Biology and the origin of life
- Thread starter Fucky McFuckerson
- Start date
Just saw this.. My main concern however, is the condition and initial habitation of the Khoisan. One must keep in mind that due to the Bantu migrations, many who happen to inhabit south Africa have either displaced, or mingled with the indigenous Khoisan. Again, it is postulated that the Bantu migrated from southwestern Nigeria, and by way of demic diffusion, settled most of southern Africa, but this does not neglect the current and historical presence of the Khoisan in the region, who still inhabit southwestern Africa.
Okay..
Okay..
This isn't the point. This here, can be seen as a matter of subjective opinion, as I believe him to be logical in some aspects, but in others too overcompensating and willing to discredit, that logic and reason goes out of the window. To call him a scientist however, is dickriding, I don't care how you twist it, I really don't. To include his name among a list of scientists is ridiculously gay..
I won't even disagree with this, but please see above on why you're delusional..
Huh????? This is my best example of cheerleading. G has never destroyed me anywhere, though I have destroyed you on several occasions in concern to the stupid shit you've said on here. I'm not a cop-out like you, if someone has a point, it is well-taken, but if they are being irrelevant and merely imposing a subjective opinion, it won't be taken into consideration.
By misleading him and including G in a list of reliable sources. G I'm sure is quite familiar with credentials and the peer review process, I doubt he has any work posted in any peer reviewed journals. Understanding the work is entirely different.
I don't chastise him for his love of science as much as for his unrelenting criticism of other people's beliefs and trying his hardest to destroy them and criticizing those who hold them. One thing good I can and have admitted about the guy, is that he's smarter than you on all fronts. Same with me, since you have no subject period that you're well versed in, you merely parrot others and appeal to false authority.
LOL! Are you serious? I follow dude nowhere. He always addresses me first when ever I make some type of theist claim, in relation to science and the compatibility of the two. I don't even consider it stalking, he's free to express his opinion. I came into this thread addressing someone else, G responded to something I said, and I even agreed. I fucked your mom created this thread so please, stop grasping at straws and accept your submissive position to the guy, which you further prove here..
Now you're on Heresy's dick, we are two entirely different people. Both of us just happen to be opinionated and have beliefs that conflict yours. And please don't reverse the argument, the jig is up! LOL! You're a clown..
ThaG said:
Make sure that you frequently check out the latest issues of Cell and Nature starting from December this year 
Are you going to include me in the list of scientists then???
Are you going to exclude from the list people like Galileo who never published anything peer-reviewed?
Are you going to include me in the list of scientists then???
Are you going to exclude from the list people like Galileo who never published anything peer-reviewed?
No,
All I wanted to say is that having published peer-reviewed papers alone does not equal being a scientist
Certainly, a person with h-index 100 is a much greater scientist than those whose h-index is 0 (as mine is right now) are
But being a scientist is a state of mind, not a profession, not a title, try to understand it
You may have your name on a fair amount of peer-reviewed papers and still not be a scientist. Examples: most engineers and plenty of lab technicians who get their names on papers for doing the grunt work. In the same time not having papers out does not mean you are not a scientist
All I wanted to say is that having published peer-reviewed papers alone does not equal being a scientist
Certainly, a person with h-index 100 is a much greater scientist than those whose h-index is 0 (as mine is right now) are
But being a scientist is a state of mind, not a profession, not a title, try to understand it
You may have your name on a fair amount of peer-reviewed papers and still not be a scientist. Examples: most engineers and plenty of lab technicians who get their names on papers for doing the grunt work. In the same time not having papers out does not mean you are not a scientist
Actually, editors have to verify your credentials and find the information to be acceptable by way of scientific standards. A non-scientist cannot publish a study in a peer-reviewed scientific journal. Common sense and actual fact.
Goes with out saying. But a scientist is one who adheres to the scientific method and bases their conclusions and results of their research from this method. Anyone with a passion for knowing can be a scientist (I didn't mean it in that context when saying that you're not a scientist), but someone with an h-index of 0, who has published and contributed nothing whatsoever to the scientific community, isn't considered a reliable source by scientific standards, even if one would consider that person to be a "scientist" by definition..
