Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

Stoddart, David; Heron, Andrew J.; Mikhailova, Ellina; Maglia, Giovanni; Bayley, Hagan

doi:10.1073/pnas.0901054106

Cited by 420 publications

(519 citation statements)

References 56 publications

(74 reference statements)

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“…It is thought that 10-15 nucleotides contribute to the current blockage signal when ssDNA translocates through the α-HL ion channel (28); thus, a single AP lesion is unlikely to be resolved from the native nucleotides via a modest change in the current amplitude. The goals of chemical modification of DNA bases are both to amplify the signal differences and to stabilize the AP site against strand breaks.…”

Section: Resultsmentioning

confidence: 99%

“…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%

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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.

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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Section: Resultsmentioning

confidence: 99%

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.

129

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“…a nanometer scale. The principal sensing zone of α-HL for the purposes of sequencing ssDNA is the β-barrel that has a constriction of 1.4 nm in diameter (31); in addition, the cis latch region recently was shown to report on the presence of base lesions in dsDNA (32).…”

Section: Significancementioning

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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“…For example, pseudo-rotaxanes formed by a DNA thread capped by a DNA processing enzyme are used in nanopore and protein sequencing applications. [15][16][17][18][19][20][21][22][23] Nanopores might also be used to study single proteins. Ionic currents through nanopores are very sensitive to the environment of the nanopore and small differences between protein homodimers 24 or isomeric protein-DNA interactions 25 can be observed by specific changes to the nanopore conductance.…”

Section: Introductionmentioning

confidence: 99%

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore

2015

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Rotaxanes, pseudorotaxanes and catenanes are supramolecular complexes with potential use in nanomachinery, molecular computing and single-molecule studies. Here we constructed a protein rotaxane in which a polypeptide thread is encircled by a Cytolysin A (ClyA) nanopore and capped by two proteins stoppers. The rotaxane could be switched between two states. At low negative applied potentials (<−50 mV) one of the protein stoppers resided inside the nanopore indefinitely. Under this configuration the rotaxane prevents the diffusion of protein molecules across the lipid bilayer and provides a useful platform for single-molecule analysis. High negative applied potentials (−100 mV) dismantled the interlocked rotaxane system by the forceful translocation of the protein stopper, allowing new proteins to be trapped inside or transported across the nanopore. The observed voltage threshold for the translocation of the protein stopper through the nanopore related well to the biphasic voltage dependence of the residence time measured for the freely diffusing protein stopper. We propose a model in which molecules translocate through a nanopore when the average dwell time decreases with the applied potential.

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Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

Cited by 420 publications

References 56 publications

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

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

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

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore

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