Amplification of RNA by an RNA polymerase ribozyme

In summary, a recent publication discusses a significant advancement in replicating RNA systems. Researchers have found an RNA enzyme that can synthesize longer RNA molecules and fully replicate shorter RNA molecules without the use of proteins. However, it cannot replicate itself or its corresponding RNA, which would be another major step. The study also highlights the importance of ensuring the fidelity of replication in these systems.
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I stumbled upon this publication (News at phys.org).

As far as I understand, it is a big step towards replicating RNA systems. They found an RNA enzyme that can synthesize longer RNA molecules, and fully replicate shorter RNA molecules - completely without proteins. It cannot replicate itself (or its corresponding RNA), that would be another huge step.
 
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Hi mfb:

You will probably find the following also of interest

Self-Sustained Replication of an RNA Enzyme
Tracey A. Lincoln, Gerald F. Joyce*
* To whom correspondence should be addressed. E-mail: gjoyce@scripps.edu
Science 27 Feb 2009:
Vol. 323, Issue 5918, pp. 1229-1232
DOI: 10.1126/science.1167856

Abstract
An RNA enzyme that catalyzes the RNA-templated joining of RNA was converted to a format whereby two enzymes catalyze each other's synthesis from a total of four oligonucleotide substrates. These cross-replicating RNA enzymes undergo self-sustained exponential amplification in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about 1 hour and can be continued indefinitely. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates, during which recombinant replicators arose and grew to dominate the population. These replicating RNA enzymes can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.

Regards,
Buzz
 
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  • #3
Here is the paper that started interest in RNA synthesis as a step in the evolution of protein synthesis and modern life.

RNA evolution and the origins of life
GF Joyce - Nature, 1989 - its.caltech.edu
... The earliest organisms may have been required to provide their own reducing power, for example
using the reduced form of nicotinamide adenine dinucleotide ... simple and efficient reaction that
it would be surprising if it did not have some relevance to the early history of life
http://www.its.caltech.edu/~bch176/Joyce1989.pdf
 
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  • #4
jim mcnamara said:
Here is the paper that started interest in RNA synthesis as a step in the evolution of protein synthesis and modern life.

Athough Jerry Joyce is an important leader in the origin of life field, I would not say that the interest in RNA synthesis started with that paper. Most point to the 1968 articles by Francis Crick and Leslie Orgel (Joyce's PhD advisor) in the Journal of Molecular Biology as some of the first published papers describing the RNA world hypothesis (Carl Woese also published a book around the same time which also proposes the RNA world hypothesis):

Crick FH (1968). "The origin of the genetic code". J Mol Biol 38: 367. doi:https://dx.doi.org/10.1016%2F0022-2836%2868%2990392-6 [Broken].
Orgel LE (1968). "Evolution of the genetic apparatus". J Mol Biol 38: 381. doi:https://dx.doi.org/10.1016%2F0022-2836%2868%2990393-8 [Broken].

For example, in Crick's article, he speculates that the first enzyme was possibly "an RNA molecule with replicase properties" (the word ribozyme did not exist yet as they were not discovered until the 1980s by Cech, Altman and Pace).

Some of the first RNA synthesizing ribozymes were created by Jack Szostak's group (see Green and Szostak 1992). Since then, many research groups have made important strides in improving the capabilities of RNA to synthesize RNA, and researchers will continue these gradual improvements as even the polymerase described by the latest article does not have all the properties one would like a primordial RNA polymerase to have.
 
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  • #5
mfb said:
I stumbled upon this publication (News at phys.org).

As far as I understand, it is a big step towards replicating RNA systems. They found an RNA enzyme that can synthesize longer RNA molecules, and fully replicate shorter RNA molecules - completely without proteins. It cannot replicate itself (or its corresponding RNA), that would be another huge step.

According to the Abstract (see below) each RNA replicates itself indirectly. They work in pairs. Each one replicates the other, so the pair replicate a copy of the pair. Taken together, the reactions are autocatalytic. Autocatalytic small-chemical chemistry is fascinating in itself. If you so much as use a different batch of the same solvent in which to dissolve the reagents, you can get wildly different results - The contaminants in the solvent - even in < ppm range of concentrations are enough to change the course of these exquisitely sensitive reactions. In the case of RNA co-replication, small deviations in the sequence of the RNA product would likely accumulate over time, bringing the whole process to a halt - an evolutionary dead end, if you will. Therefore, another significant result would be a mechanism that ensures the fidelity of replication.
 
