Catalytic RNA accelerates Diels-Alder cycloaddition

100Nl· •3'

RNA-PEG-diene terminus

Ο

ΗΝ ΝΗ

Η Η 4

Maleimide compound

Diels-Alder reaction

Cycloaddition product

N—Biotin H

terminus

PEG = polyethylene glycol 100 Ν = 100-nucleotide randomized region of RNA

library of 1014 unique RNAs, each of which contained a 100-nucleotide ran­domized region flanked by constant-sequence segments. To enhance the cat­alytic potential of the RNAs, pyridyl-mod-ified uridine was substituted for natural uridine in the RNA synthesis process. The pyridyl-modified RNAs were cou­pled to an acyclic diene (one of two cy­cloaddition reagents) via a polyethylene glycol (PEG) linker—an arrangement that made it possible for the diene to act like a free-solution species and thus interact with catalytic sites on the RNA surface.

The second Diels-Alder reagent, a male­imide compound linked to biotin, was mixed with the RNA-diene in the presence of transition-metal ions such as Cu2+, which act as cofactors in enzyme-catalyzed reactions. Products formed by cyclization of the RNA-coupled dienes with the maleimide were separated out of solution by retention on streptavidin (a bi-otin-binding reagent), and the associated

RNAs were isolated and amplified for further rounds of selection. Af­ter 12 rounds, eight RNAs were found to catalyze the cycloaddi­tion, with rate accelerations of up to 800 relative to the uncatalyzed reaction. In a subsequent study published in Chemistry & Biology [4,675 (1997)], Eaton's group also identified RNAs that catalyze amide synthesis, with rate acceler­ations of up to 100,000.

Molecular biology professor Michael J. Yarus of the University of Colorado, Boulder—who spe­cializes in selection of RNAs capa­ble of ligand binding and cataly­sis—says, "The gloves are off in the informal battle between RNA-based and protein enzymes. Though RNAs have been jokingly called 'functionally deprived' by protein fanciers impressed by the greater chemical variety of amino acid side chains, it now seems like­ly that hybrozymes—ribozymes with added functional groups-may contain any functional group in the protein repertoire. Hydrogen-bonding, metal-chelating, and hy­drophobic groups in the uridine-modified molecules may give rise to many new RNA structures and functions."

The study may have implica­tions for the identification and en­richment of small organic mole­cules from combinatorial libraries. "We are now taking small-molecule

libraries and combining them via RNA catal­ysis to make new products," says Eaton. "There's a simultaneous evolution of small-molecule libraries in conjunction with their RNA catalysts, so that the RNA and the small molecules coevolve," he says.

In addition, the findings lend support to the RNA world hypothesis. The hy­pothesis was proposed when scientists realized that proteins couldn't have evolved before DNA because DNA is needed to encode proteins, and that DNA couldn't have evolved before pro­teins because enzymes are needed to cat­alyze DNA replication. The hypothesis suggests that RNA (which is capable of both replication and catalysis) evolved first, and that DNA and proteins adopted their now-essential roles in the machin­ery of life later on. The new findings, which show that modified RNA can cata­lyze a wider range of reactions than pre­viously realized, support the hypothesis.

Stu Borman

Latest report finds EPA reforms fall short In a National Academy of Public Admin­istration (NAPA) report released Sept. 10, Congress and the Environmental Protec­tion Agency are blamed for their inability to reform an ineffective environmental regulatory system.

The report adds to a chorus of critics who are urging EPA to institute a perfor­mance-based, integrated, decentralized en­vironmental protection system (C&EN, April 21, page 12). However, NAPA panel members releasing the report also say Congress must share blame with EPA for creating conditions under which statutory reforms, such as those endorsed by the re­port, are very difficult to achieve.

The report, according to NAPA panel member William D. Ruckelshaus, chair­man of Browning-Ferris Industries and twice EPA administrator, offers an easier path for reform, a series of "stepping stones" to begin harmonizing different, multimedia environmental laws.

"Resolving the Paradox of Environmen­tal Protection" comes two years after a similar NAPA investigation of EPA proved to be a touchstone in the debate over en­vironmental protection. Called for by Con­gress, the new report finds that EPA has tried to reform itself, especially through its reinvention experiments. Overall, howev­er, the agency has fallen short of making major changes in core programs and is only "operating at the margins."

Specifically, the report says: • Congress and EPA should develop

an "integrating statute" to legally encour­age multimedia, performance-based regu­latory experiments, such as Project XL.

• EPA programs giving more authori­ty to states, such as the National Environ­mental Performance Partnership System, should be encouraged.

• New systems to better monitor and evaluate programs should be developed, giving EPA the capacity to "learn how to learn."

• EPA, states, and Congress should make better use of the Government Per­formance & Results Act to set goals, de­fine choices, and build an annual budget to achieve those aims.

