Preparation of Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing of nucleic acids

Abstract

Mycobacterium smegmatis porin A (MspA), possessing a short (~0.6 nm) and narrow (~1.2 nm) con-striction that enables its high spatial resolution of sensing, has emerged as an optimum choice for nano-pore sequencing and nano-reactive sensing. However, prepared MspA nanopores cannot be obtained from any commercial vendors. We here provide a highly simplified protocol for MspA preparation. The protocol yields ~10 mg fully oligomerized protein per liter of culture and is straightforward to follow. With the prepared MspA nanopores, discrimination of immobilized ssDNA oligonucleotides can be per-formed, which serves as a demo application to demonstrate core procedures of single molecule sensing using MspA. This method is also in principle compatible with all other MspA mutants, which might merit the need to develop a variety of MspA based nano-reactive sensors.