Tuning the Diameter, Stability, and Membrane Affinity of Peptide Pores by DNA-Programmed Self-Assembly

Protein pores recently enabled a breakthrough in bioanalytics by making it possible to sequence individual DNA and RNA strands during their translocation through the lumen of the pore. Despite this success and the overall promise of nanopore-based single-molecule analytics, protein pores have not yet reached their full potential for the analysis and characterization of globular biomolecules such as natively folded proteins. One reason is that the diameters of available protein pores are too small for accommodating the translocation of most folded globular proteins through their lumen. The work presented here provides a step toward overcoming this limitation by programmed self-assembly of alpha-helical pore-forming peptides with covalently attached single-stranded DNA (ssDNA). Specifically, hybridization of the peptide ceratotoxin A (CtxA) with N-terminally attached ssDNA to a complementary DNA template strand with 4, 8, or 12 hybridization sites made it possible to trigger the assembly of pores with various diameters ranging from approximately 0.5 to 4 nm. Hybridization of additional DNA strands to these assemblies achieved extended functionality in a modular fashion without the need for modifying the amino acid sequence of the peptides. For instance, functionalization of these semisynthetic biological nanopores with DNA-cholesterol anchors increased their affinity to lipid membranes compared to pores formed by native CtxA, while charged transmembrane segments prolonged their open-state lifetime. Assembly of these hybrid DNA-peptides by a template increased their cytotoxic activity and made it possible to kill cancer cells at 20-fold lower total peptide concentrations than nontemplated CtxA.

Automated summary

The study demonstrated a method to overcome the size limitation of protein pores for analyzing and characterizing globular biomolecules by programmably assembling peptide with single-stranded DNA to form pores of various diameters. The functionalization of these semisynthetic biological nanopores with DNA-cholesterol anchors increased their sensitivity to lipid membranes and prolonged their open-state lifetime, showing potential for enhanced cytotoxic activity against cancer cells.