Table 1.

Comparison of the kinetics and thermodynamics of three oligonucleotide pairs studied by single channel current recordings with DNA-modified αHL pores and by ensemble melting curves

Oligo	Sequence*	Values derived from nanopore recordings				Values derived from melting profiles in solution
		kon [M−1⋅s−1]†	koff [s−1]†	Kd [M]	ΔG° [kcal mol−1]‡	k′on [M−1⋅s−1]‡§	k′off [s−1]‡¶	K′d‡	ΔG′° [kcal mol−1]‡
Oligo-B	5′-GGTGAATG-3′	1.3  × 107	1.2	9.2  × 10−8	−9.2	107	0.4	3.6  × 10−8	−9.8
Oligo-D	5′-TACGTGGA-3′	2.2  × 107	3.4	1.5  × 10−7	−8.9	107	1.7	1.7  × 10−7	−8.9
Oligo-E	5′-GGTGAAT-3′	1.1  × 107	16	1.5  × 10−6	−7.7	107	8	8.3  × 10−7	−8.0

^*The sequences of the DNA oligonucleotides added to the solution are given. The sequence of the corresponding tethered DNA strands were: for oligo-B, oligo-A (5′-CATTCACC-3′); for oligo-D, oligo-C (5′-TCCACGTA-3′); for oligo-E, oligo-A. 

^† The values given are derived from the arithmetic mean of the exponential fits of at least three independent single channel current recordings performed at 20 ± 0.4°C. For oligo-E⋅oligo-A, the single exponential fits for the event lifetime, and for oligo-D⋅oligo-C, and oligo-B⋅oligo-A, the exponential fits for the dominant, longer lifetime were used. The single exponential fits for the inter-event interval were used in all cases, and the values were adjusted for the proportion factor P in the case of oligo-D⋅oligo-C and oligo-B⋅oligo-A. The association constant (k_on) for duplex formation in the DNA-nanopore was calculated from k_on = 1/(c⋅τ_on), where τ_on is the inter-event interval and c the concentration of free oligonucleotide in the cis chamber. The strand dissociation constant (k_off) was derived from the event lifetime (τ_off): k_off = 1/τ_off. The standard deviations of k_on and k_off were smaller than 15%. 

^‡ The values are for 20°C. The values for the nanopore were derived experimentally at this temperature, and the values in solution were calculated for 20°C by using the experimentally derived thermodynamic parameters (Supporting information no. 3; www.pnas.org). 

^§ The association rate constants were assumed to be 10⁷ M⁻¹⋅s⁻¹ (see Materials and Methods, Kinetic Analysis). 

^¶ The rate constants for duplex dissociation in homogeneous solution were calculated by using the relation k′_off = k′_on⋅K′_d. The equilibrium dissociation constant K′_d was experimentally derived as described in Materials and Methods, Kinetic Analysis.