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Internal vs fishhook hairpin DNA: unzipping locations and mechanisms in the α-hemolysin nanopore.

Ding Y, Fleming AM, White HS, Burrows CJ - J Phys Chem B (2014)

Bottom Line: Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins.This demonstration of different currents for immobilized and translocating DNA has not been described previously.The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.

ABSTRACT
Studies on the interaction of hairpin DNA with the α-hemolysin (α-HL) nanopore have determined hairpin unzipping kinetics, thermodynamics, and sequence-dependent DNA/protein interactions. Missing from these results is a systematic study comparing the unzipping process for fishhook (one-tail) vs internal (two-tail) hairpins when they are electrophoretically driven from the cis to the trans side of α-HL via a 30-mer single-stranded tail. In the current studies, fishhook hairpins showed long unzipping times with one deep blockage current level. In contrast, the internal hairpins demonstrated relatively fast unzipping and a characteristic pulse-like current pattern. These differences were further explored with respect to stem length and sequence context. Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins. Interestingly, these studies were able to resolve a current difference of ~6% between hairpin DNA immobilized in the nanopore waiting to unzip vs the translocating unzipped DNA, with the latter showing a deeper current blockage level. This demonstration of different currents for immobilized and translocating DNA has not been described previously. These results were interpreted as fishhook hairpins unzipping inside the vestibule, while the internal hairpins unzip outside the vestibule of α-HL. Lastly, we used this knowledge to study the unzipping of a long double-stranded DNA (>50 base pairs) outside the vestibule of α-HL. The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids.

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Unzipping time vs stem length for internal hairpins studied atdifferent voltages (trans vs cis). (A) The sequences for the variable stem length internal hairpins.(B) Corresponding unzipping time vs applied bias. Experimental conditions:1.00 M KCl, 10 mM PBS, pH 7.4, 22.0 ± 0.5 °C.
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fig4: Unzipping time vs stem length for internal hairpins studied atdifferent voltages (trans vs cis). (A) The sequences for the variable stem length internal hairpins.(B) Corresponding unzipping time vs applied bias. Experimental conditions:1.00 M KCl, 10 mM PBS, pH 7.4, 22.0 ± 0.5 °C.

Mentions: To further investigatethe unzipping of internal hairpins through an α-HL nanopore,a group of internal hairpins was designed with the same dC30 tails and loop sequence, but the length of the hairpin stem wasincreased from 9 to 15 base pairs (bp’s) in 3 bp increments(Figure 4A). All of these internal hairpinsgave the same pulse-like pattern to the blocking current, as describedabove. The measured unzipping time constant (τ) for these hairpinsdemonstrated that, as the stem length was increased, the unzippingtime increased, as expected (Figure 4B). Inthese studies, τ showed an inverse correlation with voltage;this observation supports unzipping and translocation of the hairpinsthrough α-HL (Figure S7, Supporting Information). Compared to the unzipping time of fishhook hairpins in the currentwork and those previously reported by Meller’s laboratory,22 the unzipping times of the internal hairpinswere dramatically shorter, by a factor of ∼20. Furthermore,the unzipping times of variable stem length internal hairpins weresensitive to increased voltage that is consistent with a model ofhairpin unzipping followed by translocation (Figure 4B).


Internal vs fishhook hairpin DNA: unzipping locations and mechanisms in the α-hemolysin nanopore.

Ding Y, Fleming AM, White HS, Burrows CJ - J Phys Chem B (2014)

Unzipping time vs stem length for internal hairpins studied atdifferent voltages (trans vs cis). (A) The sequences for the variable stem length internal hairpins.(B) Corresponding unzipping time vs applied bias. Experimental conditions:1.00 M KCl, 10 mM PBS, pH 7.4, 22.0 ± 0.5 °C.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234443&req=5

fig4: Unzipping time vs stem length for internal hairpins studied atdifferent voltages (trans vs cis). (A) The sequences for the variable stem length internal hairpins.(B) Corresponding unzipping time vs applied bias. Experimental conditions:1.00 M KCl, 10 mM PBS, pH 7.4, 22.0 ± 0.5 °C.
Mentions: To further investigatethe unzipping of internal hairpins through an α-HL nanopore,a group of internal hairpins was designed with the same dC30 tails and loop sequence, but the length of the hairpin stem wasincreased from 9 to 15 base pairs (bp’s) in 3 bp increments(Figure 4A). All of these internal hairpinsgave the same pulse-like pattern to the blocking current, as describedabove. The measured unzipping time constant (τ) for these hairpinsdemonstrated that, as the stem length was increased, the unzippingtime increased, as expected (Figure 4B). Inthese studies, τ showed an inverse correlation with voltage;this observation supports unzipping and translocation of the hairpinsthrough α-HL (Figure S7, Supporting Information). Compared to the unzipping time of fishhook hairpins in the currentwork and those previously reported by Meller’s laboratory,22 the unzipping times of the internal hairpinswere dramatically shorter, by a factor of ∼20. Furthermore,the unzipping times of variable stem length internal hairpins weresensitive to increased voltage that is consistent with a model ofhairpin unzipping followed by translocation (Figure 4B).

Bottom Line: Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins.This demonstration of different currents for immobilized and translocating DNA has not been described previously.The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.

ABSTRACT
Studies on the interaction of hairpin DNA with the α-hemolysin (α-HL) nanopore have determined hairpin unzipping kinetics, thermodynamics, and sequence-dependent DNA/protein interactions. Missing from these results is a systematic study comparing the unzipping process for fishhook (one-tail) vs internal (two-tail) hairpins when they are electrophoretically driven from the cis to the trans side of α-HL via a 30-mer single-stranded tail. In the current studies, fishhook hairpins showed long unzipping times with one deep blockage current level. In contrast, the internal hairpins demonstrated relatively fast unzipping and a characteristic pulse-like current pattern. These differences were further explored with respect to stem length and sequence context. Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins. Interestingly, these studies were able to resolve a current difference of ~6% between hairpin DNA immobilized in the nanopore waiting to unzip vs the translocating unzipped DNA, with the latter showing a deeper current blockage level. This demonstration of different currents for immobilized and translocating DNA has not been described previously. These results were interpreted as fishhook hairpins unzipping inside the vestibule, while the internal hairpins unzip outside the vestibule of α-HL. Lastly, we used this knowledge to study the unzipping of a long double-stranded DNA (>50 base pairs) outside the vestibule of α-HL. The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids.

Show MeSH
Related in: MedlinePlus