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RNA rules discovered based on geometry.
University of Michigan researchers have discovered the rules that dictate the three-dimensional shapes of RNA molecules, rules that are based not on complex chemical interactions but simply on geometry.
The work, done by a team led by Hashim M. Al-Hashimi, is described in the Jan. 8, 2010, issue of the journal Science.
"RNA is a very floppy molecule that often functions by binding to something else and then radically changing shape," said Al-Hashimi, who is the Robert L. Kuczkowski Professor of Chemistry and a professor of biophysics. These shape changes, in turn, trigger other processes or cascades of events, such as turning specific genes on or off.
Because of the RNA molecule's mercurial nature, "you can't really define it as having a single structure," Al-Hashimi said. "It has many possible orientations, and different orientations are stabilized under different conditions, such as the presence of particular drug molecules."
A major goal in structural biology and biophysics is to be able to predict not only the complex three-dimensional shapes that RNA assumes (which are dictated by the order of its nucleic acid building blocks), but also the various shapes RNA takes on after binding to other molecules such as proteins and small-molecule drugs. Further, researchers would like to be able to manipulate the 3-D structure and resulting activity of RNA by tweaking the drug molecules with which it interacts. But to do that, they need to understand the rules that govern the anatomy of RNA.
The quest has parallels to the study of human anatomy, Al-Hashimi said. "Your body has a specific shape that changes predictably when you are walking or when you are catching a ball; we want to be able to understand these anatomical rules in RNA."
Manipulating RNA is a much sought-after goal, given the recent explosion in vital cellular roles ascribed to RNA and the growing number of diseases that are linked to RNA malfunction. RNA performs many of its roles by serving as a switch that changes shape in response to cellular signals, prompting appropriate reactions in response. The versatile molecule also is essential to retroviruses such as HIV, which have no DNA and instead rely on RNA to both transport and execute genetic instructions for everything the virus needs to invade and hijack its host.
In earlier work, Al-Hashimi's team determined that rather than changing shape in response to encounters with drug molecules, RNA goes through a predictable course of shape changes on its own. Drug molecules simply "wait for" the right shape and attach to RNA when the RNA assumes the particular drug's preferred orientation, Al Hashimi said.
But what rules control the predictable path of shapes the RNA molecule assumes? And are those rules the same for all sorts of RNA molecules? In the current work, Al-Hashimi's team investigated those questions.
"RNA is very similar to the human body in its construction, in that it's made up of limbs that are connected at joints," Al-Hashimi said. The limbs are the familiar, ladder-like double helix structures, and the joints are flexible junctions. The prevailing view was that interactions among loopy structures at the tips of the limbs played a role in defining the molecule's overall 3-D shape, much as a handshake defines the orientation of two arms, but Al-Hashimi's group decided to look at things from a different perspective.
"We wondered if the junctions themselves might provide the definition," Al-Hashimi said. "If you look at your arm, you'll notice that you can't move it, relative to your shoulder, in just any way; it's confined to a certain pathway because of the joint's geometry. We wondered if the same thing might be true of RNA."
To investigate that possibility, the researchers turned to a database of RNA structures and found that all structures with two helices linked by a particular type of junction called a trinucleotide bulge fell along the same pathway.
The team then went on to explore structures of RNA molecules with other kinds of junctions. All were confined to similar pathways, but the precise pathway of a given RNA depended upon structural features of its junction. Just as anatomical features of our shoulders, elbows, hips and knees define the range of motion of our arms and legs, the anatomy of RNA's junctions dictates the motion of its helices.
Next, Al-Hashimi and coworkers wanted to understand how drug molecules cause RNA molecules to freeze in specific positions. In earlier work with an RNA molecule known as TAR, which is critical for replication of HIV and thus a key target for anti-HIV drugs, the researchers had found that certain drug molecules froze the RNA molecule in a nearly straight position, while others trapped the molecule in a bent conformation and still others captured positions between the two extremes. But because that project involved a wide variety of drug molecules, it was hard to figure out why certain ones preferred certain orientations.
