Kinetics of duplex formation for individual DNA strands within a single protein nanopore

Abstract

A single oligonucleotide was covalently attached to a genetically engineered subunit of the heptameric protein pore, ␣-hemolysin, to allow DNA duplex formation inside the pore lumen. Singlechannel current recording was used to study the properties of the modified pore. On addition of an oligonucleotide 8 bases in length and with a sequence complementary to the tethered DNA strand, current blockades with durations of hundreds of milliseconds occurred, representing hybridization events of individual oligonucleotides to the tethered DNA strand. Kinetic constants for DNA duplex formation at the single molecule level were derived and found to be consistent with established literature values for macroscopic duplex formation. The resultant equilibrium constant for duplex formation in the nanopore was found to be close to the experimentally derived constant for duplex formation in solution. A good agreement between the equilibrium constants for duplex formation in the nanopore and in solution was also found for two other oligonucleotide pairs. In addition, the nanopore recordings revealed details of the kinetics difficult to obtain by conventional methods, like surface plasmon resonance, which measure ensemble properties. By investigating the temperature dependence of DNA duplex formation at the single molecule level, the standard enthalpy and entropy of the interaction could be obtained. Duplex formation by complementary RNA or DNA strands is a fundamental biochemical process. The physical chemistry of DNA duplex formation has been studied thoroughly with techniques that measure bulk properties (1-4). However, despite its importance, little is known about DNA duplex formation at the single molecule level. Individual polynucleotide strands can be studied by fluorescence-based detection (5-8) and force measurements (9, 10), and both techniques have been used to detect duplex formation within or between individual DNA strands (11)(12)(13)(14). With the exception of unfolding of individual RNA molecules (14), detailed kinetic data about duplex formation at the single molecule level have not been obtained. Electrical recording is another attractive technique to study individual DNA strands (15)(16)(17)(18)(19)(20)(21)(22). Electrical recordings measure the ionic current flowing through a single membrane-embedded nanopore, and the passage or binding of individual molecules, such as DNA, can be followed by the associated change in current.In this work, single-channel current recording was used to detect and characterize the hybridization of individual DNA strands inside the lumen of the protein nanopore ␣-hemolysin. ␣-Hemolysin (␣HL) is an exotoxin produced by Staphylococcus aureus. ␣HL monomers assemble to form a heptameric pore of known structure (23). The pore resembles a mushroom 10 nm in height and up to 10 nm in width. The cap of the mushroom contains an internal cavity 4.6 nm wide with entrances of 3 nm diameter at the cis opening and 1.4 nm diameter at the narrow inner constriction (Fig. 1A). Molecular graphics reveal...

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

“…Under standard electrolyte conditions 3,35,49 at 100 mV, the blank αHL pore exhibited a conductance of 1930 ± 190 pS (n = 4, number of independent recordings), in line with literature. 49,58,59 Addition of the peptide-tagged strands R 3 -DNA, R 5 -DNA, and R 7 -DNA to the cis side gave rise to high-amplitude current blockades ( 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 modified DNA oligonucleotides (91.7%), 49 suggesting that the peptide tag almost completely blocks the inner pore by steric and/or electrostatic factors. The peptide tags also affected the translocation duration, τ off , ( Figure 2B).…”

Polymer translocation through a long nanopore

“…The motion of polymers in a confined medium is also technologically important in food and medicine production, in oil recovery and separation, and in many other industrial processes. Accordingly, the mechanisms of polymer translocation have become a subject of numerous experimental 3,4,5,6,7,8,9 and theoretical studies. 10,11,12,13,14,15,16,17 A polymer molecule moving across a nanopore faces a large entropic barrier due to the decrease in the number of available configurations for polymer segments.…”

“…In order to overcome this barrier and to speed up the motion of polymers, an external field or interaction is needed. In recent in vitro experiments, 3,4,5,6,7,8,9 DNA and RNA molecules are driven through an α-hemolysin membrane channel with the help of an external electric field. These elegant experiments are based on the following simple idea.…”

“…17 , as discussed below). The α-hemolysin membrane channel, that has been used as the nanopore in in vitro experiments, 3,4,5,6,7,8,9 has a length of approximately 5 nm for the narrow part of the channel and thus can hold up to 10-15 DNA or RNA monomers. The theoretical approach of Ambjörnsson et al 17 takes into account the nanopore length and studies both the polymer entrance into the pore and the translocation process.…”

Controlling molecular transport through nanopores

“…This enables almost a free choice over their properties down to the single amino acid and even atomic level by mutagenesis. In effect, this was used in one of the earliest demonstrations of control of DNA in a nanopore whereby a single DNA strand in the pore was used to immobilize translocating DNA [22]. A notable disadvantage, however, is most biological nanopores have diameters of less than 2 nm.…”