Did an Earlier Genetic Molecule Predate DNA and RNA?

Simpler Times: Did an Earlier Genetic Molecule Predate DNA and RNA?

In the chemistry of the living world, a pair of nucleic acids -- DNA and RNA -- reign supreme. As carrier molecules of the genetic code, they provide all organisms with a mechanism for faithfully reproducing themselves as well as generating the myriad proteins vital to living systems.

Yet according to John Chaput, a researcher at the Center for Evolutionary Medicine and Informatics, at Arizona State University's Biodesign Institute®, it may not always have been so.


Chaput and other researchers studying the first tentative flickering of life on earth have investigated various alternatives to familiar genetic molecules. These chemical candidates are attractive to those seeking to unlock the still-elusive secret of how the first life began, as primitive molecular forms may have more readily emerged during the planet's prebiotic era.

One approach to identifying molecules that may have acted as genetic precursors to RNA and DNA is to examine other nucleic acids that differ slightly in their chemical composition, yet still possess critical properties of self-assembly and replication as well as the ability to fold into shapes useful for biological function.

According to Chaput, one interesting contender for the role of early genetic carrier is a molecule known as TNA, whose arrival on the primordial scene may have predated its more familiar kin. A nucleic acid similar in form to both DNA and RNA, TNA differs in the sugar component of its structure, using threose rather than deoxyribose (as in DNA) or ribose (as in RNA) to compose its backbone.

In an article released online January 9 in the journal Nature Chemistry, Chaput and his group describe the Darwinian evolution of functional TNA molecules from a large pool of random sequences. This is the first case where such methods have been applied to molecules other than DNA and RNA, or very close structural analogues thereof. Chaput says "the most important finding to come from this work is that TNA can fold into complex shapes that can bind to a desired target with high affinity and specificity." This feature suggests that in the future it may be possible to evolve TNA enzymes with functions required to sustain early life forms.

Nearly every organism on earth uses DNA to encode chunks of genetic information in genes, which are then copied into RNA. With the aid of specialized enzymes known as polymerases, RNA assembles amino acids to form essential proteins. Remarkably, the basic functioning of the genetic code remains the same, whether the organism is a snail or a senator, pointing to a common ancestor in the DNA-based microbial life already flourishing some 3.5 billion years ago.

Nevertheless, such ancestors were by this time quite complex, leading some scientists to speculate about still earlier forms of self-replication. Before DNA emerged to play its dominant role as the design blueprint for life, a simpler genetic world dominated by RNA may have prevailed. The RNA world hypothesis as it's known alleges that ribonucleic acid (RNA) acted to store genetic information and catalyze chemical reactions much like a protein enzyme, in an epoch before DNA, RNA and proteins formed the integrated system prevalent today throughout the living world.

While the iconic double helix of DNA is formed from two complimentary strands of nucleotides, attached to each other by base pairing in a helical staircase, RNA is single-stranded. The two nucleic acids DNA and RNA are named for the type of sugar complex that forms each molecule's sugar-phosphate backbone -- a kind of molecular thread holding the nucleotide beads together.

Could a simpler, self-replicating molecule have existed as a precursor to RNA, perhaps providing genetic material for earth's earliest organisms? Chaput's experiments with the nucleic acid TNA provide an attractive case. To begin with, TNA uses tetrose sugars, named for the four-carbon ring portion of their structure. They are simpler than the five-carbon pentose sugars found in both DNA and RNA and could assemble more easily in a prebiotic world, from two identical two-carbon fragments.

This advantage in structural simplicity was originally thought to be an Achilles' heel for TNA, making its binding behavior incompatible with DNA and RNA. Surprisingly, however, research has now shown that a single strand of TNA can indeed bind with both DNA and RNA by Watson-Crick base pairing -- a fact of critical importance if TNA truly existed as a transitional molecule capable of sharing information with more familiar nucleic acids that would eventually come to dominate life.

