Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

Arnaut, Vera; Langecker, D Martin; Simmel, Friedrich C.

doi:10.1016/j.bpj.2013.07.047

Cited by 24 publications

(37 citation statements)

References 59 publications

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“…14 Beyond basic research, nanopores also hold tremendous promise in biotech applications such as DNA sequencing 11, 15-17 and biosensing. 1 Molecular detection using a single nanopore works by observing modulations in ionic current flowing through the pore during an applied potential.…”

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confidence: 99%

Resolved Single-Molecule Detection of Individual Species within a Mixture of anti-Biotin Antibodies Using an Engineered Monomeric Nanopore

2015

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Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop’s movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity – even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design while OmpG’s monomeric structure greatly simplifies nanopore production.

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confidence: 99%

Resolved Single-Molecule Detection of Individual Species within a Mixture of anti-Biotin Antibodies Using an Engineered Monomeric Nanopore

2015

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“…Another concern lies with the ability of ssDNA to fold into a variety of secondary structures that inhibit the translocation through the protein ion channel (21,22). A few studies have been performed to better understand the effect of DNA secondary structures on their electrical behaviors, mostly focusing on duplexes, hairpins, and the thrombin-binding aptamer (TBA) (23)(24)(25)(26)(27)(28)(29). Gu and coworkers (23) studied the simple two-tetrad G-quadruplex adopted by the TBA sequence being captured within the α-HL vestibule and its unraveling kinetics upon binding with different cations.…”

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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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“…These features make nanopores promising tools for screening drugs that induce conformational changes in viral RNA. Biological nanopores such as α-hemolysin have previously been utilized for studying nucleic acid conformations such as G-quadruplex structures, 6,7 aptamers to small molecules, 8,9 and DNA hairpins with chemical damage. 10,11 In addition, prior RNA detection works using nanopores include studies of the dynamics of single-stranded 12–15 and double-stranded 16 RNA translocations, microRNA and tRNA detection, 17 RNA/antibiotic interactions, 18 RNA/protein interactions, 19,20 RNA folding and secondary structures, 21 and have demonstrated nanopores sensitive enough to discriminate between different RNA homopolymers.…”

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Nanopore-Based Conformational Analysis of a Viral RNA Drug Target

et al. 2014

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Nanopores are single-molecule sensors that show exceptional promise as a biomolecular analysis tool by enabling label-free detection of small amounts of sample. In this paper, we demonstrate that nanopores are capable of detecting the conformation of an antiviral RNA drug target. The hepatitis C virus uses an internal ribosome entry site (IRES) motif in order to initiate translation by docking to ribosomes in its host cell. The IRES is therefore a viable and important drug target. Drug-induced changes to the conformation of the HCV IRES motif, from a bent to a straight conformation, have been shown to inhibit HCV replication. However, there is presently no straightforward method to analyze the effect of candidate small-molecule drugs on the RNA conformation. In this paper, we show that RNA translocation dynamics through a 3 nm diameter nanopore is conformation-sensitive by demonstrating a difference in transport times between bent and straight conformations of a short viral RNA motif. Detection is possible because bent RNA is stalled in the 3 nm pore, resulting in longer molecular dwell times than straight RNA. Control experiments show that binding of a weaker drug does not produce a conformational change, as consistent with independent fluorescence measurements. Nanopore measurements of RNA conformation can thus be useful for probing the structure of various RNA motifs, as well as structural changes to the RNA upon small-molecule binding.

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Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

Cited by 24 publications

References 59 publications

Resolved Single-Molecule Detection of Individual Species within a Mixture of anti-Biotin Antibodies Using an Engineered Monomeric Nanopore

Resolved Single-Molecule Detection of Individual Species within a Mixture of anti-Biotin Antibodies Using an Engineered Monomeric Nanopore

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

Nanopore-Based Conformational Analysis of a Viral RNA Drug Target

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