?? Who once said that scientific aspiration is a profession? Certainly not me, I'm zoning in on individual reliability.
If science isn't a profession (like engineering is), then how are these people not scientists if they use the scientific method to attain results? Not to mention enough scientific credentials to be accepted in being cited in a scientific journal? Engineering deals with mechanisms of science; applicable science. You've contradicted yourself here. Not having any papers published doesn't make you a non-scientist, but it makes you unreliable by academic standards for scientific information since no one has confirmed or denounced your results... Merely parroting the results of others is not science, it is journalism..
Goes with out saying. But a scientist is one who adheres to the scientific method and bases their conclusions and results of their research from this method. Anyone with a passion for knowing can be a scientist (I didn't mean it in that context when saying that you're not a scientist), but someone with an h-index of 0, who has published and contributed nothing whatsoever to the scientific community, isn't considered a reliable source by scientific standards, even if one would consider that person to be a "scientist" by definition..
?? Who once said that scientific aspiration is a profession? Certainly not me, I'm zoning in on individual reliability.
If science isn't a profession (like engineering is), then how are these people not scientists if they use the scientific method to attain results? Not to mention enough scientific credentials to be accepted in being cited in a scientific journal? Engineering deals with mechanisms of science; applicable science. You've contradicted yourself here. Not having any papers published doesn't make you a non-scientist, but it makes you unreliable by academic standards for scientific information since no one has confirmed or denounced your results... Merely parroting the results of others is not science, it is journalism..
You know better than this since they only do it to support their OWN conclusions, which is why I said merely..
When (notice I said "when" and not "if", since I'm not doubting you) that day comes, what am I shutting up about? This is immaterial..
anyway, when some day (hopefully in the very near future) I have papers in journals with IFs around 30, are you going to shut up
PDF: http://www.sendspace.com/file/zgkowc
Or text without the images...
___________________________________________________________
Self-Sustained Replication of an RNA Enzyme
Tracey A. Lincoln and Gerald F. Joyce*
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.
Department of Chemistry, Department of Molecular Biology, and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
* To whom correspondence should be addressed. E-mail: [email protected]
A long-standing research goal has been to devise a nonbiological system that undergoes replication in a self-sustained manner, brought about by enzymatic machinery that is part of the system being replicated. One way to realize this goal, inspired by the notion of primitive RNA-based life, would be for an RNA enzyme to catalyze the replication of RNA molecules, including the RNA enzyme itself (1–4). This has now been achieved in a cross-catalytic system involving two RNA enzymes that catalyze each other's synthesis from a total of four component substrates.
The "R3C" RNA enzyme is an RNA ligase that binds two oligonucleotide substrates through Watson-Crick pairing and catalyzes nucleophilic attack of the 3'-hydroxyl of one substrate on the 5'-triphosphate of the other, forming a 3',5'-phosphodiester and releasing inorganic pyrophosphate (5). The R3C ligase was configured to self-replicate by joining two RNA molecules to produce another copy of itself (6). This process was inefficient because the substrates formed a nonproductive complex that limited the extent of exponential growth, with a doubling time of about 17 hours and no more than two successive doublings.
The R3C ligase subsequently was converted to a cross-catalytic format (Fig. 1A), whereby a plus-strand RNA enzyme (E) catalyzes the joining of two substrates (A' and B') to form a minus-strand enzyme (E'), which in turn catalyzes the joining of two substrates (A and B) to form a new plus-strand enzyme (7, 8). This too was inefficient because of the formation of nonproductive complexes and the slow underlying rate of the two enzymes. The enzymes E and E' operate with a rate constant of only ~0.03 min–1 and a maximum extent of only 10 to 20% (9). These rates are about 10 times slower than that of the parental R3C ligase (5), and when the two cross-catalytic reactions are carried out within a common mixture the rates are even slower (7).
