NEB Podcast #56 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewee: Bill Jack, Emeritus Scientist, New England Biolabs, Inc.
Lydia Morrison:
Welcome to the Lessons from Lab & Life podcast from New England Biolabs. I'm your host, Lydia Morrison, and I hope this episode offers you some new perspective.
Today, I'm joined by Bill Jack, who's an emeritus scientist here at New England Biolabs and was previously our executive director of research. Bill has been with NEB for many years, and to say he knows a great deal about restriction enzymes would be an understatement.
Bill, thank you so much for being here today.
Bill Jack:
My pleasure.
Lydia Morrison:
So we're going to jump right into it. Today, we're doing a restriction enzyme 101 podcast, and I was wondering if you could tell our listeners what restriction enzymes are.
Bill Jack:
Well, restriction enzymes are really the foundation of a fundamental immune system for bacteria. Bacteria have a constant war going on with bacteriophages, which infect the cells, much like viruses infect people, insert their DNA, and the DNA then takes over the cell and, in many cases, kills the cell. The bacteria have developed a number of different immune systems to be able to fight that. The restriction enzyme modification system works by cutting up the incoming foreign DNA into small pieces, using an endonuclease that cuts internal to the DNA. The cellular exonucleases then chop it up into very, very small pieces, and so the phage infection is aborted.
And a fundamental part of this, though, is that the bacterial host has to have a way of distinguishing this foreign DNA from the host DNA that's there so it doesn't cut itself up. It does this by adding modifications to its own DNA that are very specifically matched to the endonucleases that cleave the foreign DNA coming in. This is the basis of restriction enzymes and was there an initial discovery as a biological phenomena, later refined to understand the biochemical characterization and characteristics, and then used as a great tool for molecular biology.
Lydia Morrison:
Well, it seems like a really effective defense mechanism for bacteria. Are there different types of restriction enzymes?
Bill Jack:
There actually are different types of restriction enzymes. They have the same fundamental properties that I just described in terms of immune defenses, but they can be distinguished based on their different biochemical properties, namely how they recognize the sites that are there, the types of sites that are recognized, and the types of cleavages that occur. The primary type is what we call type II, and that's the one that is used as the main tool for molecular biology. And there are some subtypes of restriction enzymes underneath the type II category.
Lydia Morrison:
So, historically, how have restriction enzymes been used in molecular biology?
Bill Jack:
That's a great question. The earliest uses of restriction enzymes were for mapping DNA sequences. Remember that back in that era, we didn't have DNA sequencing to be able to tell us the order of the bases in the DNA or even the construction of those. We just had long pieces of DNA, and we had to figure out what order or what was on those pieces of DNA and how we might be able to understand the functions of the genes that were there, the regulatory regions, and other interesting segments.
By taking restriction enzymes, we were able to fragment the DNA in very specific ways because the restriction enzymes each recognize a very specific sequence. For example, the enzyme EcoRI, one of the earliest restriction enzymes of type II that were characterized, recognizes the sequence GAATTC, and every time that six nucleotide sequence occurs, inevitably, there will be a cut at that site.
By using that, we can cut a piece of DNA, separate the fragments on a gel system, and estimate the sizes of the various fragments or the distances between all the EcoRI sites on a specific fragment. If we want to then know what order those fragments were in, we might be able to go back and use a second enzyme, for example, an enzyme BamHI, which recognizes GGATCC, slightly different from EcoRI, but going to cut a different distinct set of sites.
By then analyzing the fragment sizes from that digest and perhaps even doing a digest with both BamHI and EcoRI, we can then order the fragments together and have a better idea of exactly which fragments are in which order. If we can then correlate those to the functions of the genes, we then have a more complete map.
The secondary use is the one that we probably think about more today, and that was used for DNA cloning and rearranging fragments in a desired order, in a synthetic construct. This relates largely to the ability or the property of restriction enzymes. In their cleavage sites, many of the Type IIS enzymes, when they cut, leave a staggered overhang.
In other words, they nick on one strand, and then they nick a certain distance away from that on the other strand, leaving a single-stranded region. These single-stranded regions have defined nucleotides in them, which can then be used via complementarity with a similar single-stranded overhang to ligate the pieces together in a defined order. That's probably the use of restriction enzymes that we understand most today.
