June 10, 1998 Discovery Narrows Search For Precursor Of All Life Forms
New Haven, Conn. -- Yale scientists report they have synthesized molecules like those that probably gave rise to the earliest life forms on Earth nearly 4 billion years ago, thus creating a biochemist's version of "Jurassic Park" populated by exotic molecular "fossils" that have long since become extinct.
In the May 26 issue of the Proceedings of the National Academy of Sciences, the Yale biologists report the creation of one of these "fossils," an unusual hybrid molecule made up of a scaffold from deoxyribonucleic acid (DNA) with chemical "scissors" attached to it.
Ronald R. Breaker, who created the first DNA enzymes in 1994 with colleagues at The Scripps Research Institute, said he "looted the tool box of proteins" to get the amino acid "scissors," which destroy messenger ribonucleic acid (RNA) in humans and many other organisms. The feat was accomplished using a technique known as test-tube evolution.
Breaker's tailor-made enzyme is the first known nucleic acid enzyme that uses an amino acid to trigger chemical activity, and it brings scientists a step closer to finding the precursor of all life -- a single molecule containing both genetic code and an enzyme capable of triggering self-replication.
"If we can raid a protein's tool box to take one of its favored chemical groups -- in this case, a key amino acid called histidine found in a protein called RNase A -- then we should be able to raid the entire tool box and make use of anything we find there to make highly sophisticated DNA or RNA enzymes," said Breaker, who collaborated with Yale postdoctoral associate Adam Roth.
Which Came First -- DNA, RNA Or Proteins?
The discovery provides important clues to the chicken-or-egg dilemma of which came first -- DNA, RNA or proteins. Most scientists agree life as we know it cannot exist without DNA as the storehouse of genetic code, RNA as the genetic messenger, and proteins to carry out the chemistry of reproduction. Can any one of these three key molecules have existed as the precursor of the other two, serving as both chicken and egg?
Evidence is mounting that "it was an RNA World at the dawn of life as the Earth began to cool," said Breaker, who added that he and his colleagues can create dual-purpose genetic enzymes in the laboratory out of either RNA or DNA. "These genetic enzymes have the chemical sophistication, the full catalytic ability, to do many of the fundamental reactions we see in biology today. I am confident one will be created soon that can replicate itself."
He added that the new DNA enzyme he crafted destroys RNA with impressive efficiency at a rate 10 million times faster than it would decay naturally, although the protein the enzyme mimics acts much faster still.
No naturally occurring DNA enzymes have been found to date, but such a discovery would not surprise Breaker. The discovery nearly two decades ago of naturally occurring RNA enzymes, or ribozymes, earned Yale biochemist Sidney Altman and University of Colorado researcher Thomas Cech the 1989 Nobel Prize in Chemistry. In separate experiments, Altman and Cech exploded the myth that RNA is merely a passive carrier of genetic code incapable of triggering cell activity.
Referring to the dozen or more DNA and RNA enzymes created in his laboratory in recent months, Breaker said, "We believe these are like ancient molecular 'fossils' that might have been found stomping around the planet -- or more likely floating in the seas -- during the Archean Era between 3.8 and 4 billion years ago."
RNA Identified As Strongest Candidate For Precursor To All Life
While the Yale biologists created the versatile protein mimic from DNA, Breaker theorizes that a similar enzyme could be created with RNA, which many scientists believe is the strongest candidate for being the precursor of all other life forms. In addition to RNA's dual function as genetic molecule and as enzyme, RNA serves important roles in all living systems as the carrier of genetic instructions from DNA and as the orchestrator of all protein synthesis.
"This is exactly what you would expect if RNA invented these processes during the 'RNA World,'" Breaker said. "Because DNA is about a million times more stable than RNA, DNA most likely evolved later as a safe storehouse for the genetic code first found in RNA. Similarly, proteins probably evolved that were more efficient chemical catalysts, eventually driving most RNA enzymes extinct and relegating RNA to a more limited role."
The discovery that nucleic acids can raid the tool box of proteins means "the RNA World could have been a very sophisticated place," Breaker said. "The earliest RNA could have had access to all of these chemical helpers now used by proteins. Instead of working from a very primitive palette, varieties of RNA could have evolved that had a very rich chemical capability early on."
Tailoring Nucleic Enzymes To Fight Disease
Besides elucidating how life might have evolved, DNA and RNA enzymes show great promise as powerful medications. In fact, some RNA enzymes already have been developed to function as precision scissors that can snip out flawed gene segments and splice in corrected versions -- a method that has potential for treating diseases ranging from cystic fibrosis to muscular dystrophy and sickle cell anemia.
Because DNA lends itself well to test-tube evolution techniques, it can be synthesized readily in the laboratory, and different strains of enzymes can be genetically engineered for specific purposes, Professor Breaker said. For example, he and his colleagues have created self-cleaving DNA enzymes that can fold into chemically active molecules and cut themselves or other DNAs into segments. The next step is to genetically engineer a DNA enzyme that can shred the genetic code of a harmful organism like the HIV virus, rendering it harmless.
Specific DNA enzymes also could be tailor-made to break down only in the presence of target molecules, making them effective as biosensors for detecting toxic chemicals in the environment or for medical diagnostics. Working in collaboration with a Jerusalem-based firm called IntelliGene Ltd., Breaker plans to create biosensors for detecting biological or chemical warfare agents with funding from the Defense Advanced Research Projects Agency (DARPA).
"Test-Tube Evolution" Mimics Nature
Breaker sets up a system of natural selection through test-tube evolution to produce DNA sequences with the characteristics he desires. Typically, Breaker and his colleagues begin crafting an enzyme by synthesizing more than 10 trillion random DNA sequences using a computerized DNA synthesizer. Then they wash a grid containing the sequences with various compounds, in this case histidine. Rare DNA molecules that by chance fold into enzymes will break themselves free from the grid.
By cloning the DNA sequences that are washed away by the amino acid and then repeating the process several times, the Yale biochemists isolate desired enzymes. "Our latest findings not only improve our understanding about the origins of life, they also expand our skills in molecular evolution," he said. "While we may not be able to resurrect fossilized creatures like they did in 'Jurassic Park,' we very well may be able to recreate many of the ancient enzymes that were needed at the very beginning of life nearly 4 billion years ago."
Funding for this research was from the Arnold and Mabel Beckman Foundation Young Investigator Award.
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