  • #6
Mark Harder said:
According to the Abstract (see below) each RNA replicates itself indirectly. They work in pairs. Each one replicates the other, so the pair replicate a copy of the pair. Taken together, the reactions are autocatalytic. Autocatalytic small-chemical chemistry is fascinating in itself. If you so much as use a different batch of the same solvent in which to dissolve the reagents, you can get wildly different results - The contaminants in the solvent - even in < ppm range of concentrations are enough to change the course of these exquisitely sensitive reactions. In the case of RNA co-replication, small deviations in the sequence of the RNA product would likely accumulate over time, bringing the whole process to a halt - an evolutionary dead end, if you will. Therefore, another significant result would be a mechanism that ensures the fidelity of replication.

P.S. Of course, the problem of accurate replication constitutes an evolutiionary pressure to ensure a faithful process. Either the reaction mechanism itself becomes all but foolproof (a little sloppiness creates 'mutations', a few of which can be 'beneficial'), or the process enlists co-factors, perhaps other co-evolving RNA sequences, that repair replication errors.
 
  • #7
Mark Harder said:
According to the Abstract (see below) each RNA replicates itself indirectly. They work in pairs. Each one replicates the other, so the pair replicate a copy of the pair. Taken together, the reactions are autocatalytic. Autocatalytic small-chemical chemistry is fascinating in itself. If you so much as use a different batch of the same solvent in which to dissolve the reagents, you can get wildly different results - The contaminants in the solvent - even in < ppm range of concentrations are enough to change the course of these exquisitely sensitive reactions. In the case of RNA co-replication, small deviations in the sequence of the RNA product would likely accumulate over time, bringing the whole process to a halt - an evolutionary dead end, if you will. Therefore, another significant result would be a mechanism that ensures the fidelity of replication.

Regarding the 2009 study on cross-replicating ribozymes that @Buzz Bloom cited, the reaction is indeed autocatalytic, but instead of sitting in a chaotic area of phase space where the initial conditions can greatly affect the final state, the system (much like many biological systems) instead appears to reside in a more stable area of phase space. The system itself is capable of tolerating mutation and the mutations create new ribozymes with different catalytic activities. The authors set up an evolution experiment in which they combined 12 different sets of cross-replicating ribozymes into the same test tube and performed twenty rounds of replication. Notably, in these experiments, the ribozymes can recombine with others in the reaction to generate new sets of cross-replicating ribozymes. At the end of the experiment, less than 10% of the ribozymes resembled those they started with, and the most abundant ribozymes at the end of the evolution experiment showed much higher catalytic activity than the other ribozymes tested indicating that the cross-replicating ribozymes can indeed successfully undergo natural selection. Rather than being an evolutionary dead end, the paper demonstrated a simple chemical system capable of evolution.

Link to the study in Science: http://science.sciencemag.org/content/323/5918/1229.full
Paper is freely available here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652413/

Because the RNA-replicating ribozyme from the 2016 study is not self-replicating, it is not yet capable of performing such evolutionary experiments.
 
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  • #8
Ygggdrasil said:
Carl Woese also published a book around the same time which also proposes the RNA world hypothesis
Hi Ygggdrasil:

Wikipedia listed just this one Woese book, and I could find nothing else on the Internet.
Woese, Carl (1967). The Genetic Code: the Molecular Basis for Genetic Expression. New York: Harper & Row. OCLC 293697
Is this the book you were referring to? If not, I would much appreciate your posting the title, publisher, and publication date.

