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  1. Test-Tube RNA

    Based on an MIT Whitehead Institute press release

    A new RNA enzyme, or ribozyme, synthesized by David Bartel, Wendy Johnson and colleagues at MIT’s Whitehead Institute for Biomedical Research, opens a door to create a path for the earliest evolution to have happened without either DNA or proteins in the primordial soup. Since first described in the journal Science, the Whitehead ribozyme, or RNA catalyst, has filled in the picture of early chemical evolution and how life might have arisen.

    As highlighted in Sydney Altman’s 1989 Nobel laureate address entitled “RNA World”: “If very primitive life on Earth did not arise until about 3.5 billion years ago, there was, perhaps, a period of 0.5 billion years in which to sample many polymer sequences [like RNA] that originally arose through non-biochemical mechanisms and that ultimately evolved to direct the first self-replicating systems.” As discoverer of the first catalytic RNA, Altman speculated that: “neither DNA nor protein were required in such a primitive system if RNA could perform as a catalyst.”

    According to Bartel, “A fundamental question about the origin of life is what class of molecules gave rise to some of the earliest life forms?” Up until two decades ago, RNA was thought to act as a molecular go-between, translating genetic DNA to proteins and finally cells. The idea of an evolutionary time, when life replicated by RNA alone, seemed not possible, and what came to be called “RNA World” could not have sustained any reaction that could support early chemical evolution. But the discovery of enzymes that are not proteins, but RNAs themselves, showed RNA as capable of much more: both a translator and a catalyst for its own replication.

    For the scientific team, their first challenge was to create the RNA enzyme from scratch in the laboratory, not isolate it from nature. “Creating a complimentary strand of RNA is a challenging enzymatic reaction because it requires several things to happen at the same time”, says Wendy Johnston, first author on the paper and research associate in the Bartel lab.

    But once the ribozyme was created, the molecule then had to be able to copy itself from simpler building blocks, particularly if RNA reactions might serve as self-replicating or evolving reagents. The second challenge for Bartel’s group was therefore to make many RNA copies from the first template. When aligning the master and copy molecules upon themselves, they tested their fidelity to the original design. The key feature showed 95% accuracy.

    This new finding removed a limit found in previous work, where the fidelity depended on either RNA chain length or sequence order. “The reaction must be accurate in incorporating nucleotides based on the template strand, general enough that any template can be copied, and efficient enough to add on a large number of nucleotides,” says Johnston. In fact, one complete RNA helix turn, a chain length of 14 code letters (or nucleotides) was able to replicate itself.

    One key piece for a RNA World scenario is now available: a laboratory version of a master and copy molecule, 95% fidelity to the master, and independence from RNA chain length or sequence order. What the Whitehead scientists hoped for—RNA that replicated itself in self-sustaining ways— gives some of the strongest evidence yet that as life’s building blocks, RNA world could propagate itself even before the chemical advent of DNA and proteins.

    “It was a classic chicken-and-egg argument. RNA, like DNA, has the genetic information necessary to reproduce but needs proteins to catalyze the reaction. Conversely, proteins can catalyze reactions but cannot reproduce without the information supplied by RNA,” says Bartel.

    What had held back previous attempts at such test-tube evolution of molecules was in fact a fundamental challenge to evolution itself, the ability to select promising candidates from outright failures. “During the selection process, we look for RNAs that can change themselves in a special way. We can then separate them from RNA that don’t change themselves,” says Bartel. In Darwinian terms, RNA replication with selection, but without wild mutations, finally came together.

    When RNA World was first proposed, a debate centered around the lack of available RNA candidates. There are only eight known ribozymes in nature, far too few to perform selection and drive molecular evolution in any self-sustaining way. But since Bartel’s lab and others pioneered the chemical synthesis of RNA from scratch, the converse problem had arisen. Too many mutations started to inhibit the biological usefulness and most importantly, self-replication. In fact, ever since 1993, when Bartel and Jack Szostak at Harvard University first found 65 novel ribozymes from a search of more than 1000 trillion RNAs, the problem has been to make much more efficient selection. “When we subjected these ribozymes to a test-tube evolution, we found descendants that were 100 times more efficient,” says Bartel. So without wild mutations in play, the molecular efficiency sought by Bartel’s colleagues will ultimately help evolutionary biologists address questions about how life began on earth more than three billion years ago.

    What’s Next

    Prior to the discovery of catalytic RNAs, proteins were considered by many to be the only organic molecules in living matter that could function as catalysts. In this scenario, proteins are required for DNA synthesis and DNA is required for protein synthesis. However, the intriguing possibilities of RNA World and the synthesis of new ribozymes provide yet another path for earliest life. Self-replicating RNA-based systems could have arisen first, and DNA and proteins would have been added later.

    “We will never be able to prove the existence of the RNA world because we can’t go back in time, but we can examine the basic properties of RNA and see if these are compatible within the RNA world scenario,” says Bartel.

    The next targets for these molecular architects will likely extend to more than one helix turn and a veritable copy machine for keeping the integrity of their RNA designs across many chemical generations.