Figure 1 Fig. 1. Cross-replicating RNA enzymes. (A) The enzyme E' (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, whereas E catalyzes ligation of A' and B' to form E'. The two enzymes dissociate to provide copies that can catalyze another reaction. (B) Sequence and secondary structure of the complex formed between the enzyme and its two substrates (E', A, and B are shown; E, A', and B' are the reciprocal). The curved arrow indicates the site of ligation. Solid boxes indicate critical wobble pairs that provide enhanced catalytic activity. Dashed boxes indicate paired regions and catalytic nucleotides that were altered to construct various cross replicators. (C) Variable portion of 12 different E enzymes. The corresponding E' enzymes have a complementary sequence in the paired region and the same sequence of catalytic nucleotides (alterations relative to the E1 enzyme are highlighted). [View Larger Version of this Image (24K GIF file)]
The catalytic properties of the cross-replicating RNA enzymes were improved by the use of in vitro evolution, optimizing the two component reactions in parallel and seeking solutions that would apply to both reactions when conducted in the cross-catalytic format (9). The 5'-triphosphate–bearing substrate was joined to the enzyme via a hairpin loop (B' to E and B to E'), and nucleotides within both the enzyme and the separate 3'-hydroxyl–bearing substrate (A' and A) were randomized at a frequency of 12% per position. The two resulting populations of molecules were subjected to six rounds of stringent in vitro selection, selecting for their ability to react in progressively shorter times, ranging from 2 hours to 10 ms. Mutagenic polymerase chain reaction was performed after the third round to maintain diversity in the population. After the sixth round, individuals were cloned from both populations and sequenced. There was substantial sequence variability among the clones, but all contained mutations just upstream from the ligation junction that resulted in a G·U wobble pair at this position.
The G·U pair was installed in both enzymes and both 3'-hydroxyl–bearing substrates (Fig. 1B). In the trimolecular reaction (with two separate substrates), the optimized enzymes E and E' exhibited a rate constant of 1.3 and 0.3 min–1 with a maximum extent of 92 and 88%, respectively. The optimized enzymes underwent robust exponential amplification at a constant temperature of 42°C, with more than 25-fold amplification after 5 hours, followed by a leveling off as the supply of substrates became depleted (Fig. 2A). The data fit well to the logistic growth equation [E]t = a/(1 + be–ct), where [E]t is the concentration of E (or E') at time t, a is the maximum extent of growth, b is the degree of sigmoidicity, and c is the exponential growth rate. For the enzymes E and E', the exponential growth rate was 0.92 and 1.05 hour–1, respectively.
Figure 2 Fig. 2. Self-sustained amplification of cross-replicating RNA enzymes. (A) The yield of both E (black curve) and E' (gray curve) increased exponentially before leveling off as the supply of substrates became exhausted. (B) Amplification was sustained by performance of a serial transfer experiment, allowing approximately 25-fold amplification before transferring 4% of the mixture to a new reaction vessel that contained a fresh supply of substrates. The concentrations of E and E' were measured at the end of each incubation. [View Larger Version of this Image (13K GIF file)]
Exponential growth can be continued indefinitely in a serial transfer experiment in which a portion of a completed reaction mixture is transferred to a new reaction vessel that contains a fresh supply of substrates. Six successive reactions were carried out in this fashion, each 5 hours in duration and transferring 4% of the material from one reaction mixture to the next. The first mixture contained 0.1 µM E and 0.1 µM E', but all subsequent mixtures contained only those enzymes that were carried over in the transfer. Exponential growth was maintained throughout 30 hours total of incubation, with an overall amplification of greater than 108-fold for each of the two enzymes (Fig. 2B).