Regards,
Buzz
 
  • #9
Buzz Bloom said:
Wikipedia listed just this one Woese book, and I could find nothing else on the Internet.
Woese, Carl (1967). The Genetic Code: the Molecular Basis for Genetic Expression. New York: Harper & Row. OCLC 293697
Is this the book you were referring to? If not, I would much appreciate your posting the title, publisher, and publication date.
Yes, that's the book I was referring to. Joyce cites that book along with Crick's and Orgel's articles when discussing the first proposal of the RNA world hypothesis (http://cshperspectives.cshlp.org/content/4/5/a003608.full). Apparently, the term "RNA world" was not coined until two decades later by Wally Gilbert (http://www.nature.com/nature/journal/v319/n6055/abs/319618a0.html).
 
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  • #10
Ygggdrasil said:
Yes, that's the book I was referring to.
Hi Ygggdrasil:

Thank you for your prompt reply to my question.

Regards,
Buzz
 
  • #11
Ygggdrasil said:
Regarding the 2009 study on cross-replicating ribozymes that @Buzz Bloom cited, the reaction is indeed autocatalytic, but instead of sitting in a chaotic area of phase space where the initial conditions can greatly affect the final state, the system (much like many biological systems) instead appears to reside in a more stable area of phase space. The system itself is capable of tolerating mutation and the mutations create new ribozymes with different catalytic activities. The authors set up an evolution experiment in which they combined 12 different sets of cross-replicating ribozymes into the same test tube and performed twenty rounds of replication. Notably, in these experiments, the ribozymes can recombine with others in the reaction to generate new sets of cross-replicating ribozymes. At the end of the experiment, less than 10% of the ribozymes resembled those they started with, and the most abundant ribozymes at the end of the evolution experiment showed much higher catalytic activity than the other ribozymes tested indicating that the cross-replicating ribozymes can indeed successfully undergo natural selection. Rather than being an evolutionary dead end, the paper demonstrated a simple chemical system capable of evolution...

Thanks for the clarification, Yggdrasil. I'll have to dig into this some more. The study sounds even more significant than first described. Of course, I've been out of the field for some years now. Who knows what wonders have been uncovered since then?
 
  • #12
mfb said:
I stumbled upon this publication (News at phys.org).

As far as I understand, it is a big step towards replicating RNA systems. They found an RNA enzyme that can synthesize longer RNA molecules, and fully replicate shorter RNA molecules - completely without proteins. It cannot replicate itself (or its corresponding RNA), that would be another huge step.
That is another important confirmation of evolution. Many accept the concept of variation and selection changing the heritable forms of life, but think the theory is weak in explaining the "beginning" of life. RNA is the most likely theoretical bridge between non-living chemistry, and chemistry that has a heritable component, and is thus subject to the forces of evolution: variation and selection.

Glancing at that abstract, this RNA molecule provides a physical example of how an RNA molecule can be that bridge from chemistry to life:
the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA
 
  • #13
votingmachine said:
but think the theory is weak in explaining the "beginning" of life
The beginning of life is not part of the theory of evolution, it is a separate research field (although concepts of biological evolution are relevant there as well).
 
  • #14
mfb said:
The beginning of life is not part of the theory of evolution, it is a separate research field (although concepts of biological evolution are relevant there as well).
That is a narrow reading of evolution, which explains the entire concept of life evolving from chemicals to complex multi-cellular species. Evolution DOES primarily focus on the process by which life evolves across generations, by selection of heritable genes. But the origin if life is also a part of that.

Life as we know it is entirely made of cells. There is no "pre-cellular" life in the world that we know of. Yet we also expect that pre-cellular life existed. And evolved into cellular life. The RNA hypothesis is a strong one, and the evolution of RNA molecules is a powerful example of how chemistry in the lab can show a "proof-of-principle" of the pre-cellular chemistry can be heritable, and could then evolve. The chemistry is a function of the RNA structure, which is a function of its sequence, which can mutate, and within a purely lab driven system, self-replicate.
 
  • #15
Ygggdrasil said:
Joyce cites that book along with Crick's and Orgel's articles when discussing the first proposal of the RNA world hypothesis (http://cshperspectives.cshlp.org/content/4/5/a003608.full).
Hi @Ygggdrasil:

I have been reading the Woese book looking for his discussion of what much later became called "The RNA World". In particular the Joyce article says in referring to the Woese book:
It was suggested that catalysts made entirely of RNA are likely to have been important at this early stage in the evolution of life, ...​
I was not able to find this discussion in the Woese book when I scanned it, but I have not yet read it fully.

