An AFM/Rotaxane Molecular Reading Head for Sequence‐Dependent DNA Structures

Abstract

A nanomechanical molecular “tape reader” is assembled and tested by threading a β-cyclodextrin ring onto a DNA oligomer and pulling it along with an AFM tip. The formation and mechanical operation of the system is confirmed by measuring the forces required to unfold secondary structures in the form of hairpins. Unfolding induced by this 0.7 nm aperture requires 40 times more force than that reported for pulling on the ends of the DNA. A kinetic analysis shows that much less strain is required to destabilize the double helix in this geometry. Consequently, much more force is required to provide the free energy needed for opening. DNA secondary structure may prove to be a significant obstacle both for enzymes that process DNA though an orifice, and for the passage through nanopores proposed for some novel sequencing schemes.

Polymer sequencing by molecular machines: a framework for predicting the resolving power of a sliding contact force spectroscopy sequencing method

“…These authors proposed that this rather low value was due to the mobility of the bases around their point of attachment to the (deoxy)ribose backbone, allowing them to fold flat against the phosphate-deoxyribose backbone to pass through the CD pore. The same group subsequently published experimental data 10 showing force plateaus for the sliding of β-CD along DNA somewhat larger than this value, at approximately 125 pN. We report here most probable sliding forces of 63-98 pN for differently-substituted aromatic groups and 45-46 pN for a monosaccharide ( passing through α-CD), at comparable or higher loading rates (instantaneous loading rates from 400 to 8000 pN s −1 ).…”

“…A check on the applicability of the method may be made by comparing the value of ΔG sl it predicts for ssDNA with the simulated and measured values found by Lindsay and Williams. 10,23 Using calculated values of Φ and P we find predicted values of ΔG sl for the four nucleotides and β-CD to fall between 67 and 76 kJ mol −1 . This is rather larger than the values of ΔG sl calculated using the simulated forces and spring constants reported by Lindsay and Williams 10,23 (31-33 kJ mol −1 ), but as noted above, the experimental data for the sliding of β-CD along ssDNA published subsequently 10 shows force plateaus of approximately 110 pN, corresponding to a ΔG sl of 67 kJ mol −1 which agrees with our prediction.…”

“…10,23 Using calculated values of Φ and P we find predicted values of ΔG sl for the four nucleotides and β-CD to fall between 67 and 76 kJ mol −1 . This is rather larger than the values of ΔG sl calculated using the simulated forces and spring constants reported by Lindsay and Williams 10,23 (31-33 kJ mol −1 ), but as noted above, the experimental data for the sliding of β-CD along ssDNA published subsequently 10 shows force plateaus of approximately 110 pN, corresponding to a ΔG sl of 67 kJ mol −1 which agrees with our prediction. Nelson et al 18 observed two classes of behaviour for ssDNA sliding in nanopores with dimensions from 1-2 nm: so-called "frictionless" and "stick-slip".…”

“…18 The most common terminology 19 for describing the processes occurring in a rotaxane depicts a macrocyclic 'bead' shuttling between 'stations' on the polymer axle. In the SCFS method described here and previously, [8][9][10] the bead is α-or β-cyclodextrin (α-CD and β-CD hereafter) and the stations are the individual monomers comprising the polymer axle, while the AFM probe supplies the unidirectional driving force for shuttling the bead between stations (hereafter 'sliding') under a load generated by the controlled separation of probe and sample. Fig.…”

“…The introduction of a driving force in the form of an AFM probe may be expected to drive the accommodation and passage of even larger guests at the cost of frictional energy, and indeed the passage of single stranded DNA through β-CD, for which Φ > 3.3, has been experimentally observed. 10 The calculation of Φ for stations 1 to 5 is presented in ESI, † and ranges from 0.6 to 3 (Table S3 †). Likewise, the solvation component reflects the passage of the monomer from an aqueous environment, into the hydrophobic interior of the CD pore, and then back out into the aqueous phase again, so that P, the dimensionless octanol : water partition coefficient commonly used (in its log form) as a measure of hydrophobicity in drug design, can be used to describe the relative favourability of these two environments for a particular monomer.…”