It is possible to construct variants of the cross-replicating RNA enzymes that differ in the regions of Watson-Crick pairing between the cross-catalytic partners without markedly affecting replication efficiency. These regions are located at the 5' and 3' ends of the enzyme (Fig. 1B). Four nucleotide positions at both the 5' and 3' ends were varied, adopting the rule that each region contains one G·C and three A·U pairs so that there would be no substantial differences in base-pairing stability. Of the 32 possible pairs of complementary sequences for each region, 12 were chosen as a set of designated pairings (Fig. 1C). Each pairing was associated with a particular sequence within the catalytic core of both members of a cross-replicating pair. Twelve pairs of cross-replicating enzymes were synthesized, as well as the 48 substrates (12 each of A, A', B, and B') necessary to support their exponential amplification. Each replicator was individually tested and demonstrated varying levels of catalytic activity and varying rates of exponential growth (fig. S1). The pair shown in Fig. 1B (now designated E1 and E1') had the fastest rate of exponential growth, achieving about 20-fold amplification after 5 hours. The various cross-replicating enzymes shown in Fig. 1C had the following rank order of replication efficiency: E1, E10, E5, E4, E6, E3, E12, E7, E9, E8, E2, E11. The top five replicators all achieved more than 10-fold amplification after 5 hours, and all except E11 achieved at least fivefold amplification after 5 hours.
A serial-transfer experiment was initiated with a 0.1 µM concentration each of E1 to E4 and E1' to E4' and a 5.0 µM concentration of each of the 16 corresponding substrates. Sixteen successive transfers were carried out over 70 hours, transferring 5% of the material from one reaction mixture to the next (fig. S2A). Individuals were cloned from the population after the final reaction and sequenced. Among 25 clones (sequencing E' only), there was no dominant replicator (fig. S2B). E1', E2', E3', and E4' all were represented, as well as 17 clones that were the result of recombination between a particular A' substrate and one of the three B' substrates other than its original partner (or similarly for A and B). Recombination occurs when an enzyme binds and ligates a mismatched substrate. In principle, any A could become joined to any B or B', and any A' could become joined to any B' or B, resulting in 64 possible enzymes. The set of replicators were designed so that cognate substrates have a binding advantage of several kilocalories per mole as compared with noncognate substrates (fig. S2C), but once a mismatched substrate is bound and ligated, it forms a recombinant enzyme that also can cross-replicate. Recombinants can give rise to other recombinants, as well as revert back to nonrecombinants. Based on relative binding affinities, there are expected to be preferred pathways for mutation, primarily involving substitution among certain A' or among certain B components (fig. S2D).
A second serial transfer experiment was initiated with a 0.1 µM concentration each of all 12 pairs of cross-replicating enzymes and a 5.0 µM concentration of each of the 48 corresponding substrates. This mixture allowed 132 possible pairs of recombinant cross-replicating enzymes as well as the 12 pairs of nonrecombinant cross-replicators. Twenty successive reactions were carried out over 100 hours, transferring 5% of the material from one reaction mixture to the next, and achieving an overall amplification of greater than 1025-fold (Fig. 3A). Of 100 clones isolated after the final reaction (sequencing 50 E and 50 E'), only 7 were nonrecombinants (Fig. 3B). The distribution was highly nonuniform, with sparse representation of molecules containing components A6 to A12 and B5 to B12 (and reciprocal components B6' to B12' and A5' to A12'). The most frequently represented components were A5 and B3 (and reciprocal components B5' and A3'). The three most abundant recombinants were A5B2, A5B3, and A5B4 (and their cross-replication partners), which together accounted for one third of all clones.
