The direct manipulation by atomic force microscopy (AFM) of individual macrocycles within a rotaxane offers a potential route to a new sequencing tool for complex macromolecules such as polysaccharides, glycoproteins and nucleic acids. In this paper we demonstrate for the first time that a sliding contact made between a macrocycle, α-cyclodextrin, and its polymer axle by an AFM tip can be used to map the positions of specific groups along the polymer as if they were beads along a string, thereby generating sequence information. We find very good agreement (linear fit with slope = 1.03, R(2) = 0.968) between the calculated and measured positions of phenylenediamine and benzenetricarboxylic acid groups within polymers of polyethylene oxide (PEO). The rupture force profiles attributable to the dethreading interactions of phenylenediamine and benzenetricarboxylic acid differ observably from each other and, in the latter case, from the rupture of the corresponding host-guest complex. As well as opening the way to a macromolecular sequencing technique, the ability demonstrated by this method to manipulate the dethreading of a rotaxane offers a new tool for investigating the process energetics in a wide array of spontaneously forming and forced rotaxane systems.

We evaluate an AFM-based single molecule force spectroscopy method for mapping sequences in otherwise difficult to sequence heteropolymers, including glycosylated proteins and glycans. The sliding contact force spectroscopy (SCFS) method exploits a sliding contact made between a nanopore threaded over a polymer axle and an AFM probe. We find that for sliding α-and β-cyclodextrin nanopores over a wide range of hydrophilic monomers, the free energy of sliding is proportional to the sum of two dimensionless, easily calculable parameters representing the relative partitioning of the monomer inside the nanopore or in the aqueous phase, and the friction arising from sliding the nanopore over the monomer.Using this relationship we calculate sliding energies for nucleic acids, amino acids, glycan and synthetic monomers and predict on the basis of these calculations that SCFS will detect N-and O-glycosylation of proteins and patterns of sidechains in glycans. For these applications, SCFS offers an alternative to sequence mapping by mass spectrometry or newly-emerging nanopore technologies that may be easily implemented using a standard AFM.