Petra Burgstaller

An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudo-thymidine as a substrate for thermostable polymerases

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions. In combination with the scintillation proximity assay (SPA[trade]), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides. A proof of the concept is obtained using pseudo-thymidine (psiT), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psiT arising from the carbon-carbon bond between the sugar and the base make it an interesting probe for the importance of conformational restraints in the active site of polymerases during primer elongation. From a pool of commercially available thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides containing psiT. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psiT are presented. This is the first time that PCR has been performed with a C-nucleoside.

6 Did the RNA World Exploit an Expanded Genetic Alphabet

SINGLE BIOPOLYMER LIFE FORMS BASED ON RNA In terms of its macromolecular chemistry, life on Earth can be classified as a “two-biopolymer” system. Nucleic acid is the encoding biopolymer, storing information within an organism and passing it to its descendants. Nucleic acids also direct the biosynthesis of the second biopolymer, proteins. Proteins generate most of the selectable traits in contemporary organisms, from structure to motion to catalysis. The two-biopolymer strategy evidently works rather well. It has lasted on Earth for several billion years, adapting in this time to a remarkable range of environments, surviving formidable geobiological (and perhaps cosmic) events that threatened its extinction, and generating intelligence capable of exploring beyond Earth. The terrestrial version of two-biopolymer life contains a well recognized paradox, however, one relating to its origins. It is difficult enough to envision a nonbiological mechanism that would allow either proteins or nucleic acids to emerge spontaneously from nonliving precursors. But it seems astronomically improbable that both biopolymers arose simultaneously and spontaneously, and even more improbable (if that can be imagined) that both biopolymers so arose with an encoder-encoded relationship. Accordingly, a variety of “single-biopolymer” models have been proposed as forms of life that antedated the two-biopolymer system. These (presumably) could have emerged more easily than a two-biopolymer system. Such models postulate that a single biopolymer can perform the catalytic and information repository roles and undergo the Darwinian evolution that defines life (Joyce 1994). For example, Rich (1962), Woese (1967), Orgel (1968), and Crick (1968) proposed that the...

Structural characterization of a flavin-specific RNA aptamer by chemical probing

Abstract In a previous study, an RNA aptamer for the specific recognition of flavin cofactors was isolated from a combinatorial RNA-pool. We have now carried out a structural analysis of this RNA by chemical probing and identified residues protected from modification in the presence of the cofactor and modifications that interfere with its complexation. An induced fit of the RNA motif occurs upon cofactor binding. Our analysis led to a refined secondary structure model for the FMN/RNA complex.