Project Summary - Single-chain MspA for nanopore sequencing of DNA
New DNA sequencing technologies have transformed biomedical research and clinical
applications. Due to its low cost, fast sample processing time, minimal sample amount, and lack
of sample amplification, nanopore sequencing is a very promising technology to further reduce
costs and increase read length of the DNA sequencing. Essential to this method is a nanopore
which can be produced from inorganic, organic or biological materials. Despite recent
advancements significant challenges remain for nanopore sequencing of DNA. For example, the
residual current of each nucleobase in the MspA pore is determined by approximately four
nucleotides reducing the accuracy of base calling, DNA translocation through the pore is too
fast, and the stochastic activity of DNA-processing enzymes complicates data analysis. These
challenges could, in principle, be addressed by protein engineering, which, however, is
hampered by the oligomeric structure of MspA and all other currently used biological nanopores.
This proposal is based on the construction of single-chain MspA in which all eight subunits are
linked. Single-chain MspA enables us to control the subunit assembly of the MspA pore and the
chemical properties of each channel residue by site-directed mutagenesis. This technological
breakthrough provides the basis for further improvement of the sequencing capabilities of MspA.
The goals of this proposal are to characterize the channel activity of single-chain MspA and its
DNA sequencing properties and to systematically alter the pore diameter of MspA by
constructing scMspA pores with different subunit stoichiometries. This will enable us, for the first
time, to examine the influence of the pore diameter on nucleobase recognition by a biological
nanopore.