Nature Chemical Biology: Polymerase’s new language

已有 1815 次阅读 2016-6-9 09:11 |系统分类:论文交流

Polymerase’s new language

DNA aptamers are of potential use in numerous biotechnological applications, and the addition of methoxy (OMe) or fluorine substituents to C2′ of the sugar ring makes DNA resistant to nuclease-mediated degradation. However, natural DNA polymerases can neither read sequences containing these substituents nor incorporate the modified nucleotides into newly synthesized oligomers, which hampers their use in aptamer evolution. Using an improved phage-based selection system for evolved polymerases, Chen et al. identified novel thermostable DNA polymerase variants that can 'reverse transcribe' a fully C2′-OMe-modified template into a DNA product or vice versa. A third polymerase variant identified in the screen can directly PCR amplify DNA with every G, C, A, or U nucleotide modified with C2′-OMe or any combination of two nucleotides modified with C2′-F. Analysis of the mutations acquired during polymerase evolution indicates that their increased activity with unnatural substrates stems from stabilization of the interaction between the finger and thumb domains via a salt bridge. The error rates for these evolved enzymes are 10- to 100-fold greater with the modified substrates compared to Taq polymerase with a natural substrate, but this is not likely to hamper their use in many practical applications. These polymerases, and others developed through the same activity-based selection system, should be useful for streamlining the development of DNA aptamers.

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