Label-Free Detection of Solo Oligonucleotide Lesion Based on Site-Direct Mutagenized Aerolysin Nanopore

Abstract

Nanopore sensing is one of the prominent single molecule detection tools due to its ultimate spatial and time resolution. Such tool offers wide application in studying peptide and protein-protein interaction, DNA translocation monitoringand even amino acid directional movement control. Aerolysin expressed by A. hydrophila can form watered filled lumen composed by 7-fold repeats, which provides 10-14 Å confined nano-space. When reconstituted into artificial bilayer, it self-assembled as nanopore. WT Aerolysin has fulfilled the discrimination of oligonucleotides at different length, single base discrimination (XA3, X=A, G, T, C), single methylcytosine detection (CA3 and mCA3) etc. Previously, by carefully examining the Aerolysin nanopore structure, R220 is credited with the bone fide sensing responsibility whilst K238 determines the substrate sojourn. We hereby present a rationally designed Aerolysin nanopore (denoted as AL) by optimizing the amino acid at 238 position. Primarily check the AL in the case of discriminating (dX)4 (X=A, G, T, C), it has one order of magnitude longer than the WT Aerolysin. The AL has been optimized so that been capable to read the slight electrical difference among random chains (XGTA) that is not accessible from any reported biological nanopores. As expected, single damaged nucleotide base could be identified from any position of a 4-base chain, moreover, lesion occurs at duel positions (ACGCGA, AmCGmCGA, AmCGCGA) could also be sensed. Further application would be performed utilizing such label-free tool to sense DNA lesion from whole blood or even in situ diagnose in a living cell.