In nature, Darwinian evolution takes millennia or more toeffect noticeable changes in an organism. In molecular biologistGerald F. Joyce's laboratory, it took 10 generations of "one ortwo working days" each.

Today's issue of Science describes how Joyce and his colleague,Amber A. Beaudry at The Scripps Research Institute in La Jolla,Calif., caused an RNA-cleaving stretch of nucleic acid -- aribozyme -- to evolve into a DNA-cleaver.

In the beginning, they extracted and amplified a randomizedmixture of nucleic acid variants from a protozoanmicroorganism, Tetrahymena thermophila. In this mix, theyisolated the ribozyme responsible in nature for catalyzing itsown excision from precursor ribosomal RNA.

Multiplying the heterogeneous native sequence 10 trilliontimes by polymerase chain reaction (PCR), they hit upon oneenzyme-like nucleic-acid sequence in the population thatcleaved DNA rather than RNA -- but only at very hightemperature.

Selectively amplifying this misfit ribozyme in a mutagenic PCRenvironment and repeating the process 10 times, Joyce andBeaudry ended up with a molecule that splits DNA at bodytemperature almost as efficiently as its aboriginal ancestorcleaved RNA. This "full-bore, directed evolution-in-a-test-tube," Joyce told BioWorld, replicates the basic principles ofDarwinian evolution -- selection, amplification andmutagenesis.

"But like most people," he adds, "we are thinking not justphilosophically, but in a biotechnology framework; how to useour in vitro genetics to find new drugs."

Joyce said this approach is "the opposite of rational drugdesign. We don't know ahead of time what we're trying to get.We throw all three technologies at a problem -- selection,amplification, mutagenesis -- make a trillion or 10 trillionrandomized guesses, select one in a billion and repeat theprocess until we have a population enriched in the propertythat interests us."

A renowned British molecular biologist, Sydney Brenner,approvingly calls this approach "irrational drug design."

With part of his funding from the NIH's AIDS researchprogram, Joyce has put his test tube-evolved ribozyme intoEscherichia coli, where it cleaves the genome of a bacterialvirus. "Our goal is not to vaccinate a germ," he observes, butrather to inhibit a retrovirus such as HIV."

At least two U.S. biotechnology R&D companies, Gilead SciencesInc. and NeXagen Inc. are known to be seeking new drugs byselectively amplifying nucleic acids.

-- Gilead of Foster City, Calif., has created "aptamers," double-stranded DNA or single-stranded RNA molecules, from auniverse of more than a trillion molecules. They bind to andinhibit specific disease-causing protein. Several inhibitedthrombin-catalyzed fibrin-clot formation in vitro. Gileadexpects to file an IND and begin cardiovascular clinical trials bymid-1994.

-- NeXagen of Boulder, Colo., employing a proprietarytechnology it calls SELEX (for systematic evolution of ligands byexponential enrichment), makes high-affinity nucleic-acidligands targeting small peptides and proteins, such asthrombin, nerve growth factor and several involved inreplication of the AIDS virus.

"But selection and amplification are only two-thirds of a trueDarwinian evolutionary method," observes Joyce. The third is tointroduce new mutations into progeny."

NeXagen's founder, Larry Gold, remarked of the Science paper:"It's a beautiful piece of high craftsmanship that extends ourknowledge of ribozymes a small amount."

Gilead's president and CEO, Michael L. Riordan, told BioWorld: "Ifollow Joyce's work with admiration. It's an important thing inthe field."

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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