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Biophys J, December 1999, p. 2899-2901, Vol. 77, No. 6
Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021 USA
Single-channel electrophysiology provides for
high-resolution measurements of the dynamics of single macromolecules,
the channel proteins (Neher and Sakmann, 1976 In this issue of Biophysical Journal, Akeson et al. explore
the feasibility of using single ion channels, in this case the Staphylococcus aureus Do individual polynucleotides have distinct signatures that reflect
their chemical composition, and are these signatures sufficiently different that one might be able to use single-channel methods for
sequencing purposes? As shown by Akeson et al., the answers are
"yes" and "most likely." When examining blocking events caused by homopolynucleotides, one can distinguish the characteristic current
reduction (~85%) caused by the passage of polyadenylic acid (poly A)
from the current reduction (90-95%) caused by the passage of
polycytidylic acid (poly C). This difference in current levels allows
Akeson et al. to detect the passage of tandem polynucleotides, such as
a construct with 30 A and 70 C, which is very encouraging for
future developments.
But then matters get complicated. Polyuridylic acid (poly U), for
example, causes a current reduction similar to that caused by poly A. The kinetics of polynucleotide transfer through the Complications also arise because the secondary structure of the
polynucleotides influences both the magnitude and time distribution of
the current reductions. The pyrimidine cytosine, for example, is less
bulky than the purine adenosine. Even accepting the lack of a
well-defined relation between lumen geometry and conductance, e.g.,
(Finkelstein, 1985 However, if the polynucleotides have significant secondary structure,
how does the channel catalyze the unwinding of the secondary structure
at a rate sufficient to account for the observed kinetics of the
blocking events? That remains an open question. The rate constant for
the polynucleotide-induced block of the The results of Akeson et al. are very promising, but the question
remains of how close we are to polynucleotide sequencing by
single-channel methods. Two quite different limitations need to be
overcome before one can construct a prototype. First, how do we
overcome the limitations imposed by the secondary structure dependence?
It may be possible to do the experiments at temperatures sufficiently
high to disrupt the secondary structure, which will become particularly
important for "real" polynucleotides that may form double helices,
which would block polynucleotide transfer through the pores
(Kasianowicz et al., 1996 Eventually the bilayer technology is likely to become limiting, as it
will be difficult to design a bilayer-based apparatus that would be
sufficiently stable for routine sequencing purposes. Polymerizable
lipids may offer a solution, but technical problems remain to resolved
(Rhodes et al., 1994 Finally, how are other single-molecule methods for polynucleotide
sequencing progressing? Currently, no fully implemented method exists,
but the future is almost here. Eigen and Rigler (1994)
ARTICLE
). Among the measurements
that have proven fruitful have been ability to monitor, in almost real time, the interactions between the channel and a blocker, or modifier, of ion permeation (Neher and Steinbach, 1978; Prod'hom et al., 1987;
Hemsley and Busath, 1991). The emphasis usually has been on the
channel, but different blockers/modifiers may have different characteristic "fingerprints" (Hemsley and Busath, 1991), which provides for possibilities in terms of using the characteristics of the
blocking events to determine the chemical identity of the blocking species.
-hemolysin channels, to determine
the chemical identity of polynucleotides, with the aim of establishing
a method for DNA or RNA sequencing. Earlier work (Kasianowicz et al.,
1996) showed that single-stranded DNA or RNA molecules can cause brief, incomplete block of the current through -hemolysin channels when the
polarity of the applied potential is such as to drive the polynucleotides into the pore. Kinetic analysis provided evidence for
three different kinds of incomplete blocking events. When examining the
relation between polynucleotide length and block duration, the average
durations of two of the three types of blocking events varied as linear
functions of the polynucleotide length, suggesting they could be
related to polynucleotide passage through -hemolysin channels. If
so, the average block duration should decrease as the magnitude of the
applied potential increased, which was observed. To validate the
suggestion, Kasianowicz et al. (1996) used quantitative polymerase
chain reaction to demonstrate that single-stranded, but not
double-stranded, DNA could pass through -hemolysin-doped bilayers.
The flux of single-stranded DNA through the bilayer, as evaluated by
the polymerase chain reaction, could be related quantitatively to the
frequency of long-lived blocking events. Moreover, polynucleotides that
were constructed such that they would have both single- and
double-stranded stretches caused long-lived current blocks, which could
be "cleared" by reversing the polarity of the applied potential.
Finally, the frequency of blocking events caused by RNA varied
predictably when ribonuclease was added to the
polynucleotide-containing solution.
-hemolysin
channel are different, however, as the passage of poly U (~1.4
µs/nucleotide at 120 mV) is about 10-fold faster than that of poly A
(~20 µs/nucleotide) for polynucleotides of comparable length.
Because the distribution of blocking durations (polynucleotide passage
times) is nonexponential and appears similar to a normal distribution
(Kasianowicz et al., 1996), one might be able to distinguish between
poly A and poly U based simply on their characteristically different
passage times, assuming polynucleotides of equal length.
