Stochastic Detection of Motor Protein–RNA Complexes by Single‐Channel Current Recording

Astier, Yann; Kainov, Denis E.; Bayley, Hagan; Tůma, Roman; Howorka, Stefan

doi:10.1002/cphc.200700179

Cited by 33 publications

(27 citation statements)

References 47 publications

(79 reference statements)

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“…The strengths of nanopore analysis with protein pores are well illustrated by the growing body of work using ␣HL to detect and characterize ssDNA and RNA (1,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and by the recent demonstration of dADP detection with an engineered version of the porin OmpF (20). The possibility of using base-specific modulation of the ionic current for rapid, inexpensive DNA sequencing has been a central motivating factor for these investigations.…”

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confidence: 99%

“…The possibility of using base-specific modulation of the ionic current for rapid, inexpensive DNA sequencing has been a central motivating factor for these investigations. Although recent progress in recognizing and differentiating the bases and regulating DNA passage through the pore supports the feasibility of this approach (18,19,(21)(22)(23)(24), realization of de novo nanopore DNA sequencing remains elusive. The introduction of new protein pores capable of single-molecule detection and characterization of nucleic acids is of high scientific and technological relevance because it may provide the improved biophysical insight and analytical capabilities needed to realize this goal.…”

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confidence: 99%

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Single-molecule DNA detection with an engineered MspA protein nanopore

Butler

Pavlenok

Derrington

et al. 2008

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

439

494

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Nanopores hold great promise as single-molecule analytical devices and biophysical model systems because the ionic current blockades they produce contain information about the identity, concentration, structure, and dynamics of target molecules. The porin MspA of Mycobacterium smegmatis has remarkable stability against environmental stresses and can be rationally modified based on its crystal structure. Further, MspA has a short and narrow channel constriction that is promising for DNA sequencing because it may enable improved characterization of short segments of a ssDNA molecule that is threaded through the pore. By eliminating the negative charge in the channel constriction, we designed and constructed an MspA mutant capable of electronically detecting and characterizing single molecules of ssDNA as they are electrophoretically driven through the pore. A second mutant with additional exchanges of negatively-charged residues for positively-charged residues in the vestibule region exhibited a factor of Ϸ20 higher interaction rates, required only half as much voltage to observe interaction, and allowed ssDNA to reside in the vestibule Ϸ100 times longer than the first mutant. Our results introduce MspA as a nanopore for nucleic acid analysis and highlight its potential as an engineerable platform for single-molecule detection and characterization applications.DNA sequencing ͉ protein engineering ͉ bio-nanotechnology

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confidence: 99%

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confidence: 99%

Single-molecule DNA detection with an engineered MspA protein nanopore

Butler

Pavlenok

Derrington

et al. 2008

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

439

494

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“…The formation (20,21) and dissociation of nucleic acid double strands (22-24) and hairpins (25, 26) have also been examined. In addition, the interactions of individual proteins with nucleic acids have been observed (27)(28)(29)(30). Similarly, protein-ligand interactions can be examined indirectly, when the ligand is incorporated into nucleic acid strands (31).…”

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confidence: 99%

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

Maglia

Rincón-Restrepo²,

Mikhailova³

et al. 2008

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

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266

303

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Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the ␣-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at ؉120 mV by Ϸ10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event⅐s ؊1 ⅐ M ؊1 . Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification.DNA sequencing ͉ electroosmosis ͉ nanopore ͉ protein engineering ͉ single-molecule detection P ores with diameters of a few to hundreds of nanometers, ''nanopores,'' are being developed for a wide variety of analytical applications (1-5). Nanopores can be fabricated by using particle beams and etchants to treat various substrates, including silicon nitride (3, 6) and plastics, for instance poly(ethylene terephthalate) (4). Alternatively, protein pores such as ␣-hemolysin (␣HL) can be used (1, 5). In both cases, to be detected, an analyte must travel into the pore by electrodiffusion, but few systematic attempts have been made to optimize this process. In the present work, we show how the manipulation of charge on the internal surface of the ␣HL pore can be used to improve the rate of capture of an important analyte, DNA.The ␣HL protein nanopore is advantageous in sensing applications because it can be engineered with subnanometer precision with reference to the high resolution crystal structure (7). Further, the ␣HL pore is far more stable than commonly held, operating as normal at temperatures approaching 100°C (8). In stochastic sensing, a binding site for a family of analytes is formed within the pore by site-directed mutagenesis or targeted chemical modification (1). The concentration of an analyte is estimated from the number of binding events per unit time to a single pore. An analyte is identified through the signature provided by the amplitude and mean duration of the individual binding events. In this way, a great variety of analytes have been examined including: cations, anions, organic molecules, and various polymers (1). For example, all 4 DNA bases can be detected as nucleoside monophosphates by an ␣HL pore equipped with an aminocyclodextrin adapter (9). In addition, individual covalent chemical reactions occurring within the lumen of the pore can be observed (5), offering a basis for the detection of reactive molecules (10).Polymers can also be analyzed from the characteristics of transit events through the ␣HL pore (11-15). Studies of nucleic acids have been especially intensive, following the demonstration of the transit of single str...

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“…The C-terminally tagged protein (P4His) was expressed at 178C in a similar fashion to wt [47]. P4His was purified following a procedure previously described for his-tagged f8 P4 protein [6]. The molecular weight of purified protein and presence of his-tag was verified by mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

RNA Packaging Motor: From Structure to Quantum Mechanical Modelling and Sequential‐Stochastic Mechanism

Telenius

Wallin

Straka

et al. 2008

Computational and Mathematical Methods in Medicine

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The bacteriophages of the Cystoviridae family package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate, i.e. the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work.

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Stochastic Detection of Motor Protein–RNA Complexes by Single‐Channel Current Recording

Cited by 33 publications

References 47 publications

Single-molecule DNA detection with an engineered MspA protein nanopore

Single-molecule DNA detection with an engineered MspA protein nanopore

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

RNA Packaging Motor: From Structure to Quantum Mechanical Modelling and Sequential‐Stochastic Mechanism

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