Comparative biosensing of glycosaminoglycan hyaluronic acid oligo- and polysaccharides using aerolysin and [Formula: see text]-hemolysin nanopores⋆.

Seeking new tools for the analysis of glycosaminoglycans, we have compared the translocation of anionic oligosaccharides from hyaluronic acid using aerolysin and [Formula: see text]-hemolysin nanopores. We show that pores of similar channel length and diameter lead to distinct translocation behavior of the same macromolecules, due to different structural properties of the nanopores. When passing from the vestibule side of the nanopores, short hyaluronic acid oligosaccharides could be detected during their translocation across an aerolysin nanopore but not across an [Formula: see text]-hemolysin nanopore. We were however able to detect longer oligosaccharide fragments, resulting from the in situ enzymatic depolymerization of hyaluronic acid polysaccharides, with both nanopores, meaning that short oligosaccharides were crossing the [Formula: see text]-hemolysin nanopore with a speed too high to be detected. The translocation speed was an order of magnitude higher across [Formula: see text]-hemolysin compared to aerolysin. These results show that the choice of a nanopore to be used for resistive pulse sensing experiments should not rely only on the diameter of the channel but also on other parameters such as the charge repartition within the pore lumen.

Single channel planar lipid bilayer recordings of the melittin variant MelP5.

MelP5 is a 26 amino acid peptide derived from melittin, the main active constituent of bee venom, with five amino acid replacements. The pore-forming activity of MelP5 in lipid membranes is attracting attention because MelP5 forms larger pores and induces dye leakage through liposome membranes at a lower concentration than melittin. Studies of MelP5 have so far focused on ensemble measurements of membrane leakage and impedance; here we extend this characterization with an electrophysiological comparison between MelP5 and melittin using planar lipid bilayer recordings. These experiments reveal that MelP5 pores in lipid membranes composed of 3:1 phosphatidylcholine:cholesterol consist of an average of 10 to 12 monomers compared to an average of 3 to 9 monomers for melittin. Both peptides form transient pores with dynamically varying conductance values similar to previous findings for melittin, but MelP5 occasionally also forms stable, well-defined pores with single channel conductance values that vary greatly and range from 50 to 3000pS in an electrolyte solution containing 100mM KCl.

Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling.

The enzymatic degradation of long polysaccharide chains is monitored by nanopore detection. It follows a Michaelis-Menten mechanism. We measure the corresponding kinetic constants at the single molecule level. The simulation results of the degradation process allowed one to account for the oligosaccharide size distribution detected by a nanopore.

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 α-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.

Targeting specific membranes with an azide derivative of the pore-forming peptide ceratotoxin A.

Pore-forming antimicrobial peptides (AMPs) are attracting interest as cytolytic antibiotics and drug delivery agents with potential use for targeting cancer cells or multidrug-resistant pathogens. Ceratotoxin A (CtxA) is an insect-derived cytolytic AMP with 36 amino acids that is thought to protect the eggs of the medfly Ceratitis capitata against pathogens. Single channel recordings using planar lipid bilayers have shown that CtxA forms pores with well-defined conductance states resembling those of alamethicin; it also forms one of the largest pores among the group of ceratotoxins. In this work, we modified CtxA at its N-terminus with an azide group and investigated its pore-forming characteristics in planar lipid bilayer experiments. We demonstrate the possibility to target specific lipids by carrying out click reactions in-situ on lipid membranes that display a dibenzocyclooctyne (DBCO) moiety on their head group. As a result of covalent linkage of the peptides to the bilayer, pore-formation occurs at 10-fold reduced peptide concentration and with a reduced dependence on the transmembrane voltage compared to unlinked CtxA-azide peptides or native CtxA peptides.

Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore.

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.

Single molecule detection of glycosaminoglycan hyaluronic acid oligosaccharides and depolymerization enzyme activity using a protein nanopore.

Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated molecules accumulated into the arrival compartment using high-resolution mass spectrometry. Anionic oligosaccharides of various polymerization degrees were discriminated through measurement of the dwelling time and translocation frequency. This molecular sizing capability of the protein nanopore device allowed the real-time recording of the enzymatic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymatically produced oligosaccharides was carried out to monitor the depolymerization enzyme reaction at the single-molecule level.