The bulk of the book is what would be expected from the title: "The Genetic Code". It is a fascinating history of the investigations into solving the code following Watson-Crick. Chapter 7, "The Evolution of the Genetic Code", diverges from the main topic to discuss speculations regarding the pre-biotic environment. He presents an argument that any randomly generated poly-peptides would not have enzyme properties, but he still seems have the idea that cells of some kind served as the environment in which the mechanism developed that manufactured enzymatic poly-peptides based on the genetic code. He also says that DNA polymers would likely be much more common in that environment than RNA polymers.

If you have the Woese book available, I wonder if you might be able to help me find the location of the discussion about RNA acting in an catalytic manner which Joyce references.

Regards,
Buzz
 
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  • #16
Buzz Bloom said:
Hi @Ygggdrasil:

I have been reading the Woese book looking for his discussion of what much later became called "The RNA World". In particular the Joyce article says in referring to the Woese book:
It was suggested that catalysts made entirely of RNA are likely to have been important at this early stage in the evolution of life, ...​
I was not able to find this discussion in the Woese book when I scanned it, but I have not yet read it fully.

The bulk of the book is what would be expected from the title: "The Genetic Code". It is a fascinating history of the investigations into solving the code following Watson-Crick. Chapter 7, "The Evolution of the Genetic Code", diverges from the main topic to discuss speculations regarding the pre-biotic environment. He presents an argument that any randomly generated poly-peptides would not have enzyme properties, but he still seems have the idea that cells of some kind served as the environment in which the mechanism developed that manufactured enzymatic poly-peptides based on the genetic code. He also says that DNA polymers would likely be much more common in that environment than RNA polymers.

If you have the Woese book available, I wonder if you might be able to help me find the location of the discussion about RNA acting in an catalytic manner which Joyce references.

Regards,
Buzz

Unfortunately, I have not read Woese's book, so I can't be any help there.
 
  • #17
It's a bit of a quibble, but an important one: The origin of life was not a biological phenomenon; that is, it did not rely on life forms as an origin of new life forms. It must be a different kind of phenomenon; I would think it would be a matter of physics and chemistry and therefore not a form of biological evolution at all.
To take an example: for evolution to occur, the entities that mutate and out-reproduce one another must be discrete. Otherwise, you will just have pools of genomes mixed together with some RNAs out-reproducing others. Furthermore, the availability of substrates for building some ur-organism must be guaranteed to the genome responsible for generating them. It won't do to have a sort of 'primeval soup' at all. There must be a boundary between 'self' and 'other' for a given genome to guarantee is continued existence and proliferation.
All organisms known today (AFAIK) are comprised of cells around which a so-called lipid bilayer provides a barrier to the passage of water-soluble molecules; for example sodium and potassium ions and protons (which control pH). That also prevents the necessary macromolecules like RNA and proteins from drifting away. But by itself a lipid membrane is too impervious. In order to provide a suitable milieu, a cell must import and export small (and sometimes large) molecules in carefully controlled ways. It's important to modern life-forms that their cells maintain a gradient of metal ions - sodium outside and potassium inside - and protons. Fluxes of calcium and magnesium ions are important triggers of cell responses. It takes energy to maintain these energetically unfavorable gradients; and when coupled to the synthesis of energy-carrying molecules, the flow of ions down gradients creates useful chemical energy for the cell. And on and on. And the problems/opportunities membranes create for multicellular organisms? Let's not even go there. The necessity for compartmentation requires that some mechanism for accomplishing same must have been created very early in life's history. Impermeable membrane-like vesicles can be produced with the application of mechanical energy to suspensions of phospholipids in water media. So it's easy to imagine how primitive cell walls could be created without the intervention of elaborate biosynthetic chemical systems. However, it won't do to have little vesicles popping up and swallowing the right kind of RNAs at random. Whole chemical networks, including those that create more of the right kind of lipids, have to be scooped up together in approximately the right concentrations for the first living machine to come about.
I don't mean to rain on the RNA parade life here. This discovery is exciting and stimulating, but we have a tendency nowadays to reduce life to a simple matter of genetics - oh look, RNA can be a gene and catalyze the right kind of chemical reactions at the same time! - that sort of thing. My other point is that the origin of life problem won't have an answer until we can explain how things like membranes and proper packaging came about through physical and chemical means. Probably lots of other things as well.
 