In the current study, Chaput and his group use an approach known as molecular evolution to explore TNA's potential as a genetic biomolecule. Such work draws on the startling realization that fundamental Darwinian properties -- self-replication, mutation and selection -- can operate on non-living chemicals.
Extending this technique to TNA requires polymerase enzymes that are capable of translating a library of random DNA sequences into TNA. Once such a pool of TNA strands has been generated, a process of selection must successfully identify members that can perform a given function, excluding the rest. As a test case, the team hoped to produce through molecular evolution, a TNA strand capable of acting as a high-specificity, high-affinity binding receptor for the human protein thrombin.

They first attempted to demonstrate that TNA nucleotides could attach by complementary base pairing to a random sequence of DNA, forming a hybrid DNA-TNA strand. A DNA polymerase enzyme assisted the process. Many of the random sequences, however, contained repeated sections of the guanine nucleotide, which had the effect of pausing the transcription of DNA into TNA. Once random DNA libraries were built excluding guanine, a high yield of DNA-TNA hybrid strands was produced.

The sequences obtained were 70 nucleotides in length, long enough Chaput says, to permit them to fold into shapes with defined binding sites. The DNA-TNA hybrids were then incubated with the target molecule thrombin. Sequences that bound with the target were recovered and amplified through PCR. The DNA portion was removed and used as a template for further amplification, while the TNA molecules displaying high-affinity, high specificity binding properties were retained.

Additionally, the binding affinity of the evolved and selected TNA molecules was tested against two other common proteins, for which they displayed no affinity, strengthening the case that a highly specific binding molecule had resulted from the group's directed evolution procedure.

Chaput suggests that issues concerning the prebiotic synthesis of ribose sugars and the non-enzymatic replication of RNA may provide circumstantial evidence of an earlier genetic system more readily produced under primitive earth conditions. Although solid proof that TNA acted as an RNA precursor in the prebiotic world may be tricky to obtain, Chaput points to the allure of this molecule as a strong candidate, capable of storing information, undergoing selection processes and folding into tertiary structures that can perform complex functions. This result provides the motivation to explore TNA as an early genetic system.

Chaput is optimistic that major questions about the prebiotic synthesis of TNA, its role in the origin and early evolution of life on earth, and eventual genetic takeover by RNA will, over time, be answered.

Synthetic Chromosome

'Synthetic' Chromosome Permits Rapid, On-Demand 'Evolution' of Yeast; Artificial System Has Built-In Diversity Generator

In the quest to understand genomes -- how they're built, how they're organized and what makes them work -- a team of Johns Hopkins researchers has engineered from scratch a computer-designed yeast chromosome and incorporated into their creation a new system that lets scientists intentionally rearrange the yeast's genetic material.

A report of their work appears September 14 as an Advance Online Publication in the journal Nature.

"We have created a research tool that not only lets us learn more about yeast biology and genome biology, but also holds out the possibility of someday designing genomes for specific purposes, like making new vaccines or medications," says Jef D. Boeke, Ph.D., Sc.D., professor of molecular biology and genetics, and director of the High Throughput Biology Center at the Johns Hopkins University School of Medicine.

Boeke notes that yeast is probably the best-studied organism with a nucleus on the planet and is "already used for everything from medicine to biofuel," making it a good candidate for his team's focus.

In designing the synthetic yeast chromosome, Boeke says, the goal was to make it maximally useful to researchers by laying down some ground rules: First, the product could not compromise yeast survival; second, it must be as streamlined as possible; and third, it had to contain the capacity for genetic flexibility and change.

Using the already known full genetic code -- or DNA sequences -- of the yeast genome as a starting point, Johns Hopkins graduate student Sarah Richardson wrote a software program for making a series of systematic changes to the DNA sequence. The changes were planned to subtly change the code and remove some of the repetitive and less used regions of DNA between genes, and to generate a mutated "version 2.0" of a yeast cell's original 9R chromosome. The smallest chromosome arm in the yeast genome, 9R contains about 100,000 base pairs of DNA and represents about one percent of the single-celled organism's genome.

