Single Molecule Rna Base Identification With A Biological Nanopore

The sequencing of individual DNA and RNA strands with nanopores is under investigation as a rapid, low-cost platform. In this approach, bases are identified in order as the strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as α-hemolysin (αHL), are advantageous because they can be precisely manipulated by genetic modification. Previously, it has been shown that the αHL nanopore contains three recognition sites, capable of discriminating between single DNA bases when oligonucleotides are immobilized within the nanopore. Here we extend these investigations into discrimination of nucleobases in RNA. By immobilizing RNA homopolymers within the αHL pore using a terminal biotin-streptavidin complex, we achieve sharply defined current distributions that enable clear discrimination of the nucleobases. This is further extended by investigating individual RNA bases in a DNA polyC background, which gives comparable results. Additionally, with a view to “exo sequencing”, an engineered protein nanopore is successfully used to detect and discriminate all four ribonucleoside monophosphates (rNMPs) by using, am7-β-cyclodextrin as both a transient and covalent molecular adapter.