Artificial RNA enzymes: big and fast.

RNA, the molecular middleman that translates DNA into proteins, has long been like the comic Rodney Dangerfield: It just didn't "get no respect."

That scientific disdain evaporated in the last decade as researchers discovered that RNA can perform an important duty many considered the province solely of proteins. Certain RNA molecules, called ribozymes, act like enzymes, substances that enable specific chemical reactions to go forward. That discovery even led to the bold hypothesis that early life was composed only of RNA that could build copies of itself as well as other molecules.

For such an RNA world to exist, there would have to be an impressive array of ribozymes to drive all the chemical reactions needed for life. Yet investigators looking for ribozymes in nature have found only a small variety.

To help establish the feasibility of an RNA world, some researchers are striving to construct ribozymes with new functions. They argue that if they can make a ribozyme that performs an important function, such as copying itself, then that ribozyme might once have existed, only to have disappeared as evolution proceeded. "The idea is that protein [enzymes] replaced RNA and we are left with few remnants of the RNA world," says David P. Bartel of the Whitehead Institute for Biomedical Research in Cambridge, Mass.

In 1993, Bartel and Jack W. Szostak, both then at Massachusetts General Hospital in Boston, reported that they had sifted through more than 1,000 trillion different lab-made RNA molecules, each a random sequence of 220 nucleotides, the basic building blocks of RNA. They were looking for RNA molecules that could chemically attach themselves to a specific target RNA molecule that they provided. They identified 65 novel ribozymes that could perform this chemical reaction.

The two then subjected these RNA molecules to 10 generations of so-called test-tube evolution, a molecular survival-of-the-fittest strategy (SN: 8/7/93, p.90). In each cycle of this process, they created copies of the preceding generation but introduced small errors in the RNA sequences. They then selected out the ribozymes that could best attach themselves to their target RNA and sent them through another round.

In the July 21 Science, Bartel, Szostak, and Eric H. Ekland, also of the Whitehead Institute, discuss further experiments with these ribozymes. After grouping the most efficient descendants of the original 65 into seven families, the researchers determined each family's catalytic domains--the nucleotides that help the ribozyme to bond chemically to the target RNA. These domains speed the joinings but don't include the specific nucleotides that bind to the target RNA. By creating an RNA molecule composed only of a particular domain, the scientists created a smaller ribozyme that can join RNA strands more than once.

One of the most interesting ribozymes Bartel and his colleagues found had a catalytic domain 93 nucleotides long. That's a surprisingly complex domain to emerge randomly from a 220-nucleotide-long molecule, asserts Bartel.

This particular ribozyme is also speedy, they report. Through further test-tube evolution, they identified its fastest version: a ribozyme that could join RNA strands a billion times faster than they would normally join. This reaction rate, more than once a second, is faster than any other ribozyme-mediated reaction and comparable to reactions aided by protein enzymes.

"That's impressive. People have always regarded RNA enzymes as slugs compared to proteins," says Gerald F. Joyce of Scripps Research Institute in La Jolla, Calif.