Lydia Morrison:
Yeah, so how has the use of restriction enzymes in molecular cloning progressed over the years? I know that, currently, there are DNA assemblies that can be ordered and put together with lots of different fragments and sometimes in a single reaction. So which enzymes are being used for that, and how have those sticky ends grown into the ability to assemble these large DNA complexes?
Bill Jack:
Yeah. And you're really addressing one of the traditional holdbacks for use of restriction enzymes, the first class of restriction enzymes, which were ones that cut within what we call a palindromic sequence or cut within the recognition sequence. For example, EcoRI, the enzyme I mentioned earlier, cuts after the G, so G, cut, AATTC. On the other strand, would also cut between the G and the A, leaving that single-stranded overhang.
But when you assemble the fragments, you inevitably have to put that EcoRI site back in place. Later, discoveries of restriction enzymes uncovered a class which we call the type IIS enzymes, S standing for shifted sequence because the cleavage occurs outside of the recognition sequence, leaving again a single-stranded overhang.
In this case, since the single-stranded overhang is not defined by the restriction site, it can be essentially any set of nucleotides, and so, there's a great ability to order the type of sequence or the type of overhang that will be there to allow assembly. That also allows us to, by putting the restriction site outside of the fragment of interest, be able to make a cut and then reassemble without including that recognition sequence, giving us a lot more flexibility in the final constructs that we could make.
A great deal of effort has been done here at New England Biolabs to understand what types of overhangs will best ligate together and give the greatest specificity. And indeed, the Golden Gate assembly process now uses the type IIS enzymes to assemble... In our case, we've assembled up to 52 fragments together by using various overhangs and varying the overhang between the different fragments that are assembled.
Lydia Morrison:
So I know that this type of DNA assembly has also been used in vaccine production. How does that work?
Bill Jack:
There are a couple of different applications of restricted enzymes in vaccine production. Perhaps the one that's closest to our minds at the moment is the messenger RNA vaccines that were created in response to the Covid infection and epidemic.
In that case, we have to have a DNA construct, which can be made using these assembly methods, which accurately encodes for the messenger RNA that wants to be delivered. Most frequently and most often are grown up on plasmid circles of DNA that contain the coding sequences and other elements that are required for replication within bacteria.
However, once the messenger RNA is made, we want a very distinct three prime end to that message. That is created by cutting with a restriction enzyme to make a linear DNA from which the messenger RNA can be created. That's probably the most prominent use of restriction enzymes in the production of the messenger RNA vaccines that are available today.
Lydia Morrison:
And what about the use of nicking enzymes in diagnostics for flu or Covid detection?
Bill Jack:
Nicking enzymesare really interesting because those are restriction enzymes that have been engineered or have been modified so that instead of cutting two strands of the DNA only cut one strand of the DNA. That nick site then becomes an entry point for DNA polymerization, which, of course, has to initiate an existing three prime end on a template.
A variety of methods have now been developed to couple that nicking activity along with extension to amplify different products that come from that extension as a detection method. So use of nicking enzymes in detection of DNA, for example, a bacterial infection or a viral infection, anything where there's a DNA that needs to be detected amidst many other DNA sequences, one would typically use a synthetic DNA oligonucleotide that contained the nicking enzyme's recognition site but also contained sequences that would be on the DNA that you wanted to detect. Those would be annealed, which would then allow the nicking enzyme to act.
Once the nicking enzyme is nicked, typically, then there is a DNA extension by DNA polymerase to be able to displace the DNA in front of it and create a new copy of the detected DNA. Once that occurs, it creates a new site for nicking, and that continual process can create more and more strands that have copied the DNA that desires to be... That can be detected.
With a clever arrangement of other oligonucleotides in the system, that amplification can be amplified and become exponential rather than just linear in terms of amplification. The creation of all that new DNA is then used as a detection tool, somewhat similar to PCR, the way PCR might be used, but again, can be a very powerful technique for detecting foreign DNA in a sample.