To explore the issue more methodically, Al-Hashimi's group used a series of aminoglycosides (antibiotics that are known to target RNA) that systematically differed from one another in charge, size and other chemical properties. Size turned out to be the key: bigger aminoglycosides froze RNA in more bent positions; smaller ones favored straighter RNA structures. Looking more closely, the researchers discovered that the aminoglycoside molecule nestles between two helices and acts like wedge, forcing the helices apart. Examination of other RNA structures bound to small molecules revealed that this rule is not specific to TAR but a more general feature of RNA-small molecule interactions.
"With these findings, it now should be possible to predict gross features of RNA 3-D shapes based only on their secondary structure, which is far easier to determine than is 3-D structure," Al-Hashimi said. "This will make it possible to gain insights into the 3-D shapes of RNA structures that are too large or complicated to be visualized by experimental techniques such as X-ray crystallography and NMR spectroscopy. The anatomical rules also provide a blueprint for rationally manipulating the structure and thus the activity of RNA, using small molecules in drug design efforts and also for engineering RNA sensors that change structure in user-prescribed ways."
Al-Hashimi's coauthors on the Science paper were graduate students Maximillian Bailor and Xiaoyan Sun. Funding was provided by the Michigan Economic Development Corporation, the Michigan Technology Tri-Corridor, the W.F. Keck Foundation, the National Science Foundation and the National Institutes of Health.
Source:
Nancy Ross-Flanigan
University of Michigan
The work, done by a team led by Hashim M. Al-Hashimi, is described in the Jan. 8, 2010, issue of the journal Science.
"RNA is a very floppy molecule that often functions by binding to something else and then radically changing shape," said Al-Hashimi, who is the Robert L. Kuczkowski Professor of Chemistry and a professor of biophysics. These shape changes, in turn, trigger other processes or cascades of events, such as turning specific genes on or off.
Because of the RNA molecule's mercurial nature, "you can't really define it as having a single structure," Al-Hashimi said. "It has many possible orientations, and different orientations are stabilized under different conditions, such as the presence of particular drug molecules."
A major goal in structural biology and biophysics is to be able to predict not only the complex three-dimensional shapes that RNA assumes (which are dictated by the order of its nucleic acid building blocks), but also the various shapes RNA takes on after binding to other molecules such as proteins and small-molecule drugs. Further, researchers would like to be able to manipulate the 3-D structure and resulting activity of RNA by tweaking the drug molecules with which it interacts. But to do that, they need to understand the rules that govern the anatomy of RNA.
The quest has parallels to the study of human anatomy, Al-Hashimi said. "Your body has a specific shape that changes predictably when you are walking or when you are catching a ball; we want to be able to understand these anatomical rules in RNA."
Manipulating RNA is a much sought-after goal, given the recent explosion in vital cellular roles ascribed to RNA and the growing number of diseases that are linked to RNA malfunction. RNA performs many of its roles by serving as a switch that changes shape in response to cellular signals, prompting appropriate reactions in response. The versatile molecule also is essential to retroviruses such as HIV, which have no DNA and instead rely on RNA to both transport and execute genetic instructions for everything the virus needs to invade and hijack its host.
In earlier work, Al-Hashimi's team determined that rather than changing shape in response to encounters with drug molecules, RNA goes through a predictable course of shape changes on its own. Drug molecules simply "wait for" the right shape and attach to RNA when the RNA assumes the particular drug's preferred orientation, Al Hashimi said.
But what rules control the predictable path of shapes the RNA molecule assumes? And are those rules the same for all sorts of RNA molecules? In the current work, Al-Hashimi's team investigated those questions.
"RNA is very similar to the human body in its construction, in that it's made up of limbs that are connected at joints," Al-Hashimi said. The limbs are the familiar, ladder-like double helix structures, and the joints are flexible junctions. The prevailing view was that interactions among loopy structures at the tips of the limbs played a role in defining the molecule's overall 3-D shape, much as a handshake defines the orientation of two arms, but Al-Hashimi's group decided to look at things from a different perspective.
"We wondered if the junctions themselves might provide the definition," Al-Hashimi said. "If you look at your arm, you'll notice that you can't move it, relative to your shoulder, in just any way; it's confined to a certain pathway because of the joint's geometry. We wondered if the same thing might be true of RNA."