  • #18
Mark Harder said:
It's a bit of a quibble, but an important one: The origin of life was not a biological phenomenon; that is, it did not rely on life forms as an origin of new life forms. It must be a different kind of phenomenon; I would think it would be a matter of physics and chemistry and therefore not a form of biological evolution at all.
To take an example: for evolution to occur, the entities that mutate and out-reproduce one another must be discrete. Otherwise, you will just have pools of genomes mixed together with some RNAs out-reproducing others. Furthermore, the availability of substrates for building some ur-organism must be guaranteed to the genome responsible for generating them. It won't do to have a sort of 'primeval soup' at all. There must be a boundary between 'self' and 'other' for a given genome to guarantee is continued existence and proliferation.
All organisms known today (AFAIK) are comprised of cells around which a so-called lipid bilayer provides a barrier to the passage of water-soluble molecules; for example sodium and potassium ions and protons (which control pH). That also prevents the necessary macromolecules like RNA and proteins from drifting away. But by itself a lipid membrane is too impervious. In order to provide a suitable milieu, a cell must import and export small (and sometimes large) molecules in carefully controlled ways. It's important to modern life-forms that their cells maintain a gradient of metal ions - sodium outside and potassium inside - and protons. Fluxes of calcium and magnesium ions are important triggers of cell responses. It takes energy to maintain these energetically unfavorable gradients; and when coupled to the synthesis of energy-carrying molecules, the flow of ions down gradients creates useful chemical energy for the cell. And on and on. And the problems/opportunities membranes create for multicellular organisms? Let's not even go there. The necessity for compartmentation requires that some mechanism for accomplishing same must have been created very early in life's history. Impermeable membrane-like vesicles can be produced with the application of mechanical energy to suspensions of phospholipids in water media. So it's easy to imagine how primitive cell walls could be created without the intervention of elaborate biosynthetic chemical systems. However, it won't do to have little vesicles popping up and swallowing the right kind of RNAs at random. Whole chemical networks, including those that create more of the right kind of lipids, have to be scooped up together in approximately the right concentrations for the first living machine to come about.
I don't mean to rain on the RNA parade life here. This discovery is exciting and stimulating, but we have a tendency nowadays to reduce life to a simple matter of genetics - oh look, RNA can be a gene and catalyze the right kind of chemical reactions at the same time! - that sort of thing. My other point is that the origin of life problem won't have an answer until we can explain how things like membranes and proper packaging came about through physical and chemical means. Probably lots of other things as well.
The theme of evolution is variation and selection can lead to differentiation of populations across generations. And the variation is specifically the RANDOM variation of genetic errors. I think that the idea is precisely that the beginning of life WAS an accidental, random event, not that it required a creator. The objections you raise ... that it seems improbable are ones that are frequently discussed.

It is important to recognize that a complex system did not have to arise instantaneously. That would indeed be improbable. The complex cell membrane was likely preceded by a less complex boundary system, which may have been preceded by a poorly bound system (adsorption to clays has been suggested).

I don't doubt that the pathway for the origin of life from non-living systems will be an open question for a long time. But it seems to me that a beginning point in pre-cellular life would likely be an RNA molecule. It satisfies the dual requirements of genotype and phenotype.
 
  • #19
votingmachine said:
The theme of evolution is variation and selection can lead to differentiation of populations across generations. And the variation is specifically the RANDOM variation of genetic errors. I think that the idea is precisely that the beginning of life WAS an accidental, random event, not that it required a creator. The objections you raise ... that it seems improbable are ones that are frequently discussed.

It is important to recognize that a complex system did not have to arise instantaneously. That would indeed be improbable. The complex cell membrane was likely preceded by a less complex boundary system, which may have been preceded by a poorly bound system (adsorption to clays has been suggested).

I don't doubt that the pathway for the origin of life from non-living systems will be an open question for a long time. But it seems to me that a beginning point in pre-cellular life would likely be an RNA molecule. It satisfies the dual requirements of genotype and phenotype.

