Characterization of individual polynucleotide molecules using a membrane channel

^{Biotechnology Division, National Institute of Science and Technology, 222/A353, Gaithersburg, MD 20899;}^{Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; and}^{Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064}

^{To whom reprint requests should be addressed. e-mail:}ude.dravrah@notnarbd.

Daniel Branton

Accepted 1996 Sep 5.

Abstract

We show that an electric field can drive single-stranded RNA and DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer membrane. Because the channel diameter can accommodate only a single strand of RNA or DNA, each polymer traverses the membrane as an extended chain that partially blocks the channel. The passage of each molecule is detected as a transient decrease of ionic current whose duration is proportional to polymer length. Channel blockades can therefore be used to measure polynucleotide length. With further improvements, the method could in principle provide direct, high-speed detection of the sequence of bases in single molecules of DNA or RNA.

Abstract

Measurement of ionic current passing through single ion channels in biological membranes or planar lipid bilayers is routine in neurobiology and biophysics (1, 2). Although most such channels undergo voltage- or ligand-dependent gating (3, 4), several relatively large ion channels, including Staphylococcus aureus α-hemolysin (5, 6) and the mitochondrial voltage-dependent anion channel (7, 8), can remain open for extended periods, thereby allowing continuous ionic current to flow across a lipid bilayer (9, 10). We reasoned that a transmembrane voltage applied across a continuously open channel of appropriate size should draw polyanionic DNA or RNA molecules through the channel as extended linear chains whose presence would detectably reduce or block normal ionic flow. Such blockages should make it possible to use single channel recordings to characterize the length and, possibly, other characteristics of the polymer.

Random coil polymers have previously been used to investigate channel pore dimensions (8) and single channel current recordings show that small poly(ethylene glycol) polymers partition into channels formed by alamethicin (11) or S. aureus α-hemolysin (12, 13). Recent investigations show that proteins can also traverse a lipid bilayer by moving through protein translocating channels, presumably as unfolded, extended chains (14, 15, 16, 17, 18). While translocating a protein, such channels are electrically silent; they regain their ion permeability only after the translocating polypeptide has been cleared from the channel (18). The ionic conductance of large nuclear pore complexes is similarly reduced during translocation of transcription factors, although in this case the macromolecules are assumed to move through the nuclear pore in a folded state (19, 20).

To determine whether nucleic acid polymers can be detected by single channel measurements, S. aureus α-hemolysin was used to form a single channel across a lipid bilayer separating two buffer-filled compartments (21). The α-hemolysin monomers spontaneously insert into lipid bilayers (9) and assemble to form heptameric transmembrane channels (6) that are 2.6 nm in diameter (L. Song, M. R. Hobaugh, C. Shustak, S. Cheley, H. Bayley, and J. E. Gouaux, personal communication). Channels with this dimension should be sufficiently large to accommodate the diameter of an extended, single-stranded polynucleotide.

The number of blockades/min is compared with the number of molecules/min that moved to the trans chamber during experiments in which the cis chamber contained 0.27 mg/ml of 150-nt single-stranded DNA and an equivalent number of 100-nt strands of double-stranded DNA. Multiple channels were used in these experiments to show the proportionality between channel number and DNA movement. Because frequent current reversals were required during the experiments (see Materials and Methods), the precision of the blockade rate calculation is limited to ±30%. The number of molecules transported to the trans chamber per minute was calculated from the total number of molecules found in the trans chamber at the end of each experiment, as measured by competitive PCR analysis which can reliably discriminate 2-fold differences in copy number (25).

Acknowledgments

We thank H. Bayley for the generous gift of α-hemolysin and H. Berg, J. Golovchenko, and D. Nelson for their encouragement and suggestions. We also thank A. Schroder, R. Li, and S. Lee for technical assistance. This research was supported by grants from the National Aeronautics and Space Administration (D.W.D.), the National Science Foundation (D.B.), the National Institutes of Health (D.W.D.), and a National Academy of Sciences/National Research Council Research Associateship (J.J.K.).

Acknowledgments

Footnotes

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

^{In closely related experiments, F. Roth, R. Baldarelli, and G. M. Church have modeled the movement of DNA through the pore formed by the LamB receptor. Their model shows that DNA can cause conductance changes that should be sensitive to the structure of the different bases (personal communication).}

Footnotes

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