Figure 3 Fig. 3. Self-sustained amplification of a population of cross-replicating RNA enzymes, resulting in selection of the fittest replicators. (A) Beginning with 12 pairs of cross-replicating RNA enzymes (Fig. 1C), amplification was sustained for 20 successive rounds of ~20-fold amplification and 20-fold dilution. The concentrations of all E (black) and E' (gray) molecules were measured after each incubation. (B) Graphical representation of 50 E and 50 E' clones (dark and light columns, respectively) that were sequenced after the last incubation. The A and B (or B' and A') components of the various enzymes are shown on the horizontal axes, with nonrecombinant enzymes indicated by shaded boxes along the diagonal. The number of clones containing each combination of components is shown on the vertical axis. (C) Comparative growth of E1 (circles) and A5B3 (squares) in the presence of either their cognate substrates alone (solid symbols) or all substrates that were present during serial transfer (open symbols). (D) Growth of A5B3 (black curve) and B5'A3' (gray curve) in the presence of the eight substrates (A5, B2, B3, B4, B5', A2', A3', and A4') that comprise the three most abundant cross-replicating enzymes. [View Larger Version of this Image (38K GIF file)]
In the presence of their cognate substrates alone, E1 remained the most efficient replicator, but in the presence of all 48 substrates, the most efficient replicator was A5B3 (Fig. 3C). When the A5B3 replicator was provided with a mixture of substrates corresponding to the components of the three most abundant recombinants, its exponential growth rate was the highest measured for any replicator (Fig. 3D). The fitness of a pair of cross-replicating enzymes depends on several factors, including their intrinsic catalytic activity, exponential growth rate with cognate substrates, ability to withstand inhibition by other substrates in the mixture, and net rate of production through mutation among the various cross-replicators. The A5B3 recombinant and its cross-replication partner B5'A3' have different catalytic cores (Fig. 1C), and both exhibit comparable activity, accounting for their well-balanced rate of production throughout the course of exponential amplification (Fig. 3D). The selective advantage of this cross-replicator appears to derive from its relative resistance to inhibition by other substrates in the mixture (Fig. 3C) and its ability to capitalize on facile mutation among substrates B2, B3, and B4 and among substrates A2', A3', and A4' that comprise the most abundant recombinants (fig. S2D).
Populations of cross-replicating RNA enzymes can serve as a simplified experimental model of a genetic system with, at present, two genetic loci and 12 alleles per locus. It is likely, however, that the number of alleles could be increased by exploiting more than four nucleotide positions at the 5' and 3' ends of the enzyme and by relaxing the rule that these nucleotides form one G·C and three A·U pairs. In order to support much greater complexity, it will be necessary to constrain the set of substrates, for example, by using the population of newly formed enzymes to generate a daughter population of substrates (9). An important challenge for an artificial RNA-based genetic system is to support a broad range of encoded functions, well beyond replication itself. Ultimately, the system should provide open-ended opportunities for discovering novel function, something that probably has not occurred on Earth since the time of the RNA world but presents an increasingly tangible research opportunity.
References and Notes
* 1. F. H. C. Crick, J. Mol. Biol. 38, 367 (1968). [CrossRef] [ISI] [Medline][UC-eLinks]
* 2. J. W. Szostak, D. P. Bartel, P. L. Luisi, Nature 409, 387 (2001). [CrossRef] [Medline][UC-eLinks]
* 3. G. F. Joyce, Nature 418, 214 (2002). [CrossRef] [Medline][UC-eLinks]
* 4. L. E. Orgel, Crit. Rev. Biochem. Mol. Biol. 39, 99 (2004). [CrossRef] [ISI] [Medline][UC-eLinks]
* 5. J. Rogers, G. F. Joyce, RNA 7, 395 (2001).[Abstract]
* 6. N. Paul, G. F. Joyce, Proc. Natl. Acad. Sci. U.S.A. 99, 12733 (2002).[Abstract/Free Full Text]
* 7. D.-E. Kim, G. F. Joyce, Chem. Biol. 11, 1505 (2004). [CrossRef] [ISI] [Medline][UC-eLinks]
* 8. K.-S. Kim, S. Oh, S. S. Yea, M.-Y. Yoon, D.-E. Kim, FEBS Lett. 582, 2745 (2008). [CrossRef] [ISI] [Medline][UC-eLinks]
* 9. Materials and methods are available as supporting material on Science Online.
* 10. The authors thank L. Orgel for many stimulating discussions. This research was supported by grants from NASA (NNX07AJ23G) and NIH (R01GM065130) and by the Skaggs Institute for Chemical Biology.
Or text without the images...
___________________________________________________________
Self-Sustained Replication of an RNA Enzyme
Tracey A. Lincoln and Gerald F. Joyce*
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.