I agree with all the above. However, I want it to be clear that I'm not questioning the notion that life could arise through random mechanisms involving some sort of trial-and-effort/success selection. Like you say, it's still an open question. I was trying to inject a note of caution into the discussion here, one that wasn't raised in any of my - admittedly limited - reading of the subject, and to raise one of the yet-to-be explained requirements for any mechanism for the genesis of life as we now know it. Another example would be the origin of the chirality of RNA and its precursors.
 
  • #20
Mark Harder said:
Another example would be the origin of the chirality of RNA and its precursors.
Hi Mark:

If you are interested in reading some material that deals with some of your issues, I recommend the following:
Genesis: The Scientific Quest for Life's Origin by Robert M. Hazen (2005)
Vital Dust: Life as a Cosmic Imperative by Christian De Duve (1996)
Singularities: Landmarks on the Pathways of Life by Christian De Duve (2005)​
Genesis is a personal history of various laboratory work related to origin issues. It includes a discussion of chirality.
Christian De Duve shared the 1974 Nobel prize for physiology and medicine, and he also explored many issues regarding origin. Vital Dust includes a very clear explanation of "The RNA World". Singularities presents a technically detailed discussion of a plausible sequence of important milestones leading to the origin.

Hope you enjoy these book as much as I did.

Regards,
Buzz
 
  • #21
Mark Harder said:
It's a bit of a quibble, but an important one: The origin of life was not a biological phenomenon; that is, it did not rely on life forms as an origin of new life forms. It must be a different kind of phenomenon; I would think it would be a matter of physics and chemistry and therefore not a form of biological evolution at all.
Do biological organisms not also operate under the rules of physical and chemistry?

In general I agree with your post—we currently do not understand all aspects of abiogenesis. In fact, we may never know how abiogenesis occurred because it's impossible to go back in time and observe what happened. What scientists can do, however, is try to come up with plausible scenarios to explain how life might have arose on early Earth (with the ultimate goal of perhaps coaxing a new origin of life in the lab). However, we are still very far away from that point. Despite many decades of work on RNA self-replication, even the article cited in the OP is nowhere close to providing a plausible mechanism for how self-replicating RNAs might have given rise to an RNA world. There is clearly still a lot of work to be done to understand how the origin of life occurred, and RNA replication is only one small piece of the puzzle. But just as there are smart people working on understanding the RNA world, there are also smart people thinking about proto-cells and how they might form, replicate, and integrate with genetic material.
 
  • #22
Thanks, Buzz.
 
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  • #23
Ygggdrasil said:
Do biological organisms not also operate under the rules of physical and chemistry?

Sorry about the confusion. No, the laws of chemistry and physics are not canceled in living systems. I was trying to say that the biological paradigms probably won't be of much use in understanding abiogenesis (good term, that.).
 

1. What is RNA amplification and how is it achieved?

RNA amplification is the process of increasing the amount of RNA molecules in a sample. This can be achieved by using an RNA polymerase ribozyme, which is an enzyme that can synthesize RNA using a DNA or RNA template.

2. How does an RNA polymerase ribozyme work?

An RNA polymerase ribozyme works by binding to a DNA or RNA template and using nucleotides to synthesize a complementary RNA strand. This process is similar to how a traditional protein-based RNA polymerase works, but the ribozyme is composed entirely of RNA molecules.

3. What are the advantages of using an RNA polymerase ribozyme for RNA amplification?

One advantage is that ribozymes are more stable and can function in a wider range of conditions compared to protein-based enzymes. Additionally, using an RNA polymerase ribozyme can eliminate the need for expensive protein-based enzymes and reduce the risk of contamination.

4. Is RNA amplification by an RNA polymerase ribozyme specific to a certain type of RNA?

No, RNA polymerase ribozymes can amplify a variety of RNA molecules, including messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

5. How is RNA amplification by an RNA polymerase ribozyme used in scientific research?

This technique is commonly used in studies that require a large amount of RNA for further analysis, such as gene expression studies and RNA sequencing. It is also used in diagnostic tests for detecting certain RNA viruses and in the production of RNA-based therapeutics.

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