Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter.
Individual nucleic acid molecules might be sequenced by the identification of nucleoside 5'-monophosphates as they are released by processive exonucleases. Here, we show that single molecule detection with a modified protein nanopore can be used to identify ribonucleoside and 2'-deoxyribonucleoside 5'-monophosphates, thereby taking a step along this path. Distinct levels of current block are observed for each of the four members of a set of nucleoside 5'-monophosphates when the molecules bind within a mutant alpha-hemolysin pore, (M113R)(7), equipped with the molecular adapter heptakis-(6-deoxy-6-amino)-beta-cyclodextrin. While our results compare favorably with alternative approaches, further work will be required to improve the accuracy of identification of the nucleic acid bases, to feed each released nucleotide into the pore, and to ensure that every nucleotide is captured by the adapter.
Astier
Yann
Y
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, OX1 3TA, Oxford, UK.
Braha
Orit
O
Bayley
Hagan
H
D004247:DNA / Q000032:analysis*; D003854:Deoxyribonucleotides / Q000032:analysis*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D045333:Nanotubes, Peptide / Q000737:chemistry*; D015202:Protein Engineering; D012265:Ribonucleotides / Q000032:analysis*; D017422:Sequence Analysis, DNA / Q000379:methods*; D047392:beta-Cyclodextrins / Q000737:chemistry*
D003854:Deoxyribonucleotides; D006460:Hemolysin Proteins; D045333:Nanotubes, Peptide; D012265:Ribonucleotides; D047392:beta-Cyclodextrins; D004247:DNA