Controlling the Translocation of Single-Stranded DNA through α-Hemolysin Ion Channels Using Viscosity

Kawano, Ryuji; Schibel, Anna E. P.; Cauley, Christopher; White, Henry S.

doi:10.1021/la803556p

Cited by 91 publications

(95 citation statements)

References 24 publications

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“…2D, Left). To rule out the possibility that the high viscosity of the electrolyte was slowing the diffusion of the DNA molecules to the ion channel leading to an absence of events (38), the same amount of a control sequence (poly-dC 87 ) was added to the same solution, and after being gently mixed, much higher event frequencies were observed with deep current blockages (Fig. 2D, Right, and SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Fleming

Middleton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

102

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Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structuredependent electrical signatures through DNA-protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology.α-hemolysin nanopore | single-molecule detection N ucleic acids can fold into a myriad of secondary structures that depend on the primary sequence as well as the physical conditions in which the structures are prepared and characterized. One prime example of a multistructural sequence is human telomeric DNA comprising the repeat sequence 5′-TTAGGG-3′. This guanine-rich single-stranded sequence is known to fold into highly ordered nanostructures in the form of G-quadruplexes that feature the coordination of two alkali cations to three layers of G-tetrads formed by Hoogsteen hydrogen-bonded assemblies of four guanine bases ( Fig. 1A) (1, 2). G-quadruplexes provide a fascinating case in which the cation and physical context play critical roles in defining the overall structural topology as well as the stability of the fold. Guanine-rich sequences also are known to present challenges to PCR amplification and sequencingby-synthesis methods that require processive analysis of singlestranded DNA (ssDNA) because of the high thermodynamic stability of these folded structures.The following studies highlight the cation and contextdependent conditions in which the topology of the natural human telomere sequence 5′-TAGGG(TTAGGG) 3 TT-3′ is affected. In NaCl solution, NMR studies revealed a structure referred to as the basket fold (Fig. 1A) (4). Key features of this structure include an antiparallel strand arrangement with alternating syn and anti orientations of the guanosine glycosidic bonds and three tetrads linked by two edgewise loops and one diagonal loop. In contrast, NMR-based studies in KCl solution show that the same sequence folds predominantly to the hybrid-1 and hybrid-2 structures (Fig. 1A) (5-8). Characteristics of this structure are an antiparallel strand orientation with a 3+1 core of syn and anti G nucleotides yielding three tetrads. The loop topology of the hybrid fold consists of a double-chain reversal loop and two edgewise loops, in which hybr...

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Section: Resultsmentioning

confidence: 99%

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Fleming

Middleton

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

102

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“…The high speed (1-20 μs∕nucleotide) at which ssDNA is driven through the ion channel above the threading voltage promises rapid and long reads; however, it is this very feature of free translocation that prohibits resolution of single bases that exhibit characteristic signatures of less than a few picoamperes current difference using available electronics (14). Most efforts to approach these challenges are devoted to engineering the protein (28)(29)(30), embedding adapters (31), synthesizing peptide-conjugated nucleotides (32), controlling DNA movement with molecular motors (33,34), and exploring experimental conditions (35,36). Among these studies, the stable AP analog tetrahydrofuran (THF) has been used as a marker to inspect DNA polymerase activities and to map the recognition sites of the α-HL ion channel (24)(25)(26); the native AP lesion has never been studied in α-HL or related ion channels.…”

Section: Alpha-hemolysin | Crown Ethers | Single-molecule Detection |mentioning

confidence: 99%

Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

Fleming

White

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

129

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DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na þ produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li þ , Na þ or K þ ) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a singlemolecule level are provided by the nanopore measurement.alpha-hemolysin | crown ethers | single-molecule detection | DNA damage

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“…In terms of the environment surrounding the nanopore, both biological and solid-state nanopore researchers have experimented with reducing solvent temperature [29,30] and increasing viscosity [31][32][33]. Solid-state researchers have further explored the use of alternate salts [14,34] and the introduction of a salt concentration gradient [35,36] or counter pressure [37,38], achieving close to 10-fold translocation rate reductions by these means [38].…”

Section: Introductionmentioning

confidence: 99%

Interfacing solid‐state nanopores with gel media to slow DNA translocations

et al. 2015

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We demonstrate the ability to slow DNA translocations through solid‐state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores—presently one of the largest hurdles facing nanopore‐based analysis—without affecting the signal quality or capture efficiency.

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Controlling the Translocation of Single-Stranded DNA through α-Hemolysin Ion Channels Using Viscosity

Cited by 91 publications

References 24 publications

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

Interfacing solid‐state nanopores with gel media to slow DNA translocations

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