An Efficiently Extended Class of Unnatural Base Pairs

摘要

The four natural nucleotides of DNA form two base pairs that are capable of encoding complex genetic information as well as functional nucleic acids.Expansion of the genetic alphabet to include a third base pair,formed between two identical or different unnatural nucleotides,referred to as self-pairs and hetero-pairs,respectively,would expand the informational and functional potential of DNA.While the pairing of the natural nucleo-bases is mediated by hydrogen-bonding (H-bonding)complementarity,it has become clear that,like proteins,hydrophobic forces are also capable of mediating these interactions.Indeed,a variety of nucleotides bearing predominantly hydrophobic nu-cleobase analogues form pairs that are both stable in duplex DNA and synthesized by the exonuclease-deficient Klenow fragment of DNA Pol I (Kf),by incorporation of an unnatural dNTP opposite the unnatural base in the template,with reasonable efficiency and selectivity.However,extension of the newly synthesized unnatural base pair,by incorporation of the next correct dNTP,has consistently limited the enzymatic synthesis of unnatural DNA.Only recently have efforts begun to focus on the determinants of efficient extension;however,they remain largely unknown.This has limited the rational design of viable unnatural base pairs.