Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules

Vercoutere, W.; Winters‐Hilt, Stephen; DeGuzman, Veronica S.; Deamer, David W.; Ridino, S.; Rodgers, Joseph T.; Olsen, Hugh E.; Marziali, Andre; Akeson, Mark

doi:10.1093/nar/gkg218

Cited by 143 publications

(163 citation statements)

References 35 publications

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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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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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“…Although several essential features of translocation are richly documented in systems such as mRNP complex through nuclear pores (1-3), a simple system has only recently been identified for following the single-file passage of one isolated polymer through one channel (4)(5)(6)(7)(8)(9)(10)(11). In this system, the channel is constituted by self-assembling heptamers of the Staphylococcus aureus ␣-hemolysin (␣HL) protein.…”

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Simulation of polymer translocation through protein channels

Muthukumar

Kong

2006

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

128

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A modeling algorithm is presented to compute simultaneously polymer conformations and ionic current, as single polymer molecules undergo translocation through protein channels. The method is based on a combination of Langevin dynamics for coarse-grained models of polymers and the Poisson-Nernst-Planck formalism for ionic current. For the illustrative example of ssDNA passing through the ␣-hemolysin pore, vivid details of conformational fluctuations of the polymer inside the vestibule and ␤-barrel compartments of the protein pore, and their consequent effects on the translocation time and extent of blocked ionic current are presented. In addition to yielding insights into several experimentally reported puzzles, our simulations offer experimental strategies to sequence polymers more efficiently.T ranslocation of polymers through biological channels is very complex involving many machineries and is a fundamental step in many life processes. Although several essential features of translocation are richly documented in systems such as mRNP complex through nuclear pores (1-3), a simple system has only recently been identified for following the single-file passage of one isolated polymer through one channel (4-11). In this system, the channel is constituted by self-assembling heptamers of the Staphylococcus aureus ␣-hemolysin (␣HL) protein. The channel is assembled in a phospholipid bilayer, which offers a physical barrier, and the channel has an opening diameter of Ϸ1.4 nm at the narrowest constriction (12). A single-stranded polynucleotide, such as poly(deoxyadenylate) and poly(deoxycytidylate), is pulled through the channel by an externally applied voltage gradient across the channel in a solution of a strong electrolyte. The idea is that the ionic current through the channel caused by the passage of small ions of the electrolyte is blocked to a certain extent during the event of translocation of the polymer. It has been hoped that the extent and duration of the current blockade are unique signatures of the identity of the polymer, both in terms of the polymer's chemical characteristics and physical length.Even this simplest setup, where identical molecules undergo translocation, has generated several puzzling results. The distribution, P( ), of the duration of blockade of ionic current I b is very broad and appears to exhibit at least two peaks. In addition, there are several levels of ionic current blockade I b for the same molecule. It is standard practice in experimental investigations to combine the histograms of and I b (8). The resultant scatter plots always yield two groups of data even for monodisperse homopolymers.To gain insight into these puzzles, we have developed the following simulation. It is complementary to a flurry of theoretical activity (13-21), based on entropic barrier dynamics (22), all of which lead to a generic P( ) unlike in experiments. Although it is indeed desirable to perform the computation ab initio, the size of the system to be simulated is forbiddingly large to enable such a compu...

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“…INTRODUCTION The migration of biopolymers through nanopores plays a key role in several biological processes, including the transport of mRNA molecules from the nucleus to the cytoplasm following transcription, the transport of proteins to and from the nucleus, and the injection of viral DNA into a host cell 1 . Biopolymer translocation further has technological applications in the development of biosensors for polynucleotide analysis and sequencing 2,3, 4,5,6 .…”

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Polymer translocation through pores with complex geometries

Mohan

Kolomeisky

Pasquali

2010

The Journal of Chemical Physics

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We propose a method for the theoretical investigation of polymer translocation through composite pore structures possessing arbitrarily specified geometries.Translocation through each constituent part of the composite is treated as being analogous to the diffusion of the translocation coordinate over the free energy landscape derived from the chain configurations within the pore. The proposed method accounts for possible reverse motions of the leading chain end at the interface between constituent parts of a composite pore, a possibility that has been neglected in prior studies. As an illustration of our method, we study the translocation of a Gaussian chain between two spherical compartments connected by a cylindrical pore, and by a composite pore consisting of two connected cylinders of different diameters, which is structurally similar to the α-hemolysin membrane channel. We demonstrate that reverse chain motions between the pore constituents may contribute significantly to the total translocation time. Our results further establish that translocation through a two-cylinder composite pore is faster when the chain is introduced into the pore on the cis (wide) side of the channel rather than the trans (narrow) side.

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Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules

Cited by 143 publications

References 35 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

Simulation of polymer translocation through protein channels

Polymer translocation through pores with complex geometries

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