U.S. patent application number 11/129917 was filed with the patent office on 2005-09-22 for biosensor compositions and methods of use.
This patent application is currently assigned to The Texas A&M University System. Invention is credited to Bayley, Hagan P., Howorka, Stefan G., Movileanu, Liviu.
Application Number | 20050208574 11/129917 |
Document ID | / |
Family ID | 22667045 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208574 |
Kind Code |
A1 |
Bayley, Hagan P. ; et
al. |
September 22, 2005 |
Biosensor compositions and methods of use
Abstract
Provided are pore-subunit polypeptides covalently linked to one
or more sensing moieties, and uses of these modified polypeptides
to detect and/or measure analytes or physical characteristics
within a given sample.
Inventors: |
Bayley, Hagan P.; (College
Station, TX) ; Howorka, Stefan G.; (College Station,
TX) ; Movileanu, Liviu; (Bryan, TX) |
Correspondence
Address: |
Janelle D. Waack
Howrey LLP
2941 Fairview Park Drive, Box 7
Falls Church
VA
22042
US
|
Assignee: |
The Texas A&M University
System
|
Family ID: |
22667045 |
Appl. No.: |
11/129917 |
Filed: |
May 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11129917 |
May 16, 2005 |
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09781697 |
Feb 12, 2001 |
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6916665 |
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60182097 |
Feb 11, 2000 |
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Current U.S.
Class: |
435/6.19 ;
436/518; 530/350 |
Current CPC
Class: |
C12Q 1/6874 20130101;
C12Q 2565/607 20130101; C12Q 2565/607 20130101; C12Q 2565/607
20130101; C12Q 2565/607 20130101; G01N 33/5438 20130101; C12Q
1/6874 20130101; C12Q 1/6837 20130101; C07K 14/31 20130101; C12Q
1/6837 20130101; C12Q 1/6834 20130101; G01N 33/92 20130101; Y10S
530/825 20130101; G01N 33/531 20130101; C12Q 1/6816 20130101; C12Q
1/6816 20130101; C12Q 1/6834 20130101 |
Class at
Publication: |
435/006 ;
436/518; 530/350 |
International
Class: |
C12Q 001/68; G01N
033/543; C07K 014/31 |
Goverment Interests
[0002] The U.S. government owns rights in the present invention
pursuant to grant number DE-FG0397ER20271 from the Department of
Energy, grant number C98-00656 from the Air Force Office of
Scientific Research, Multi-Disciplinary Research Program of the
University Research Initiative (AFOSR, MURI), grant number
N00014-99-1-0717 from the Office of Naval Research,
Multi-Disciplinary Research Program of the University Research
Initiative (ONR, MURI), and grant number DAPT6397-C-001 5 from the
Defense Advanced Research Projects Agency (DARPA).
Claims
1. A modified pore-subunit polypeptide comprising a pore-subunit
polypeptide covalently linked to at least a first sensing moiety,
wherein said modified pore-subunit polypeptide assembles into an
oligomeric pore assembly in the presence of a plurality of
pore-subunit polypeptides.
2. The modified polypeptide of claim 1, wherein said sensing moiety
is a functional group.
3. The modified polypeptide of claim 2, wherein said functional
group is an analyte-binding functional group.
4. The modified polypeptide of claim 2, wherein said functional
group is a synthetic molecule.
5. The modified polypeptide of claim 4, wherein said functional
group is a calixarene or a crown ether.
6. The modified polypeptide of claim 2, wherein said functional
group is a naturally occurring molecule.
7. The modified polypeptide of claim 6, wherein said functional
group is an enzyme inhibitor, a hapten, a nucleotide, an amino
acid, a lipid, a toxin, a saccharide, a chelator or a
cyclodextrin.
8. The modified polypeptide of claim 1, wherein said sensing moiety
is a polymer.
9. The modified polypeptide of claim 8, wherein said polymer is
polyethylene glycol (PEG).
