Alpha-Helical Fragaceatoxin C Nanopore Engineered for Double-Stranded and Single-Stranded Nucleic Acid Analysis.
Nanopores are used in single-molecule DNA analysis and sequencing. Herein, we show that Fragaceatoxin C (FraC), an α-helical pore-forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin-free standard planar lipid bilayers. We engineered FraC for DNA analysis and show that the funnel-shaped geometry allows tight wrapping around single-stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches. Remarkably, despite the 1.2 nm internal constriction of FraC, double-stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through the deformation of the α-helical transmembrane region of the pore. Therefore, FraC nanopores might be used in DNA sequencing and dsDNA analysis.
Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9747 AG, Groningen, The Netherlands.
Carsten
0000-0003-0487-3311
C
Wloka
Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9747 AG, Groningen, The Netherlands.
Natalie Lisa
NL
Mutter
Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9747 AG, Groningen, The Netherlands. m.soskine@rug.nl.
Misha
0000-0002-2198-9466
M
Soskine
Chemical Biology I, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, 9747 AG, Groningen, The Netherlands. g.maglia@rug.nl.
Giovanni
G
Maglia
D015374:Biosensing Techniques; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis*; D055664:Electrochemical Techniques; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D058608:Nanopores; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017422:Sequence Analysis, DNA
D003064:Cnidarian Venoms; D004277:DNA, Single-Stranded; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; C545670:fragaceatoxin C; D004247:DNA
DNA analysis; DNA sequencing; fragaceatoxins; nanopores; single-molecule studies