Department of Chemistry, Department of Molecular Biology, and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
* To whom correspondence should be addressed. E-mail: [email protected]
A long-standing research goal has been to devise a nonbiological system that undergoes replication in a self-sustained manner, brought about by enzymatic machinery that is part of the system being replicated. One way to realize this goal, inspired by the notion of primitive RNA-based life, would be for an RNA enzyme to catalyze the replication of RNA molecules, including the RNA enzyme itself (1–4). This has now been achieved in a cross-catalytic system involving two RNA enzymes that catalyze each other's synthesis from a total of four component substrates.
The "R3C" RNA enzyme is an RNA ligase that binds two oligonucleotide substrates through Watson-Crick pairing and catalyzes nucleophilic attack of the 3'-hydroxyl of one substrate on the 5'-triphosphate of the other, forming a 3',5'-phosphodiester and releasing inorganic pyrophosphate (5). The R3C ligase was configured to self-replicate by joining two RNA molecules to produce another copy of itself (6). This process was inefficient because the substrates formed a nonproductive complex that limited the extent of exponential growth, with a doubling time of about 17 hours and no more than two successive doublings.
The R3C ligase subsequently was converted to a cross-catalytic format (Fig. 1A), whereby a plus-strand RNA enzyme (E) catalyzes the joining of two substrates (A' and B') to form a minus-strand enzyme (E'), which in turn catalyzes the joining of two substrates (A and B) to form a new plus-strand enzyme (7, 8). This too was inefficient because of the formation of nonproductive complexes and the slow underlying rate of the two enzymes. The enzymes E and E' operate with a rate constant of only ~0.03 min–1 and a maximum extent of only 10 to 20% (9). These rates are about 10 times slower than that of the parental R3C ligase (5), and when the two cross-catalytic reactions are carried out within a common mixture the rates are even slower (7).
Figure 1 Fig. 1. Cross-replicating RNA enzymes. (A) The enzyme E' (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, whereas E catalyzes ligation of A' and B' to form E'. The two enzymes dissociate to provide copies that can catalyze another reaction. (B) Sequence and secondary structure of the complex formed between the enzyme and its two substrates (E', A, and B are shown; E, A', and B' are the reciprocal). The curved arrow indicates the site of ligation. Solid boxes indicate critical wobble pairs that provide enhanced catalytic activity. Dashed boxes indicate paired regions and catalytic nucleotides that were altered to construct various cross replicators. (C) Variable portion of 12 different E enzymes. The corresponding E' enzymes have a complementary sequence in the paired region and the same sequence of catalytic nucleotides (alterations relative to the E1 enzyme are highlighted). [View Larger Version of this Image (24K GIF file)]
The catalytic properties of the cross-replicating RNA enzymes were improved by the use of in vitro evolution, optimizing the two component reactions in parallel and seeking solutions that would apply to both reactions when conducted in the cross-catalytic format (9). The 5'-triphosphate–bearing substrate was joined to the enzyme via a hairpin loop (B' to E and B to E'), and nucleotides within both the enzyme and the separate 3'-hydroxyl–bearing substrate (A' and A) were randomized at a frequency of 12% per position. The two resulting populations of molecules were subjected to six rounds of stringent in vitro selection, selecting for their ability to react in progressively shorter times, ranging from 2 hours to 10 ms. Mutagenic polymerase chain reaction was performed after the third round to maintain diversity in the population. After the sixth round, individuals were cloned from both populations and sequenced. There was substantial sequence variability among the clones, but all contained mutations just upstream from the ligation junction that resulted in a G·U wobble pair at this position.
The G·U pair was installed in both enzymes and both 3'-hydroxyl–bearing substrates (Fig. 1B). In the trimolecular reaction (with two separate substrates), the optimized enzymes E and E' exhibited a rate constant of 1.3 and 0.3 min–1 with a maximum extent of 92 and 88%, respectively. The optimized enzymes underwent robust exponential amplification at a constant temperature of 42°C, with more than 25-fold amplification after 5 hours, followed by a leveling off as the supply of substrates became depleted (Fig. 2A). The data fit well to the logistic growth equation [E]t = a/(1 + be–ct), where [E]t is the concentration of E (or E') at time t, a is the maximum extent of growth, b is the degree of sigmoidicity, and c is the exponential growth rate. For the enzymes E and E', the exponential growth rate was 0.92 and 1.05 hour–1, respectively.
