Gabriel Friedman

October 28, 2010

The search for a Diels-Alderase

Investigations into the origin of life have centered on the “RNA World”

hypothesis, which suggests that ribonucleic acids were the precursor to the DNA that

enables our existence. In order to substantiate this claim, it is important to show that

RNA is capable of catalyzing a wide variety of reactions, including the crucial formation

of carbon-carbon bonds. This operation, known as the Diels-Alder cycloaddition, is

crucial to anabolic processes. In a recent paper titled, “A small catalytic RNA motif with

Diels-Alderase activity,” Burckhard Seelig and Andres Jäschke, from the Institute for

Biochemistry at Freie University in Berlin, have characterized a ribozyme that vastly

accelerates the formation of carbon-carbon bonds.

Since the 1980s, researchers have known that RNA can act as a catalyst in

biological reactions. In 1997, two years before this paper was published, a team at

NeXstar Pharmaceuticals in Colorado was able to catalyze a Diels-Alder reaction using a

ribozyme, although the RNA had to be modified. Led by Theodore Tarasow, the

researchers substituted 5-pyridylmethylcarboxamid-UTP13 in placed of the unmodified

nucleotide uridine triphosphate. The purpose of this alteration was to increase the amount

of hydrogen bonding, provide additional hydrophobic and dipolar interactions, and to

supply metal coordination sites that could not exist with natural RNA. The group ended

up finding a reaction acceleration rate of 800 for a Diels-Alder reaction between an

acyclic diene and a maleimide dienophile. While this finding was notable, it still was

insufficient to support the “RNA world” hypothesis because of the substituted nucleotide.
 The authors of this present paper first created a library of randomized 120-

nucleotide RNA conjugates which were then connected to anthracene via a polyethylene

glycol (PEG) unit. For the reaction between biotin maleimide and they first measured the

uncatalyzed rate constant and then added the antracene-PEG-RNA. This can be

visualized in the following reaction scheme. After an hour they were able to isolate the

Diels-Alder products by immobilizing them on streptavidin agarose.

The elegant methodology of the researchers must be noted. By including so many

RNA variants, they ensured to find a possible Diels-Alderase if one existed. And by

immobilizing the reacted cycloaddition products, they were able to efficiently determine

which RNA variants showed promise and which ones didn’t. After each round, they RNA

conjugates from the reacted products were reverse-transcribed and amplified via

polymerase chain reaction (PCR) and added to the next reaction cycle. As one can see

from the following diagram, after round 5 it was clear that dramatic reaction acceleration

rates were taking place.

From rounds 6-10, the amount of substrate and reaction time was diminished in order to

increase the selection pressure and find the motifs that were truly acting as catalysts. All

in all, the researchers found 42 different catalytic motifs, with 13 sequences that gave

acceleration rates of at least 15,000-fold.

They then examined the motifs and found that 90% of them shared a

pentanucleotide region with the sequence UGCCA and a hexanucleotide region with the

sequence AAUACU. Based on structural analysis, they proposed that the catalytic motif

contained a “bulge” that acted as the active site:

 To bolster this claim, they found when there was a non-complementary

“mismatch” at the top of the bulge, which would effectively increase the size of it, they

found an increased reaction rate. The researchers also examined whether the Diels-

Alderase could act as a true catalyst in a cleavage reaction and found that it was capable

of an uninspiring rate of 1.9 turnovers per hour, but it is still notable that such a turnover

could take place with a ribozyme. Overall, the researchers devised a clever experimental

scheme and undertook a rigorous analysis of their results, resulting in the fascinating

conclusion that ribozymes are capable of catalyzing a Diels-Alder reaction.

In the future, there are several steps the researchers could take to learn more about

Diels-Alder ribozymes. For example, they could alter the dienes and dienophiles used in

order to better understand the structural conformation of the active site. They could also

use selective mutations and compensatory double mutations in order to see which

nucleotides or pairs of nucleotides are truly integral to the functioning of the ribozyme.

Lastly, they could begin looking for a natural Diels-Alderase to see how such a ribozyme

would function in nature and how it compares to the catalytic motif that has now been

characterized.
References

Seelig, Burckhard, and Andres Jäschke. "A Small Catalytic RNA Motif with Diels-

Alderase Activity." Chemistry & Biology 6.3 (1999): 167-76.

Tarasow, T.M., S.L. Tarasow, & B.E. Eaton (1997). RNA-catalysed carbon-carbon bond

formation. Nature, 389: 54-57.