To investigate that possibility, the researchers turned to a database of RNA structures and found that all structures with two helices linked by a particular type of junction called a trinucleotide bulge fell along the same pathway.
The team then went on to explore structures of RNA molecules with other kinds of junctions. All were confined to similar pathways, but the precise pathway of a given RNA depended upon structural features of its junction. Just as anatomical features of our shoulders, elbows, hips and knees define the range of motion of our arms and legs, the anatomy of RNA's junctions dictates the motion of its helices.
Next, Al-Hashimi and coworkers wanted to understand how drug molecules cause RNA molecules to freeze in specific positions. In earlier work with an RNA molecule known as TAR, which is critical for replication of HIV and thus a key target for anti-HIV drugs, the researchers had found that certain drug molecules froze the RNA molecule in a nearly straight position, while others trapped the molecule in a bent conformation and still others captured positions between the two extremes. But because that project involved a wide variety of drug molecules, it was hard to figure out why certain ones preferred certain orientations.
To explore the issue more methodically, Al-Hashimi's group used a series of aminoglycosides (antibiotics that are known to target RNA) that systematically differed from one another in charge, size and other chemical properties. Size turned out to be the key: bigger aminoglycosides froze RNA in more bent positions; smaller ones favored straighter RNA structures. Looking more closely, the researchers discovered that the aminoglycoside molecule nestles between two helices and acts like wedge, forcing the helices apart. Examination of other RNA structures bound to small molecules revealed that this rule is not specific to TAR but a more general feature of RNA-small molecule interactions.
"With these findings, it now should be possible to predict gross features of RNA 3-D shapes based only on their secondary structure, which is far easier to determine than is 3-D structure," Al-Hashimi said. "This will make it possible to gain insights into the 3-D shapes of RNA structures that are too large or complicated to be visualized by experimental techniques such as X-ray crystallography and NMR spectroscopy. The anatomical rules also provide a blueprint for rationally manipulating the structure and thus the activity of RNA, using small molecules in drug design efforts and also for engineering RNA sensors that change structure in user-prescribed ways."
Al-Hashimi's coauthors on the Science paper were graduate students Maximillian Bailor and Xiaoyan Sun. Funding was provided by the Michigan Economic Development Corporation, the Michigan Technology Tri-Corridor, the W.F. Keck Foundation, the National Science Foundation and the National Institutes of Health.
Source:
Nancy Ross-Flanigan
University of Michigan
Hi merry,
Thanks for the reply. I believe virus structure was predicted by a mathemetician before it was ever seen, so go easy on the numbers guys . I think biophysics has emerged as arguably the most important weapon in the fight against HCV and other viruses..I've always thought that the intertwining of various scientific disciplines (biology,math,physics,medicine) that were once solitary areas of study is what it will take to eventually unravel the secrets of viruses.
I don't know what the implications are for bent vs. straight RNA ? I have more to read and hopefully I will find something that throws more light on the topic. Anything you might know about this topic of course would be appreciated.
ML
Thanks for the reply. I believe virus structure was predicted by a mathemetician before it was ever seen, so go easy on the numbers guys . I think biophysics has emerged as arguably the most important weapon in the fight against HCV and other viruses..I've always thought that the intertwining of various scientific disciplines (biology,math,physics,medicine) that were once solitary areas of study is what it will take to eventually unravel the secrets of viruses.
I don't know what the implications are for bent vs. straight RNA ? I have more to read and hopefully I will find something that throws more light on the topic. Anything you might know about this topic of course would be appreciated.
ML
Hi Trish,
Thanks for the reply.
Most of the vaccine work being done these days is for therapeutic vaccines, as opposed to preventative. Either way you could just about put all I know about both on a pinhead.
"If they are able to determine why RNA forms the shapes it does"
I am still trying to determine the significance of the cyclical nature of the geometric changes to the RNA as well as finding why it assumes these particular geometric shapes . (see post to Willing) So, in short I don't yet know the answer.