Figure 2 Fig. 2. Self-sustained amplification of cross-replicating RNA enzymes. (A) The yield of both E (black curve) and E' (gray curve) increased exponentially before leveling off as the supply of substrates became exhausted. (B) Amplification was sustained by performance of a serial transfer experiment, allowing approximately 25-fold amplification before transferring 4% of the mixture to a new reaction vessel that contained a fresh supply of substrates. The concentrations of E and E' were measured at the end of each incubation. [View Larger Version of this Image (13K GIF file)]
Exponential growth can be continued indefinitely in a serial transfer experiment in which a portion of a completed reaction mixture is transferred to a new reaction vessel that contains a fresh supply of substrates. Six successive reactions were carried out in this fashion, each 5 hours in duration and transferring 4% of the material from one reaction mixture to the next. The first mixture contained 0.1 µM E and 0.1 µM E', but all subsequent mixtures contained only those enzymes that were carried over in the transfer. Exponential growth was maintained throughout 30 hours total of incubation, with an overall amplification of greater than 108-fold for each of the two enzymes (Fig. 2B).
It is possible to construct variants of the cross-replicating RNA enzymes that differ in the regions of Watson-Crick pairing between the cross-catalytic partners without markedly affecting replication efficiency. These regions are located at the 5' and 3' ends of the enzyme (Fig. 1B). Four nucleotide positions at both the 5' and 3' ends were varied, adopting the rule that each region contains one G·C and three A·U pairs so that there would be no substantial differences in base-pairing stability. Of the 32 possible pairs of complementary sequences for each region, 12 were chosen as a set of designated pairings (Fig. 1C). Each pairing was associated with a particular sequence within the catalytic core of both members of a cross-replicating pair. Twelve pairs of cross-replicating enzymes were synthesized, as well as the 48 substrates (12 each of A, A', B, and B') necessary to support their exponential amplification. Each replicator was individually tested and demonstrated varying levels of catalytic activity and varying rates of exponential growth (fig. S1). The pair shown in Fig. 1B (now designated E1 and E1') had the fastest rate of exponential growth, achieving about 20-fold amplification after 5 hours. The various cross-replicating enzymes shown in Fig. 1C had the following rank order of replication efficiency: E1, E10, E5, E4, E6, E3, E12, E7, E9, E8, E2, E11. The top five replicators all achieved more than 10-fold amplification after 5 hours, and all except E11 achieved at least fivefold amplification after 5 hours.
A serial-transfer experiment was initiated with a 0.1 µM concentration each of E1 to E4 and E1' to E4' and a 5.0 µM concentration of each of the 16 corresponding substrates. Sixteen successive transfers were carried out over 70 hours, transferring 5% of the material from one reaction mixture to the next (fig. S2A). Individuals were cloned from the population after the final reaction and sequenced. Among 25 clones (sequencing E' only), there was no dominant replicator (fig. S2B). E1', E2', E3', and E4' all were represented, as well as 17 clones that were the result of recombination between a particular A' substrate and one of the three B' substrates other than its original partner (or similarly for A and B). Recombination occurs when an enzyme binds and ligates a mismatched substrate. In principle, any A could become joined to any B or B', and any A' could become joined to any B' or B, resulting in 64 possible enzymes. The set of replicators were designed so that cognate substrates have a binding advantage of several kilocalories per mole as compared with noncognate substrates (fig. S2C), but once a mismatched substrate is bound and ligated, it forms a recombinant enzyme that also can cross-replicate. Recombinants can give rise to other recombinants, as well as revert back to nonrecombinants. Based on relative binding affinities, there are expected to be preferred pathways for mutation, primarily involving substitution among certain A' or among certain B components (fig. S2D).