Lydia Morrison:
Well, that's really interesting, and I know, obviously, detection of viruses and bacterial infection is really important. I think this really highlights one of the things that I love the most about science and, in particular, about molecular biology, which is that our initial observations and understandings about restriction enzymes led individuals to develop techniques to further explore.
And the more we find, the more new ways we find to apply those restriction enzymes that have been discovered, like the discovery of type IIS restriction enzymes has clearly enabled a lot of progress in terms of creation of, shall we say, designer DNA. And basically, that has allowed us to really further scientific understanding and detection methods and be able to produce things like different types of vaccines, new types of vaccines.
I think it's just a great example of how the knowledge of science builds on itself and the curiosity and the continued exploration of what nature has to offer in terms of molecules and how we can think about innovating our technologies around that understanding really enables the advancement of science.
Bill Jack:
No, Lydia, I think that's a great point. And with the exploration of different restriction systems, we've just looked around for things that cut DNA initially. Out of that discovery process, we found the type II enzymes, we found other enzymes, and when they would come up, we'd go, "Wow, this just isn't right. This isn't the way other enzymes have worked."
And initially, I think a lot of times, we say, "Well, that oddball, I don't know what we're going to be able to do with it." We have found, though, that once we publicize the fact that these enzymes exist and have these peculiar properties, that somebody will find an application for it. And there are a lot of times you just look at it and go, "Wow, this is really an interesting enzymes... I have no idea how we're going to use it."
Lydia Morrison:
Right?
Bill Jack:
But somebody else does figure out a good way to use it. Restriction enzymes, as you pointed out, is an excellent point. Excellent way to look at that.
Lydia Morrison:
Yeah, I mean, science. It's pretty great.
Bill Jack:
It's the best.
Lydia Morrison:
We think it's the best. Is there anything new happening in the world of restriction enzymes?
Bill Jack:
One of the things that we've concentrated on and looking at, there's been a lot of discovery on modifications that occur on both DNA and RNA and trying to understand how those modifications, particularly in higher organisms, affect gene expression and the patterns of growth and development.
The areas of epigenetic modification have been of great interest as we've discovered that there are modified DNA bases in higher organisms, there are modified RNA bases, and restriction enzymes have been discovered that can help us to map and understand exactly where those modifications occur so that we can correlate those with biological properties.
So I think that that is an area in which we will continue to expand and grow in understanding how the restrict enzymes can be used as tools for understanding the biology that's going on. We have, in our own sense, looked at the ways that restricted enzymes can be even more user-friendly, both by engineering the enzymes to have more tolerant properties, by exploring what conditions the enzymes can operate in and be effective in.
And one of the problems that we've had in early restriction enzyme digest and doing them was that we had problems understanding exactly how much enzyme to put in and how long for the digest to go. We have been able to, through a series of buffer modifications, enzyme modifications, and careful studies, been able to make it so that these are all more user-friendly, that you don't have to be worried that the digest was too short or too long, that there's a greater tolerance between those, that you don't have to do sequential digests.
In the old days, we did one digest, then we'd have to kill the reaction and then do the second digest. Again, by careful exploration and a more tolerant set of enzymes, we can do those double or even triple digests at the same time without one reaction interfering with another. I think that those are some of the variations that have come off with restriction enzymes that make them new and exciting, even though they've been around for, what have we got now, 50 years, 60 years?
Lydia Morrison:
Yeah. In case our listeners don't know, New England Biolabs was started as a restriction enzyme company, and many still know us as such, although we offer many other products to support molecular biology research and genetics research at this point. So, since NEB has been working on restriction enzymes for such a long time, I know our scientists and our information technology teams have developed lots of tools to support this kind of research. Can you tell us about some of those tools that are available to the public?
Bill Jack:
New England Biolabs has developed a variety of tools to make it easier to use the restriction enzymes. I remember days in which we made up our own buffers, in which we went through the catalog page by page trying to figure out which enzymes cut where, which ones could be used in what order, and exactly how much to use.
Those have all been systematically arranged in tools so that you can... For example, if you've got a double digest you need to do, it'll tell you what conditions to use for the enzymes, whether they can be used together, whether they need to be done sequentially, and if so, what order to do them in and how to change the conditions between the reactions, which enzymes can be heat inactivated, which ones need to be extracted using other methods.