10. The modified polypeptide of claim 9, wherein said polymer is
polyethylene glycol (PEG)-biotin.
11. The modified polypeptide of claim 8, wherein said polymer is an
analyte-binding polymer.
12. The modified polypeptide of claim 11, wherein said polymer is
an oligonucleotide, an oligosaccharide or a peptide.
13. The modified polypeptide of claim 1, wherein said sensing
moiety binds to a metal, metal ion, a toxin, an enzyme, a
nucleotide, an oligonucleotide, an amino acid, a peptide, a
saccharide, a hapten, a lipid or an antibody or antigen-binding
fragment thereof.
14. The modified polypeptide of claim 1, wherein said sensing
moiety responds to a change in the type or amount of a biological
or chemical constituent in the environment of said oligomeric pore
assembly.
15. The modified polypeptide of claim 1, wherein said sensing
moiety responds to a change in the physical environment of said
oligomeric pore assembly.
16. The modified polypeptide of claim 15, wherein said sensing
moiety responds to a change in pH, light, voltage or
temperature.
17. The modified polypeptide of claim 1, wherein said polypeptide
is covalently linked to at least a first and at least a second
sensing moiety.
18. The modified polypeptide of claim 17, wherein said at least a
first sensing moiety is distinct from said at least a second
sensing moiety.
19. The modified polypeptide of claim 17, wherein said at least a
first sensing moiety is the same as said at least a second sensing
moiety.
20. The modified polypeptide of claim 1, wherein said polypeptide
is a staphylococcal hemolysin polypeptide, a porin, a complement
pore polypeptide, a hemolysin C polypeptide or a streptolysin O
polypeptide.
21. The modified polypeptide of claim 20, wherein said polypeptide
is a staphylococcal alpha hemolysin polypeptide.
22. The modified polypeptide of claim 21, wherein said polypeptide
is a mutant staphylococcal alpha hemolysin polypeptide comprising
at least a first heterologous amino acid.
23. The modified polypeptide of claim 22, wherein said mutant
staphylococcal alpha hemolysin polypeptide comprises a cysteine
residue in place of serine at position 106 of the wild-type
staphylococcal alpha hemolysin polypeptide or a cysteine residue in
place of lysine at position 8 of the wild-type staphylococcal alpha
hemolysin polypeptide.
24-43. (canceled)
Description
[0001] The present application claims priority to U.S. provisional
application Serial No. 60/182,097, filed Feb. 11, 2000, the entire
specification, claims and drawings of which are incorporated herein
by reference without disclaimer.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to detection of one
or more analytes in a sample and/or the magnitude of or changes in
physical properties of a sample. More particularly, it concerns
pore-subunit polypeptides covalently linked to one or more sensing
moieties, and the use of these modified polypeptides to detect
and/or measure analytes or certain physical characteristics within
a given sample.
[0005] 2. Description of Related Art
[0006] The examination and manipulation of individual molecules is
a thriving area of research. Single molecule detection methods,
which include electrical recording (Hladky and Haydon, 1970;
Sakmann and Neher, 1995), optical spectroscopy (Moerner and Orrit,
1999; Weiss, 1999) and force measurements (Mehta et al., 1999), can
provide structural and functional information that is often
difficult or impossible to obtain by conventional techniques, which
measure the properties of large ensembles of molecules. Recent
accomplishments include observations of the movement of individual
atoms and small molecules (Gimzewski and Joachim, 1999), the
movement of linear and rotary motor proteins (Mehta et al., 1999),
the turnover of individual enzymes (Xie and Lu, 1999) and the
unfolding and refolding of proteins (Mehta et al., 1999).