A second serial transfer experiment was initiated with a 0.1 µM concentration each of all 12 pairs of cross-replicating enzymes and a 5.0 µM concentration of each of the 48 corresponding substrates. This mixture allowed 132 possible pairs of recombinant cross-replicating enzymes as well as the 12 pairs of nonrecombinant cross-replicators. Twenty successive reactions were carried out over 100 hours, transferring 5% of the material from one reaction mixture to the next, and achieving an overall amplification of greater than 1025-fold (Fig. 3A). Of 100 clones isolated after the final reaction (sequencing 50 E and 50 E'), only 7 were nonrecombinants (Fig. 3B). The distribution was highly nonuniform, with sparse representation of molecules containing components A6 to A12 and B5 to B12 (and reciprocal components B6' to B12' and A5' to A12'). The most frequently represented components were A5 and B3 (and reciprocal components B5' and A3'). The three most abundant recombinants were A5B2, A5B3, and A5B4 (and their cross-replication partners), which together accounted for one third of all clones.
Figure 3 Fig. 3. Self-sustained amplification of a population of cross-replicating RNA enzymes, resulting in selection of the fittest replicators. (A) Beginning with 12 pairs of cross-replicating RNA enzymes (Fig. 1C), amplification was sustained for 20 successive rounds of ~20-fold amplification and 20-fold dilution. The concentrations of all E (black) and E' (gray) molecules were measured after each incubation. (B) Graphical representation of 50 E and 50 E' clones (dark and light columns, respectively) that were sequenced after the last incubation. The A and B (or B' and A') components of the various enzymes are shown on the horizontal axes, with nonrecombinant enzymes indicated by shaded boxes along the diagonal. The number of clones containing each combination of components is shown on the vertical axis. (C) Comparative growth of E1 (circles) and A5B3 (squares) in the presence of either their cognate substrates alone (solid symbols) or all substrates that were present during serial transfer (open symbols). (D) Growth of A5B3 (black curve) and B5'A3' (gray curve) in the presence of the eight substrates (A5, B2, B3, B4, B5', A2', A3', and A4') that comprise the three most abundant cross-replicating enzymes. [View Larger Version of this Image (38K GIF file)]
In the presence of their cognate substrates alone, E1 remained the most efficient replicator, but in the presence of all 48 substrates, the most efficient replicator was A5B3 (Fig. 3C). When the A5B3 replicator was provided with a mixture of substrates corresponding to the components of the three most abundant recombinants, its exponential growth rate was the highest measured for any replicator (Fig. 3D). The fitness of a pair of cross-replicating enzymes depends on several factors, including their intrinsic catalytic activity, exponential growth rate with cognate substrates, ability to withstand inhibition by other substrates in the mixture, and net rate of production through mutation among the various cross-replicators. The A5B3 recombinant and its cross-replication partner B5'A3' have different catalytic cores (Fig. 1C), and both exhibit comparable activity, accounting for their well-balanced rate of production throughout the course of exponential amplification (Fig. 3D). The selective advantage of this cross-replicator appears to derive from its relative resistance to inhibition by other substrates in the mixture (Fig. 3C) and its ability to capitalize on facile mutation among substrates B2, B3, and B4 and among substrates A2', A3', and A4' that comprise the most abundant recombinants (fig. S2D).
Populations of cross-replicating RNA enzymes can serve as a simplified experimental model of a genetic system with, at present, two genetic loci and 12 alleles per locus. It is likely, however, that the number of alleles could be increased by exploiting more than four nucleotide positions at the 5' and 3' ends of the enzyme and by relaxing the rule that these nucleotides form one G·C and three A·U pairs. In order to support much greater complexity, it will be necessary to constrain the set of substrates, for example, by using the population of newly formed enzymes to generate a daughter population of substrates (9). An important challenge for an artificial RNA-based genetic system is to support a broad range of encoded functions, well beyond replication itself. Ultimately, the system should provide open-ended opportunities for discovering novel function, something that probably has not occurred on Earth since the time of the RNA world but presents an increasingly tangible research opportunity.
References and Notes
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* 9. Materials and methods are available as supporting material on Science Online.
* 10. The authors thank L. Orgel for many stimulating discussions. This research was supported by grants from NASA (NNX07AJ23G) and NIH (R01GM065130) and by the Skaggs Institute for Chemical Biology.
