By Sean Henahan, Access Excellence

SANTA CRUZ, CA - Hitherto unrecognized properties of RNA add further support to the idea that RNA was the central molecule in the origin of life, report researchers.

RNA is the only known macromolecule that can both encode genetic information and also act as a biocatalyst. This observation forms the basis for the 'RNA world' model which suggests that both the genetic and enzymatic components of early cells were RNA molecules. However, this model requires that ribozymes be able to catalyze a variety of chemical reactions- whereas known natural ribozymes seem relatively limited in the regard.

This led a team of Harvard University scientists to design an experiment which would search through trillions of bits of RNA in search of the missing ribozymes. The researchers used a new method called "in vitro evolution" to screen 500 trillion bits of random RNA.

The first stage involved searching for RNA capable of binding to a desired substrate (biotin), but not necessarily possessing catalytic activity. The researchers then used repeated rounds of affinity chromatography and amplification to generate a second pool of RNA which contained the selected binding domain sequence. The RNA segments isolated by this process were subjected to another round of in vitro evolution. Repeated rounds of in vitro transcription produced one RNA molecule with the desired attributes.

The researchers then sought to amplify and modify that RNA molecule so it could catalyze the carbon-nitrogen bond with no help from proteins. They randomly changed some of the RNA's genetic units, so that each one had a 30 percent chance of being different from the corresponding genetic unit in the starting framework. This led to another huge pool of 80 trillion slightly different RNAs. Next, the researchers explored the abilities of these mutated RNAs to stick to the non-RNA substance and to catalyze the carbon-nitrogen alkylation.

Seven more screening steps led to one ribozyme that could both bind to the non-RNA substrate and catalyze alkylation. Next the investigators improved the efficiency of this molecule 100-fold by mutating it further and subjecting it to another round of screens. However, the ribozyme did not perform its catalyst role with the speed of proteins that typically do the same job.

"The slowness of this ribozyme might reflect the intrinsic incompetence of RNA to carry out the reactions that proteins do so well. That may be why the RNA world evolved into a protein world--proteins outcompeted RNA in these functions. But we don't know that our ribozyme is the best one possible. We could optimize it further," commented molecular biologist Charles Wilson, now at the UC Santa Cruz.

To the researchers' surprise, the structure of their final ribozyme looks like that of a well-known RNA molecule in the cells of all living things: the cloverleaf-shaped "transfer RNA." Wilson now is trying to determine why this shape arose in the experiment and how it works. "It's striking to me that we started with an enormous random pool with no bias," he says, "but we ended up with basically the only RNA whose structure we really know."

The discovery of this ribozyme reveals a far broader set of chemical skills than scientists previously had credited to RNA, he added:

"All other known ribozymes carry out the same basic reaction: breaking and making bonds in the RNA backbone between phosphorus and oxygen. Some RNAs cut and paste themselves together, but that involves similar reactions. To test the idea of an RNA world, we needed to look for other reactions beyond this backbone chemistry."

The reaction targeted in this research, the carbon-bonding process called "alkylation," would have been critical to the success of RNA as life arose. "This basic reaction comes up in all types of biological situations," Wilson says. "Our results suggest that RNA could have reacted with other molecules that didn't look like RNA. That's the first step of evolution toward a non-RNA world."

In today's organisms, nearly all chemical reactions are catalyzed by proteins, powerful enzymes that have evolved to master a bewildering array of tasks. However, proteins cannot carry a cell's genetic information. That deed is the domain of the nucleic acids: double-stranded DNA and its single-stranded relative, RNA. Although DNA is more widely known, scientists have found that RNA is far more complex and versatile.

Modern proteins and nucleic acids need each other to exist and function. However, it is extremely unlikely that these two complex types of molecules evolved together on the young earth. Within the last decade, biologists have turned to RNA as a way out of this chicken-and-egg paradox. In addition to acting as a genetic librarian and stenographer, RNA can mimic proteins by triggering certain reactions. But until now, that class of reactions had seemed limited.

For more information on this research see Nature, Vol. 374, 4/27/95, Wilson et al.