"what creates wedges between the helixes"
I think this discovery was a byproduct of the experiment to determine what froze the RNA in a bent or straight shape. They noticed that the molecule being used was found wedged between two helices. Theoretically, if you could find the correct molecule to wedge between the exact nucleotides on the helix that are needed to initiate transcription of RNA then replication could not occur. By separating the two specific nucleotides apart the bond between the them (which are held together by hydrogen and electrostatic bonds) is broken which stops the process necessary for replication of RNA to initiate. I have some more on this topic I will read and post later. Somewhere in the process maybe I will have an epiphany and will be able to provide a more complete understanding of the significance of the findings above.
ML
Thanks for the reply.
Most of the vaccine work being done these days is for therapeutic vaccines, as opposed to preventative. Either way you could just about put all I know about both on a pinhead.
"If they are able to determine why RNA forms the shapes it does"
I am still trying to determine the significance of the cyclical nature of the geometric changes to the RNA as well as finding why it assumes these particular geometric shapes . (see post to Willing) So, in short I don't yet know the answer.
"what creates wedges between the helixes"
I think this discovery was a byproduct of the experiment to determine what froze the RNA in a bent or straight shape. They noticed that the molecule being used was found wedged between two helices. Theoretically, if you could find the correct molecule to wedge between the exact nucleotides on the helix that are needed to initiate transcription of RNA then replication could not occur. By separating the two specific nucleotides apart the bond between the them (which are held together by hydrogen and electrostatic bonds) is broken which stops the process necessary for replication of RNA to initiate. I have some more on this topic I will read and post later. Somewhere in the process maybe I will have an epiphany and will be able to provide a more complete understanding of the significance of the findings above.
ML
"What I don't totally understand is the significance of the RNA changing its geometry in a predictable cycle. I'm still trying to digest this so any insight would be welcome. shapes "
To me, the difference between morphing predictably vs morphing UNpredictably to date means that they'll be able to know what pattern to disrupt, whereas in the past the pattern was unknown. Too simplistic of an interpretation?
To me, the difference between morphing predictably vs morphing UNpredictably to date means that they'll be able to know what pattern to disrupt, whereas in the past the pattern was unknown. Too simplistic of an interpretation?
Hi Willing--
I don't know if you remember the Heptazyme trials or not but their drug was an artificial ribozyme that was designed to stop viral replication by targeting the IRES region and cutting up the rna in such a way that it could not develop the translational proteins that it needed. It was somewhat effective in lowering vl but it had some serious toxicity issues and the trial was halted in stage II.
What I don't totally understand is the significance of the RNA changing its geometry in a predictable cycle. I'm still trying to digest this so any insight would be welcome. shapes
I don't know if you remember the Heptazyme trials or not but their drug was an artificial ribozyme that was designed to stop viral replication by targeting the IRES region and cutting up the rna in such a way that it could not develop the translational proteins that it needed. It was somewhat effective in lowering vl but it had some serious toxicity issues and the trial was halted in stage II.
What I don't totally understand is the significance of the RNA changing its geometry in a predictable cycle. I'm still trying to digest this so any insight would be welcome. shapes
If they are able to determine why RNA forms the shapes it does and also what creates wedges between the helixes, etc. - is this a good step towards the vaccine that has been elusive due to the way the HCV RNA is always morphing? Or not?
thanks for posting! very interesting.
I'm not sure whether structures for any other fragment of the HCV genome other than the IRES (eg PDB id 2PN4) are available. However alpha-helix loops are a significant part of that structure so it'd be interesting to check how applicable these methods are for that case.
I'm not sure whether structures for any other fragment of the HCV genome other than the IRES (eg PDB id 2PN4) are available. However alpha-helix loops are a significant part of that structure so it'd be interesting to check how applicable these methods are for that case.
so, in laymaese all we need is the right monkey wrench ; )
or something large to get our hcv bent?? ; )
glad to see mathematicians take up the challenge. after all, its not just interupting the transcription phase of RNA that's needful, it understanding how and why, when and what it most effective.
next step would then be to find one that interupts viral RNA without doing the same to our healthy cells. now that's one we can all hope will be discovered sooner rather than later.
or something large to get our hcv bent?? ; )
glad to see mathematicians take up the challenge. after all, its not just interupting the transcription phase of RNA that's needful, it understanding how and why, when and what it most effective.
next step would then be to find one that interupts viral RNA without doing the same to our healthy cells. now that's one we can all hope will be discovered sooner rather than later.
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