April 20, 2012
Researchers Create Synthetic DNA/RNA That Can Evolve
Researchers have created artificial genetic material known as Xenonucleic acids, or XNAs, that can store information and evolve over generations in a comparable way to DNA.
The research, reported Friday in the journal Science, has implications for the fields of molecular medicine and biotechnology, and sheds new light on how molecules first replicated and assembled into life billions of years ago.
Living systems owe their existence to the information-carrying molecules DNA and RNA. These fundamental chemical forms have two features essential for life: they display heredity, meaning they can encode and pass on genetic information, and they can adapt over time.
Whether these traits could be performed by molecules other than DNA and RNA has been a long-debated issue.
For the current study, an international team of researchers developed chemical procedures to convert DNA and RNA into six genetic polymers known as XNAs. The process switches the deoxyribose and ribose (the "d" and "r" in DNA and RNA) for other molecules.
The researchers demonstrated for the first time that all six XNAs could form a double helix with DNA, and were more stable than natural genetic material. Moreover, one of these XNAs, a molecule known as anhydrohexitol nucleic acid, or HNA, was capable of undergoing directed evolution and folding into biologically useful forms.
Philipp Holliger of MRC Laboratory of Molecular Biology in Cambridge, the study´s senior author, said the work demonstrated that heredity and evolution were possible using alternatives to natural genetic material.
"There is nothing Goldilocks about DNA and RNA," he told Science.
"There is no overwhelming functional imperative for genetic systems or biology to be based on these two nucleic acids."
Both RNA and DNA embed data in their sequences of four nucleotides. This information is vital for conferring hereditary traits and for supplying the coded recipe essential for building proteins from the 20 naturally occurring amino acids. However, precisely how and when this system began remains one of the most perplexing and hotly contested areas of biology.
According to one hypothesis, the simpler RNA molecule preceded DNA as the original informational conduit. The RNA world hypothesis proposes that the earliest examples of life were based on RNA and simple proteins. Because of RNA's great versatility–it is not only capable of carrying genetic information but also of catalyzing chemical reactions like an enzyme–it is believed by many to have supported pre-cellular life.
Nevertheless, the spontaneous arrival of RNA through a sequence of purely random mixing events of primitive chemicals was, at the very least, an unlikely occurrence.
"This is a big question," said study leader John Chaput, a researcher at Arizona State University´s Biodesign Institute.
"If the RNA world existed, how did it come into existence? Was it spontaneously produced, or was it the product of something that was even simpler than RNA?"
This pre-RNA world hypothesis has been gaining ground, primarily through study of XNAs, which provide plausible alternatives to the current biological system and could have acted as chemical stepping-stones to the eventual emergence of life.
Threose nucleic acid, or TNA, for example, is one candidate for this critical intermediary role.
"TNA does some interesting things," Chaput said, noting the molecule's capacity to bind with RNA through antiparallel Watson-Crick base pairing.
"This property provides a model for how XNAs could have transferred information from the pre-RNA world to the RNA world."
Nucleic acid molecules, including DNA and RNA, consist of 3 chemical components: a sugar group, a triphosphate backbone and combinations of the four nucleic acids. By manipulating these structural elements, researchers can engineer XNA molecules with unique properties.
However, in order for any of these molecules to have acted as a precursor to RNA in the pre-biotic epoch, they would need to have been able to transfer and recover their information from RNA. To do this, specialized enzymes, known as polymerases are required.
And while nature has made DNA and RNA polymerases capable of reading, transcribing and reverse transcribing normal nucleic acid sequences, no naturally occurring polymerases exist for XNA molecules.
So the researchers, led by Holliger, painstakingly evolved synthetic polymerases that could copy DNA into XNA, and other polymerases that could copy XNA back into DNA.
Ultimately, polymerases were found that transcribe and reverse-transcribe six different genetic systems: HNA, CeNA, LNA, ANA, FANA and TNA. The experiments demonstrated that these unnatural DNA sequences could be rendered into various XNAs when the polymerases were fed the appropriate XNA substrates.
Using these enzymes as tools for molecular evolution, the team evolved the first example of an HNA aptamer through iterative rounds of selection and amplification. Starting from a large pool of DNA sequences, a synthetic polymerase was used to copy the DNA library into HNA. The pool of HNA molecules was then incubated with an arbitrary target. The small fraction of molecules that bound the target were then separated from the unbound pool, reverse transcribed back into DNA with a second synthetic enzyme, and amplified by PCR. After many repeated rounds, HNAs were generated that bound HIV trans-activating response RNA (TAR) and hen egg lysosome (HEL), which were used as binding targets.
"This is a synthetic Darwinian process," Chaput said.
"The same thing happens inside our cells, but this is done in vitro."
The method for producing XNA polymerases draws on Holliger´s pervious, path-breaking work, and uses cell-like synthetic compartments of water/oil emulsion to conduct directed evolution of enzymes, particularly polymerases.
By isolating self-replication reactions from each other, the process greatly improves the accuracy and efficiency of polymerase evolution and replication.
"What nobody had really done before," Chaput said, "is to take those technologies and apply them to unnatural nucleic acids. "
Chaput said the study advances the case for a pre-RNA world, while revealing a new class of XNA aptamers capable of fulfilling many useful roles.
And while many questions surrounding the origins of life remain, he is optimistic that solutions are coming into view.
"Further down the road, through research like this, I think we'll have enough information to begin to put the pieces of the puzzle together."
In an article accompanying the study in the journal Science, Gerald Joyce of the Scripps Research Institute wrote that "the work heralds the era of synthetic genetics, with implications for exobiology (life elsewhere in the Universe), biotechnology, and understanding of life itself".
However, he stressed that the work does not yet represent a full synthetic genetics platform. For that, a self-replicating system that does not require the DNA intermediary must be developed.
If that happens, "construction of genetic systems based on alternative chemical platforms may ultimately lead to the synthesis of novel forms of life".