G F Joyce — Page 5 | PubMed Health

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In vitro evolution of a ribozyme that contains 5-bromouridine.

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

Animals↗

PubMedDate not supplied

Chiral selection in poly(C)-directed synthesis of oligo(G).

Theories of the origin of optical asymmetry in living systems place fundamental importance on the amplification of optical asymmetry by an autocatalytic process. The replication of a polynucleotide is one obvious choice for such an autocatalytic growth mechanism. If an optically homogeneous polynucleotide could replicate by directing the polymerization of monomers of the same handedness, while excluding monomers of the opposite handedness, its chiral descendants would come to dominate what was once an achiral environment. Recently, two highly efficient template-directed reaction systems have been developed for the oligomerization of activated guanosine mononucleotides (Fig. 1) on a poly(C) template. The synthesis of L-guanosine 5'-mononucleotide makes it possible to study chiral selection in these systems. We report here that poly(C)-directed oligomerization of activated guanosine mononucleotides proceeds readily if the monomers are of the same optical handedness as the template, and is indeed far less efficient if the monomers are of the opposite handedness. However, in template-directed reactions with a racemic mixture, monomers of the opposite handedness to the template are incorporated as chain terminators at the 2'(3') end of the products. This inhibition raises an important problem for many theories of the origin of life.

Chromatography, High Pressure Liquid↗