), one therefore would expect a larger current
reduction when poly A traverses the channel than when poly C does so,
which is contrary to what is observed. Under "normal" conditions,
however, poly C occurs primarily as single-stranded helices, with a
diameter (~1.3 nm) that is close to the luminal diameter of the
-hemolysin pore (~1.4 nm; Song et al., 1996). One therefore might
surmise that the more organized single-stranded structure could cause a
lower conductance and a longer passage time per nucleotide, ~6 µs,
than that of the more disorganized poly U. That appears to be the case,
as polydeoxycytidylic acid (poly dC), which has less secondary
structure, causes an only 85-90% current reduction and traverses the
channel at a rate of ~1 µs per nucleotide. The concern about
secondary structure becomes even more acute in the case of poly A,
which again forms single-stranded helix, with a diameter of ~2 nm. In
this case, the poly A structure would need to unfold for the
polynucleotide to traverse the channel, which might account for the
much longer passage times observed for poly A. In fact, poly dA
traverses the channel at a rate of ~3 µs per nucleotide, about
5-fold faster than poly A, consistent with the notion that the rate of
polynucleotide passage (and maybe even the magnitude of the current
reductions) may be biased by the secondary structure of the polynucleotide.
-hemolysin channels has yet
to be determined; however, Kasianowicz et al. (1996) reported that the
blocking rate for equimolar polynucleotide concentrations was faster
for shorter (~200 nucleotides) than for longer (~1000 nucleotides)
polynucleotides, suggesting that polynucleotide unfolding and
orientation is important. Given the results in Table 1 of Kasianowicz
et al. (1996) the bimolecular rate constant for passage of
polydeoxynucleotides with ~150 nucleotides is ~104
M
1·s1, which should be comfortably below
the diffusion-controlled limit and thus allow for significant
"processing" of the polynucleotide as it associates with the pore
entrance. The time course of any such processing should depend of the
orientation of the partially structured polynucleotide in relation to
the pore entrance, which might provide insights into why the
distribution of passage times through the pore is much broader than
predicted from "simple" polymer theory (Lubensky and Nelson, 1999).
). Gawrisch et al. (1992), in fact, could form
bilayers from dipalmitoylphosphatidylcholine at ~50°C, which might
be sufficient to eliminate the secondary structure problem. Second, how
do we deconvolute the current signal to give information about the
underlying nucleotide sequence? Given appropriate reference data,
similar to those provided by Akeson et al., it should be possible to
use computational methods similar to those that have been developed for
extracting idealized single-channel current traces from noisy current
records (Venkataramanan et al., 1998). Even then, however, one probably
would need to slow down the polynucleotide transfer through the pore in
order to get a satisfactory signal, which creates demands that seem to
be orthogonal to the need for disrupting the secondary structure, unless one could use the slowing-down caused by unwinding the secondary
structure productively. A saving grace may be that the atomic
resolution structure of the -hemolysin channel is known, which means
that one may be able to introduce bulky residues that would slow down
the passage through the pore, and maybe even catalyze the
unraveling of any secondary structures.
). Alternatively, one might be able to use
nano-scale pores etched in irradiated micro-fabricated materials, as
this allows for pore diameters close to what would be needed (Bean et
al., 1970) and has proven useful for the sizing of viruses (DeBlois and
Wesley, 1977).
proposed a
method for DNA sequencing based on single-molecule detection using
fluorescence correlation microscopy using exonuclease digestion of DNA
that was synthesized using fluorescence-labeled nucleotides, and Kinjo
et al. (1998) showed how one can monitor the restriction enzyme
cleavage of such fluorescent DNA fragments. Recently, at the 13th
International Biophysics Congress in New Delhi, India, M. Gösch
(Gösch et al., 1999) reported on the controlled digestion of
labeled DNA, which was fixed by laser tweezers in a micro-structure
with fast hydrodynamic flow, and detection of the released nucleotides
by their fluorescent signature. Work needs to be done, but a practical
implementation is likely to appear in the near term. These different
methods are likely to be complementary, however, and the
electrophysiological approach proposed by Kasianowicz et al. (1996) and
Akeson et al. allows, in principle, sequencing of unmodified
polynucleotide fragments.
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FOOTNOTES |
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Received for publication 18 October 1999 and in final form 26 October 1999.
Address reprint requests to Olaf S. Andersen, Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Ave., Rm. LC-501, New York, NY 10021-4896. Tel.: 212-746-6350; Fax: 212-746-8369; E-mail: sparre{at}mail.med.cornell.edu.
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REFERENCES |
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-hemolysin, a heptameric transmembrane pore.
Science.
274:1859-1866[Abstract/Full Text].
Biophys J, December 1999, p. 2899-2901, Vol. 77, No. 6
© 1999 by the Biophysical Society 0006-3495/99/12/2899/03 $2.00
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