A "nonemotional window of rationali­ty" may now exist, according to Ruck­elshaus, that could allow alternatives to move forward. He also notes that a large, diverse committee he chairs, Enterprise for the Environment, will issue its own report in a couple of weeks. But for ei-

10 SEPTEMBER 15, 1997 C&EN

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Once a soda bottle, now a car? Chrysler unveiled its plastic-bodied Composite Concept Vehicle (CCV) at the Frankfurt International Auto Show last week. The recyclable polyethylene terephthalate contains 15% crushed glass fiber and additives for UV protection and to aid molding. The car is made from four panels that are glued together with polyurethane adhesive on top of a steel chassis. Whereas a conven­tional car has 4,000 components, CCV consists of a mere 1,100 parts and needs no paint. It takes only six-and-a-half hours to build (a Neon takes 19 hours) in one-third the space of a conventional car. At 1,200 lb— less than half the weight of a comparable metal sedan— CCV is also environmentally sound, managing 50 mpg. And the low-cost plastic panels can include up to 20% recycled resin and can themselves be recycled. Chrysler says it may have the plastic vehicle available for sale by 2000. The company is positioning the car for develop­ing countries, where it would sell for around $6,000. Chrysler hopes to move the technology into main­stream auto construction.

Elisabeth Kirschner

ther of the reports to succeed, he says, Congress must get "actively engaged" and a "climate of fear" that has existed between EPA and Congress must end.

"It is very difficult for EPA to try risky initiatives because if they fail staff will be dragged to a hearing," Ruckelshaus says. Members of Congress should not sit like "King Farouk looking down on the poor people from EPA," he says. Instead, mem­bers of Congress and EPA should sit at the same table, discussing "the nature of these problems and how best to deal with them."

Ruckelshaus stresses that building trust is difficult in Washington. "There is a whole army of people in this town with a vested interest in the status quo," he notes. "They make their money fighting, and if you get something that works and the fight stops, they're in trouble."

Jeff Johnson

Record penalties levied for '87 chemical fire After 10 years of litigation, a New Or­leans jury has responded to plaintiffs' complaints that they were harmed by a butadiene railcar fire that occurred in their neighborhood: They awarded a record $3.4 billion in punitive and com­pensatory damages to be shared by 8,000 plaintiffs and their lawyers. The damages are to be paid by nine companies named as defendants in the suit:

The incident involved a railcar carry­ing butadiene that leaked and caught fire while parked in a New Orleans rail yard in September 1987. About 1,000 nearby residents were evacuated for 36 hours while the fire was allowed to burn itself out. No serious injuries, deaths, or major property damage were reported at the time of the incident.

The plaintiffs complained of physical and mental anguish, which they attribute to the incident. Compensatory damages of $2 million, plus 10 years' interest, were assessed against all defendants and awarded to 20 plaintiffs.

All 8,000 plaintiffs are eligible to share the $3.4 billion punitive damage award, if they prove to the court that they were harmed by the incident. Put­ting up the money are the five parties involved in transporting the hazardous cargo—$2.5 billion from CSX Transpor­tation, the owner and operator of the rail yard where the incident occurred; $175 million each from Illinois Central and Norfolk Southern Railroads, the op­erators of rail lines traversed at earlier points during the car's trip to New Or­leans; $375 million from Mitsui & Co., lessee of the railcar; and $190 million from GATX Corp., loader and inspector of the railcar.

Under Louisiana law, only companies that handle, transport, or store hazardous substances can be held liable for punitive damages. The four remaining defendants are responsible for compensatory damag-

I es only: Phillips Petroleum, the previous

owner of the railcar; Polysar Latex, own­er of the butadiene; North American Tank Car Corp., manufacturer of the rail-car; and General American Transporta­tion Corp., the lessor of the railcar. Poly­sar Latex is now part of Canada's Nova Chemicals.

In its 1988 report on the incident, the National Transportation Safety Board stat­ed that the probable cause of the inci­dent was an improperly sealed manhole cover on the railcar and cited North American Tank Car and Phillips for im­proper procedures. The report also not­ed that the emergency was exacerbated by a lack of adequate information at the scene to quickly assess and resolve the problem.

This award represents the highest penalty ever levied against a railroad and comes at a particularly difficult time for CSX and Norfolk Southern as they seek regulatory approval for their purchase of Conrail (C&EN, March 10, page 10). The award is unusually high compared with the $470 million assessed against Union Carbide for the 1984 disaster in Bhopal, India, and is almost as much as the $5 bil­lion assessed against Exxon for the Val-dez oil spill in Alaska.

At press time, the presiding judge was reviewing the case and could set aside or reduce the jury award and determine the extent of defendant liability. All of the defendant companies have vowed to ap­peal the case depending on how the judge proceeds.

Paige Morse

SEPTEMBER 15, 1997 C&EN 11