Building the actual chromosome started with stringing individual bases of DNA together that were then assembled into longer segments. Large segments of about 10,000 base pairs were finally put into live yeast cells and essentially swapped for the native counterpart in the chromosome, a process for which yeast are naturally adept. In addition to 9R, the team also made a smaller piece of the chromosome 6L. Yeast cells containing the synthetic chromosomes were tested for their ability to grow on different nutrients and in different conditions, and in each case came out indistinguishable from natural yeast.

The Hopkins teams says what distinguishes this constructed chromosome from the native version -- and sets it apart from other synthetic genome projects -- is an "inducible evolution system" called SCRaMbLE, short for Synthetic Chromosome Rearrangement and Modification by Lox-P mediated Evolution.

"We developed SCRaMbLE to enable us to pull a mutation trigger -- essentially causing the synthetic chromosome to rearrange itself and introducing changes similar to what might happen during evolution, but without the long wait," explains Boeke. Why build in the scrambling system? To change multiple things at once, says Boeke, which is anathema among experimental scientists who traditionally change only one variable at a time, Nature is never that well controlled, he says.

The team activated SCRaMbLE in yeast containing both the synthetic 9R and 6L chromosomes, then analyzed the DNA from the yeast cells. Testing this population of SCRaMbLEd yeast fed various nutrients they found some grew fast, some grew slowly and others really slowly, and some of the fast-growing ones had very specific defects resulting from specific gene loss, showing that SCRaMbLE does indeed introduce random variation. When the team analyzed the molecular structure of the synthetic 9R and 6L chromosomes from this SCRaMbLEd population, they found chromosomes with small deletions, rearrangements, and other alterations, at wildly varying locations.

"If you think of the yeast genome as a deck of cards, we now have a system by which we can shuffle it and/or remove different combinations of 5000 of those cards to get lots of different decks from the same starter deck," Boeke says. "While one derivative deck might yield good hands for poker, another might be better suited for pinochle. By shuffling the DNA according to our specifications, we hope to be able to custom design organisms that perhaps will grow better in adverse environments, or maybe make one percent more ethanol than native yeast."

Boeke says the 9R and 6L experiments are "the beginning of a big project, whose ultimate goal is to synthesize the whole yeast genome (about 6000 genes) and SCRaMbLE the 5000 likely to be individually dispensable. And he wants to make the tool available to anyone who wants to use it, without intellectual property protection.

Major support for this study came from the National Science Foundation, with other contributions from Microsoft, Department of Energy, and Fondation pour la Recherche Médicale.

In addition to Boeke, Johns Hopkins scientists who contributed to the Nature study are Jessica S. Dymond, Sarah M. Richardson, Candice E. Coombes, Timothy Babatz, Joy Wu Schwerzmann, Héloïse Müller, Narayana Annaluru, Annabel C. Boeke, Junbiao Dai, Srinivasan Chandrasegaran, and Joel S. Bader.

Also, William J. Blake of Codon Devices; and Derek L. Lindstrom, and Daniel E. Gottschling of Fred Hutchinson Cancer Research Center.

Blood Mystery Solved: Two New Blood Types Identified

Two New Blood Types Identified

You probably know your blood type: A, B, AB or O. You may even know if you're Rhesus positive or negative. But how about the Langereis blood type? Or the Junior blood type? Positive or negative? Most people have never even heard of these.

Yet this knowledge could be "a matter of life and death," says University of Vermont biologist Bryan Ballif.

While blood transfusion problems due to Langereis and Junior blood types are rare worldwide, several ethnic populations are at risk, Ballif notes. "More than 50,000 Japanese are thought to be Junior negative and may encounter blood transfusion problems or mother-fetus incompatibility," he writes.

But the molecular basis of these two blood types has remained a mystery -- until now.

