It wasn’t exactly a command performance. It was the result of persistent coaxing, or rather a delicate negotiation by means of directed evolution. In the end, though, all the effort was worthwhile, for the result was a performance that must have inspired cries of “Bravo!” in the laboratory. A particularly challenging biochemical symphony, one that included movements in both RNA synthesis and RNA replication, had been played through, possibly for the first time in four billion years.
According to a study that appeared August 15 in the Proceedings of the National Academy of Sciences (PNAS), scientists based at The Scripps Research Institute (TSRI) created a ribozyme that succeeded in replicating short lengths of RNA efficiently, and in performing transcription on even longer RNAs to make functional RNA molecules with complex structures. This virtuoso performance comes close to what scientists imagine an “RNA World” RNA replicator would have been capable of doing to support life before modern biology, where protein enzymes now handle gene replication and transcription.
The study, which was led by TSRI’s Gerald F. Joyce, Ph.D., and David P. Horning, Ph.D., culminated in the PNAS article, which was entitled, “Amplification of RNA by an RNA Polymerase Ribozyme.” The article describes how the TSRI scientists used test-tube evolution techniques to tackle the decades-old challenge of creating an enzyme that could both replicate and transcribe RNA and thus support an RNA world.
The team started with an enzyme that had been developed and improved upon by other researchers since the early 1990s. The class I RNA polymerase ribozyme, as it has come to be known, can perform the basic task of RNA synthesis—required for transcribing an RNA template into a functional RNA molecule—by binding to a strand of RNA and using it as a template to stitch together a complementary RNA strand.
The team reported that it developed an improved polymerase ribozyme that can synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even transfer RNA (tRNA).
“The polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR,” wrote the authors of the PNAS article. “Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins.”
Prior forms of the ribozyme had been very limited in the RNA sequences they could handle and couldn't transcribe RNAs that have even moderately complex structures. Because of those limitations, they also could not perform full replication of RNA, which requires the transcription of a complementary strand back into a copy of the original.
“We found that the new ribozyme can handle most sequences and all but the most difficult structures, so we can use it to make a variety of functional RNA molecules,” said Dr. Joyce.
Even when synthesizing the limited RNA sequences that the original class I RNA polymerase ribozyme could handle, the improved ribozyme, called 24-3, proved capable of stitching them together about 100 times faster than its ancestor could.
Turning to the much harder task of replication, the TSRI researchers found that ribozyme 24-3 could copy RNAs of up to two dozen nucleotides, achieving what biologists call “exponential replication” and creating as many as 40,000 copies of a target RNA within 24 hours.
The 24-3 ribozyme is thus the first ever to combine the two basic capabilities—RNA synthesis and RNA replication—necessary for a pre-protein, pre-DNA world of RNA life.
To generate and sustain a true “RNA world,” the new ribozyme will have to be improved further to enable the replication of longer, more complex RNA molecules—crucially including the polymerase ribozyme itself. The Joyce laboratory is now driving its ribozyme toward that goal with further test-tube evolution experiments.
“A polymerase ribozyme that achieves exponential amplification of itself will meet the criteria for being alive,” Dr. Joyce concluded. “That's a summit that's now within sight.”