How is a selective enzyme made even more selective? With a combinatorial approach to redesign an enzyme motif (see figure; red=O, blue=N), the mismatch extension selectivity of DNA polymerases was increased. The results impart new and deeper insight into the mechanisms of DNA polymerases and provide new tools for applications such as efficient genotyping.
In addition to their potential as diagnostic and therapeutic agents, modified oligonucleotides have also been shown to be highly valuable tools for examination of complex biological processes. Carefully designed nucleotide analogues have therefore found considerable application in investigations of DNA polymerase function and mechanism. To examine the contribution of primarily steric constraints on DNA polymerase selectivity, we have developed a new functional strategy based on the use of modified nucleotide analogues that differ primarily in their steric demand. Here we report the efficient synthesis of modified thymidine analogues bearing 4Ј-alkyl groups with varying steric demand, the effects of 4Ј-alkylation on sugar puckering, and the incorporation of
C8 Arylamine dG adducts were converted into their corresponding 5' O DMTr 3' O phosphorami dite C8 arylamine dG derivatives. These compounds were used for the automated synthesis of site specifically modified oligonucleotides. The oligonucleotides were studied for their CD properties, T m values, and their effects on primer extension assays using human DNA polymerase b.
Transmission of the genetic information from the parental DNA strand to the offspring is crucial for the survival of any living species. In nature this process is catalyzed by the replication machinery in which DNA polymerases are essential for the entire DNA synthesis. Recently, a wealth of valuable new insights into DNA polymerase mechanisms were gained through application of carefully designed synthetic nucleotides and oligonucleotides in functional enzyme studies. The applied analogues exhibit features that differ in certain aspects from their natural counterparts and thus, allow investigation of the involvement and efficacy of a chosen particular aspect on the entire complex enzyme mechanism. This review will focus on a depiction of the synthetic efforts that were undertaken towards the targeted synthesis of nucleotide analogues with carefully altered properties. The synthetic endeavors will be discussed in the context of the motivation and the problem under investigation.
Employing modified oligonucleotides that are 4'-alkylated site-specifically we investigated the involvement of DNA minor groove hydration on DNA duplex stability and helix conformation.
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