In the February issue of Nature Genetics, Ballif and his colleagues report on their discovery of two proteins on red blood cells responsible for these lesser-known blood types.

Ballif identified the two molecules as specialized transport proteins named ABCB6 and ABCG2.

"Only 30 proteins have previously been identified as responsible for a basic blood type," Ballif notes, "but the count now reaches 32."

The last new blood group proteins to be discovered were nearly a decade ago, Ballif says, "so it's pretty remarkable to have two identified this year."

Both of the newly identified proteins are also associated with anticancer drug resistance, so the findings may also have implications for improved treatment of breast and other cancers.

As part of the international effort, Ballif, assistant professor in the biology department, used a mass spectrometer at UVM funded by the Vermont Genetics Network. With this machine, he analyzed proteins purified by his longtime collaborator, Lionel Arnaud at the French National Institute for Blood Transfusion in Paris, France.

Ballif and Arnaud, in turn, relied on antibodies to Langereis and Junior blood antigens developed by Yoshihiko Tani at the Japanese Red Cross Osaka Blood Center and Toru Miyasaki at the Japanese Red Cross Hokkaido Blood Center.

After the protein identification in Vermont, the work returned to France. There Arnaud and his team conducted cellular and genetic tests confirming that these proteins were responsible for the Langereis and Junior blood types. "He was able to test the gene sequence," Ballif says, "and, sure enough, we found mutations in this particular gene for all the people in our sample who have these problems."

Transfusion Troubles

Beyond the ABO blood type and the Rhesus (Rh) blood type, the International Blood Transfusion Society recognizes twenty-eight additional blood types with names like Duffy, Kidd, Diego and Lutheran. But Langereis and Junior have not been on this list. Although the antigens for the Junior and Langereis (or Lan) blood types were identified decades ago in pregnant women having difficulties carrying babies with incompatible blood types, the genetic basis of these antigens has been unknown until now.

Therefore, "very few people learn if they are Langereis or Junior positive or negative," Ballif says.

"Transfusion support of individuals with an anti-Lan antibody is highly challenging," the research team wrote in Nature Genetics, "partly because of the scarcity of compatible blood donors but mainly because of the lack of reliable reagents for blood screening." And Junior-negative blood donors are extremely rare too. That may soon change.

With the findings from this new research, health care professionals will now be able to more rapidly and confidently screen for these novel blood group proteins, Ballif wrote in a recent news article. "This will leave them better prepared to have blood ready when blood transfusions or other tissue donations are required," he notes.

"Now that we know these proteins, it will become a routine test," he says.

A better match

This science may be especially important to organ transplant patients. "As we get better and better at transplants, we do everything we can to make a good match," Ballif says. But sometimes a tissue or organ transplant, that looked like a good match, doesn't work -- and the donated tissue is rejected, which can lead to many problems or death.

"We don't always know why there is rejection," Ballif says, "but it may have to do with these proteins."

The rejection of donated tissue or blood is caused by the way the immune system distinguishes self from not-self. "If our own blood cells don't have these proteins, they're not familiar to our immune system," Ballif says, so the new blood doesn't "look like self" to the complex cellular defenses of the immune system. "They'll develop antibodies against it," Ballif says, and try to kill off the perceived invaders. In short, the body starts to attack itself.

"Then you may be out of luck," says Ballif, who notes that in addition to certain Japanese populations, European Gypsies are also at higher risk for not carrying the Langereis and Junior blood type proteins.

"There are people in the United States who have these challenges too," he says, "but it's more rare."

Other Proteins

Ballif and his international colleagues are not done with their search. "We're following up on more unknown blood types," he says. "There are probably on the order of 10 to 15 more of these unknown blood type systems -- where we know there is a problem but we don't know what the protein is that is causing the problem."

Although these other blood systems are very rare, "if you're that one individual, and you need a transfusion," Ballif says, "there's nothing more important for you to know."