Scalable Production of DNA-Probe-Functionalized Heteromeric MspA Nanopores for Biosensing

Biological nanopores enable single-molecule sensing but often lack specificity when analytes produce similar current signatures. Covalent tethering of recognition elements improves selectivity, yet site-specific functionalization of symmetric, multimeric pores like <i>Mycobacterium smegmatis</i> porin A (MspA) remains challenging. Existing gel-based strategies for isolating heteromeric pores are labor-intensive and low-throughput and can compromise protein functionality. Here, we report a modular strategy for producing hetero-octameric MspA nanopores with a single site-specific modification. By coexpressing MspA and a D56C MspA mutant bearing a C-terminal D16 tag in <i>Escherichia coli</i> (<i>E. coli</i>), we directed the assembly of asymmetric pores with defined subunit composition. To isolate the modified pores, we developed a rapid, nondenaturing purification method that integrates magnetic bead capture with toehold-mediated DNA strand displacement, demonstrating selective enrichment of functionalized pores in under 3.5 h while preserving structural integrity. Using this platform, we generated MspA nanopores functionalized with single DNA probes targeting dopamine, microRNA, and thrombin. Single-channel recordings demonstrated distinct current signatures upon target recognition, enabling label-free and selective detection. This approach offers a robust and scalable framework for engineering functionalized nanopores for diverse molecular sensing.