impossible as recently as like two years ago. She's been doing that for a burn rate of $100,000 a month. HOST: Is that true? ANDREW: It is comical how little money produces how much in her hands. I'm not talking shit on sonogenetics, but we could do a lot with $250 million. Holy crap. You know what? $2 million? It's not that expensive. Protein engineering is cheap as hell. A few smart, motivated undergrads and some training. The instrument I built that she's done all this protein engineering with — $20,000. MARIA: I am excited about sonogenetics. I think — give us a little bit of time and we can catch up, and then we can both — ANDREW: I think in some categories there's catching up to do. I think in other categories you're already ahead. Do they have an enzyme? You know what I mean? I think we have the potential to do better. MARIA: Yeah. HOST: Do they have an antibody? ANDREW: Well, Maria and Andrew, thank you so much. MARIA: Thank you. ANDREW: Goddamn — my head. That was fun. --- Whiteboard lecture *[After the main interview, the hosts walk through a cartoon of the LOV-domain photo cycle.]* ANDREW: So we just told a lot of stories. We referred to a lot of characters in the story. I kind of want to sketch out the characters in the story. So I'm going to show a cartoon — a very simplified cartoon — of the photo cycle of the LOV domain. And you'll help me get it right, because you're the biochemist; I'm just a physicist. I like to think of the LOV domain as a little Pac-Man. It is capable of changing shape when you shine light on it. So if I shine light on the LOV domain, it can absorb the light, and the most common thing for a fluorescent protein to do when you shine light on it is, it absorbs the light and it goes into the excited singlet state. That is an enormous amount of energy for the molecule to be holding, and it tends to only hold onto that energy for a few nanoseconds. So the singlet lasts for a few nanoseconds. The most common thing for the singlet to do is relax back down to the ground state. So the energy flies away — the photon comes in and another photon goes out. And that is fluorescence. Energy goes in, hangs out for a nanosecond, energy comes out. Fluorescence — the singlet state. HOST: And when that energy comes out, it's a different wavelength, a different energy level than the photon that entered, right? ANDREW: Exactly right. HOST: Where does that extra energy go? ANDREW: The Stokes shift — the jargony name for the fact he's referring to. When you go into the excited singlet state, you start in a state which is neither electronically nor vibrationally excited. That's jargony, but it's universal jargon at least. You go to a state which has an electronic excitation and a vibrational excitation. A vibrational excitation is like the atoms in the molecule are wiggling around. That vibrational excitation tends to last for picoseconds. So you lose a little bit of energy. Exactly as you said: the photon that comes out is not the same color as the photon that comes in. It is a lower-energy photon. For photons, lower energy means redder. So in the case of the LOV domain, bluish light comes in — 450? — 450-nanometer light? What's the absorption peak? MARIA: 450. ANDREW: And the light that comes out is greenish, 510, 515 for the LOV domain? MARIA: Great. ANDREW: And 510, 515 — the units there are nanometers. That's the color of the light. However, there is another thing that can happen to a singlet. Your photon comes in, you make an excited state, but instead of lasting for a few nanoseconds, it converts into a triplet. The lifetime of a triplet for the LOV domain is tens of microseconds. There are a thousand nanoseconds in a microsecond, right? This is staggeringly longer-lived than the singlet. And depending on which protein we're talking about, the lifetime of the triplet state can actually be millions of nanoseconds. So triplet states are super-long-lived compared to singlet states. Still really short on a human-perceptible timescale, but damn long on a fluorescence timescale. So, apparently, according to the literature, in the LOV domain the yield of triplets is really high. I'm used to triplet yields in coral fluorescent proteins or jellyfish fluorescent proteins that are like 1%. In the LOV domain, there have been literature claims on the order of like 50%, 60%. This is accurate as you recall it? I'm not saying that's true. I'm saying I've read it, and I have no reason to disbelieve it. Anyway, neither this process nor this process, to our knowledge, has any dependence on the magnetic field. However, one of the things the triplet can do is participate in charge transfer. And in the case of the LOV domain, this charge transfer would be an electron literally moving from one location in the protein to another location in the protein. If you want to know which locations, don't ask a physicist, but — MARIA: Ask a biochemist. It's very likely a lot like what happens in cryptochromes — taking electrons from a tryptophan close by. So the flavin becomes very electron-withdrawing — ANDREW: And the key point here is that the lifetime of the charge-transferred state is potentially enormous — on the scale of seconds or even minutes, as opposed to the scale of nanoseconds or microseconds. HOST: What do you mean by charge transfer? It means that the electron has already moved away from the triplet towards another thing, and then that other thing just sits in that charge state for one to a hundred seconds? MARIA: Yeah. So in a sense, we think that if it's like cryptochromes, the electron comes from a tryptophan to the flavin. ANDREW: So next up — this charge transfer, and I'm actually conflating two things a little bit. Associated with the charge transfer, as I understand it, is a bond. And so following this charge transfer, there can be the breakage of the bond. Is that accurate? MARIA: In the normal photo cycle, yes — right away. It reacts with the cysteine. ANDREW: Maybe I shouldn't focus so much on the lifetime of the charge transfer. I should say the charge transfer leads pretty directly to the breakage of a bond. MARIA: Yes. ANDREW: And the breakage of the bond leads to: Pac-Man opens his mouth. MARIA: Yeah. Pretty much, you make triplet — and this can only happen when it goes to the triplet state, because the way the electrons move breaks a bond that opens up the protein. Which is really nice, because it makes the triplet state the thing that connects the conformational change to potential discharge transfer and magnetic-field control. So I think it's possible to bring back the conformational change and put it under control of the magnetic field, if I can make that system less reactive — which would be awesome. ANDREW: It would. So I'm conflating these two things a little bit. I'm not saying I know the lifetime of this separately from the lifetime of this. What we actually observe is: if you look at fluorescence, fluorescence is cycling between the ground state and the singlet state and getting photons. Every time you end up in one of these states, you