All of these tools have made it much easier to figure out what to do, exactly where to cut, what fragments are going to be created, what it will look like on an agarose gel when you separate the fragments. These tools are available both as mobile apps, and also, a more extensive set of tools are available through the NEB website.
Lydia Morrison:
Yeah. And these tools can be so helpful because I remember looking through the NEB catalog when I was in graduate school.
Bill Jack:
Which is a great resource, but it doesn't do as much as the tools do.
Lydia Morrison:
Absolutely. And I think it can be a huge time-saver, and it's really like having a more knowledgeable partner at the bench who can give you some guidance about the best way to proceed with your experiment. So I think they're super valuable. Everybody should check them out.
Bill Jack:
And then don't yell at you if you do something wrong.
Lydia Morrison:
That's the best part about them.
Bill Jack:
That's the best part.
Lydia Morrison:
So I know that NEB's restriction enzymes have undergone a change in the last couple of years in regards to the inclusion of recombinant albumin as a stabilizing factor.
Bill Jack:
Right. The recombinant albumin has traditionally been put into a variety of different enzymes, and it's not completely clear what it does. It does stabilize the enzyme for whatever reason, whether that's keeping the enzyme from absorbing to the sides of the tubes or just helping it to stick together with friendly molecules. Nonetheless, the albumin has been an important part of things.
There have been some concerns, though, because traditionally, the albumin that was used was derived from a bovine source, and so, with some of the fears of mad cow disease, there have been restrictions in certain countries on using them. We have, therefore, switched to a recombinant albumin, which avoids any potential contamination from those sources, and we have extensively characterized our restriction enzymes to make sure that that recombinant albumin has the same properties, if not better properties, than did the original bovine serum albumin that was traditionally used.
Lydia Morrison:
Okay. Thanks for explaining that change to our listeners. So you touched on methylation of DNA. Are there any ways that that methylation affects enzyme cleavage?
Bill Jack:
Oh, that's a really good point because methylation is actually the part of the immune system for restriction modification systems. It's the modification that occurs in DNA. And so, when a host wants to protect its own DNA against its restriction enzyme, it will put a methyl group in the restriction recognition site that blocks the restriction enzyme from binding to that site and cleaving that side.
This is great, but it can occasionally present a complication because there are methylation events that occur naturally in other organisms. For example, the most prominent example is that when we are digesting DNA from higher eukaryotes, it often contains CpG modifications, methylation of that cytosine residue. If that CpG occurs either within a restriction enzyme site or overlapping a restriction enzyme site, that potentially can block or modulate the activity of that restriction enzyme.
And so we do have an extensive mapping and database that tells what effects methylation has on cleavage by all the enzymes that we sell. And so that's just one point that needs to come up and you need to think about when you're cutting particularly DNA from a host that does have methylation occurring within it. Part of the tools that we have identify which methylation events can block cleavage by specific enzymes and which enzymes are immune to any kind of methylation events.
Lydia Morrison:
Oh, thanks for that explanation. I think that'll be really helpful and is also a great tool for our listeners as they begin their journey with the restriction enzymes.
Bill Jack:
And hopefully continue their journeys with restriction enzymes.
Lydia Morrison:
Absolutely. Thanks so much for taking time out of your schedule to be here with me today, Bill. I've really enjoyed our conversation.
Bill Jack:
Oh, this has been a lot of fun. Thank you.
Lydia Morrison:
Thank you. See you soon.
Bill Jack:
Okay. Bye.
Lydia Morrison:
Thank you for joining us for this episode of the Lessons from Lab & Life podcast. I'd like to take a moment to remind you where to find the helpful resources we mentioned in our conversation. Head over to our website, neb.com, to find tools, buffer tables, and much more to support your use of restriction enzymes.
Also, please join us for our next episode, when I interview Dr. Jo Handelsman from the University of Wisconsin-Madison, who was selected as this year's Don Comb Memorial Lecturer for her experience in microbiome research and her work promoting diversity in STEM fields through changes in the classroom.
