A Nanosensor for Transmembrane Capture and Identification of Single Nucleic Acid Molecules

Nakane, Jonathan; Wiggin, Matthew; Marziali, Andre

doi:10.1529/biophysj.104.040212

Cited by 147 publications

(159 citation statements)

References 27 publications

(34 reference statements)

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“…Recently, individual modified nucleotide bases have been observed ''on the fly'' (19), but these structures were very bulky. When DNA is immobilized within the ␣HL pore, by using a 5Ј-or 3Ј-terminal hairpin or biotin-streptavidin complex, better resolution of homopolymer sequences can be achieved because of the prolonged observation time (13,(28)(29)(30)(31) and in the present work we have extended the biotin-streptavidin approach.…”

Section: Resultsmentioning

confidence: 97%

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

Stoddart

Heron

Mikhailova

et al. 2009

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

414

492

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The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as ␣-hemolysin (␣HL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane ␤-barrel of an engineered ␣HL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.␣-hemolysin ͉ DNA sequencing ͉ genomics ͉ protein engineering ͉ protein pore

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Section: Resultsmentioning

confidence: 97%

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

Stoddart

Heron

Mikhailova

et al. 2009

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

414

492

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“…6), increased electroosmotic flow (51,52) or a combination of the two that depends on the applied potential. The newly engineered pores or derivatives of them will be useful for enhancing the sensitivity of ␣HL as a biosensor of nucleic acids (2,(17)(18)(19)(20)(21)(22)(23)(24)(25). An additional potential application is in nanopore sequencing, where the bases in a single DNA strand are read off one by one during translocation (16,30,(32)(33)(34)(35)(36)(37).…”

Section: Discussionmentioning

confidence: 99%

“…The examination of mean transit times and current amplitudes during transit have allowed the analysis of single-strand nucleic acid length (2), base composition (17), phosphorylation state (18) and secondary structure (19). The formation (20,21) and dissociation of nucleic acid double strands (22)(23)(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).…”

mentioning

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.

254

296

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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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“…In nanopore analytics, single molecules are detected via the passage-induced changes in ionic current. [2][3][4][5][6][7] The approach can be implemented with protein 4,[8][9][10] and solid-state nanopores [10][11][12][13][14][15][16] and has gained popularity 17 due to its simplicity, the wide range of accessible analytes, 2,13,[18][19][20] and the prospect of DNA sequencing [21][22][23] based on nucleic acid sensing. [24][25][26][27] The translocation of protein and DNA strands through protein channels is also a biologically relevant process in pathogenic bacteria and human cells.…”

Section: Introductionmentioning

confidence: 99%

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

et al. 2013

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The voltage-driven passage of biological polymers through nanoscale pores is an analytically, technologically, and biologically relevant process. Despite various studies on homopolymer translocation there are still several open questions on the fundamental aspects of the pore transport. One of the most important unresolved issues revolves around the passage of biopolymers which vary in charge and volume along their sequence. Here we exploit an experimentally tunable system to disentangle and quantify electrostatic and steric factors. This new, fundamental framework facilitates the understanding of how complex biopolymers are transported through confined space and indicates how biopolymer translocation can be slowed down to enable future sensing methods.SYNOPSIS: The voltage-driven translocation of complex biopolymers through a protein nanopore is biophysically modeled to disentangle and quantify electrostatic and steric factors which can oppose electrophoretic movement.

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A Nanosensor for Transmembrane Capture and Identification of Single Nucleic Acid Molecules

Cited by 147 publications

References 27 publications

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

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

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

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

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