are now — in the case of the LOV domain — dimmer. You do not participate as well. I don't know if it's that you stop participating entirely, or if you just don't participate as well — but basically you're not as good at going into the singlet state, relaxing back to the ground state, and giving a photon. So what we actually observe is, the fluorophore just gets dimmer over time. And you might think, "Oh, you're destroying it." But the cool part is, if you turn the light off and walk away and come back later, it'll recover back to the original state. So we believe that over time, something happens that — I think involves oxygen, we were recently taught — but anyway, something happens where, yeah, Pac-Man will close his mouth, the bond will reform, the protein is there for you to do it again. That's the photo cycle of the LOV domain. As we understand it, this step — the charge-transfer step, upstream of the super-long-lived state that has super-different properties than the default state — this is the thing that depends on whether or not the magnetic field is on. That is often attributed to the radical-pair mechanism, which I'm not an authority on. I have no reason to disbelieve. We have produced no data that supports that model. We've produced no data that contradicts that model. What we've seen is, it seems like this step depends on the magnetic field. Among the evidence for that — this scientist Jonathan Woodward, that Maria referred to earlier — he's shown, I believe, that in systems like this (not the LOV domain necessarily, although I think he might study that in the future, we hope), if you turn the magnetic field on during the triplet lifetime, you affect whether or not charge transfer occurs. If you wait more than a few triplet lifetimes for the triplets to go away, and you turn the magnetic field on, it doesn't do anything. That's our basis for belief that it's this step — whether or not the triplet participates in charge transfer — that the magnetic field affects. And then in Maria's case, in the LOV domain, this step is magnetosensitive, but you really gotta squint to see it. Holy cow, is it a small effect. But what Maria engineered is probably some combination of how much the magnetic field affects the probability of the charge transfer, and what the consequences of the charge transfer are — how observable the impact is. What she selected for was: you see the brightness wiggle up and down as you turn the magnetic field on and off. She selected for that being bigger. It worked like a stinking charm. And then every other magnetoresponsive system that we've ever engineered — we haven't proven, dissected the mechanism in any of them, but we strongly suspect the mechanism in all of them is this charge-transfer step. In addition to this internal charge transfer within the LOV domain, we have evidence — for example, when she fused the LOV domain to a red fluorescent protein, we see the brightness of the red protein remembers what you did to the LOV domain. We think that this charge transfer, instead of being internal to the LOV domain, it can also hop to something nearby. So if there was a red fluorescent protein sitting over here, you can transfer charge over to the red fluorescent protein, and it'll remember you did it for like a minute. So that's an example of how you can control the function of a partner protein by manipulating the charge transfer from this protein. HOST: And what is the reason you think that the deletion of a cysteine changes this? What is happening to this sort of test of characters when you get rid of the cysteine? MARIA: So, yeah, so if you have the cysteine, the process of reaction — once you get to the triplet state, the reaction with the cysteine is a very fast process. So it has no chance — ANDREW: That's the charge transfer. MARIA: Yeah. So the triplet has no chance to sit there and take electrons from the tryptophan. ANDREW: Actually — you've done some work, correct me if I'm wrong, where you've just deleted the entire jaw of the Pac-Man and given it no possible way for this bond to form or exist. And yet this charge-donation process — you hook it to another protein, look how the function of this protein changes based on the illumination — this protein still works. Is that accurate, for example, in the case of luminescence? MARIA: It's true. Yeah. So we don't need the jaw — the part that's doing the conformation. ANDREW: That's a nice example of, you could call it, basic science that emerged from a very mechanism-agnostic engineering effort. Delete the whole goddamn thing — goddamn, that makes it clear how important he was to the process. So is there more important things we should get to? These are really the characters in the story: singlet, triplets, charge transfer, conformational change — with conformational change perhaps a red herring, because, you know what I mean, whether or not the jaw opens might not matter if you can just delete the jaw. HOST: Is the cysteine where — just to walk me through it — the triplet — what amino acid catches the triplet? Is that the cysteine? MARIA: Yes. HOST: And then the cysteine dumps it to another amino acid, and that's the charge transfer. MARIA: So, no — so the triplet can take electrons from the cysteine. I associate the triplet with the flavin itself. Would you agree? ANDREW: Yes. MARIA: So in a sense, physically what it looks like is, the flavin becomes covalently bonded to that cysteine for a period of a controllable amount of time, depending on the mutation. But that reaction with the cysteine is super fast. So if you want that triplet to take electrons from something else, that cysteine is there and it's very happy to interact with it. So you need to make that cysteine less reactive. HOST: So when you delete the cysteine, the triplet time increases from 10 microseconds to something higher? MARIA: Yes, actually, it would. That makes sense. ANDREW: I don't think we observed that. That makes total sense. MARIA: But it happens. I agree. HOST: How do you know? MARIA: I think there is transient — yes. ANDREW: Okay, you're saying literature. MARIA: We would expect it. It's a good question. But there are literature observations. We didn't measure that ourselves. And it's actually so fast that — I think at least one paper that I remember, it's almost impossible to measure that radical. It exists for such a short period of time. So otherwise — the moment, yeah, it just happens too fast. Otherwise, it sits there. ANDREW: And then — I guess I should probably draw the arrow. If there was some target protein over here where you wanted to turn his function on or off — this charge transfer, which I've kind of drawn as some internal charge transfer — if it was an external charge transfer (giving or taking an electron from the protein you're attached to) — yeah, the flavin wants an electron. It's going to take it from the cysteine if it's close by, real quick, because it's very well-positioned for it. If it's not there, then it's going to take it from something else, and that is a longer process.