[0007] In the area of biosensors, progress has been made in
developing protein channels and pores as sensor elements (Ziegler
and Gopel, 1998; Bayley, 1999). According to this concept, analyte
molecules modulate the ionic current passing through the pores
under a transmembrane potential. For example, binding sites can be
engineered into pores expressly for capturing analyte molecules,
which act as partial channel blockers. Stochastic sensing, which
uses currents from single pores, is an especially attractive
prospect (Braha et al., 1997; Gu et al., 1999). The approach yields
both the concentration and identity of an analyte, the latter from
its distinctive current signature. Using certain types of
stochastic sensing, the inventors have succeeded in detecting
divalent metal ions (Braha et al., 1997) and a variety of organic
molecules (Gu et al., 1999).
[0008] Despite the initial development of stochastic sensing, there
remains in the art a need for sensing elements and systems that
respond to a wider variety of analytes. The development of
stochastic sensing components and systems that permit interactions
with sensor elements that would not normally occur with the
existing methodology would represent a significant advance in the
art.
SUMMARY OF THE INVENTION
[0009] The present invention overcomes one or more of the foregoing
and other shortcomings in the art by providing compositions and
methods comprising improved, versatile and adaptable responsive
sensing moieties for use in sensing applications, including
stochastic sensing. The invention particularly provides modified
pore-forming polypeptides in which a pore-subunit polypeptide is
covalently linked to at least a first sensing moiety; oligomeric
and polymeric pore assemblies and biosensors thereof, and methods
of using such covalently modified polypeptides, pore assemblies and
biosensors.
[0010] The "modified" polypeptides of the invention are "sensing"
polypeptides that generally comprise a "pore-forming" or
"pore-subunit" polypeptide that is covalently linked to at least a
first sensing moiety. "Pore-forming" and "pore-subunit"
polypeptides, as used herein, are polypeptides that are capable of
forming a pore and/or those that are capable of assembling into an
oligomeric or polymeric pore assembly in the presence of a
plurality of pore-forming or pore-subunit polypeptides.
[0011] A "pore-forming" polypeptide for use in the invention may
therefore be a polypeptide that forms a pore as a single unit or
monomer. Examples of such pore-forming polypeptides include certain
porins and channel proteins and polypeptides. Where the
pore-forming polypeptide requires association with other
pore-forming polypeptides to form a pore, the term "pore-subunit"
polypeptide may be preferred, although the terms "pore-forming
polypeptide" and "pore-subunit polypeptide" are used
interchangeably herein unless otherwise stated or clear from the
scientific context.
[0012] The "covalent attachment" of one or more sensing moieties to
pore-forming or pore-subunit polypeptides to create the "modified,
pore-forming, sensing pore-subunit polypeptides" of the present
invention means that at least a first "exogenous" sensing moiety is
covalently attached to the polypeptide. This differs from
pore-subunit polypeptides in which the only modification(s) is one
or more mutations within the amino acid sequence of the polypeptide
itself. Although the sensing moiety is engineered into such
polypeptides, in contrast to the native polypeptide sequence, such
engineered, modified or "mutant" polypeptides still comprise an
"endogenous" sensing moiety.
[0013] In contrast, the present invention provides modified
pore-forming or pore-subunit polypeptides that at least comprise
one exogenous sensing moiety that is covalently linked to the
pore-subunit polypeptide. Those of ordinary skill in the art will
understand that engineered, mutant or variant pore-subunit
polypeptides may well be used in the invention so long as they are
further "covalently attached" to at least a first sensing moiety.
That is, so long as any existing amino acid mutation is not solely
relied upon to provide the sensing means.
[0014] In using one or more engineered, mutant or variant
pore-forming or pore-subunit polypeptides in the present invention,
the modified, mutated or "heterologous" amino acid(s) may, in fact,
form the point of attachment for one or more of the covalently
attached sensing moieties. As such, the pore-subunit polypeptide
may be engineered to produce at least a first new or heterologous
"attachment site", to which the sensing moiety or moieties are
subsequently covalently attached. Equally, the invention includes a
range of engineered, mutant or variant pore-subunit polypeptides
that comprise at least one modified, mutated or "heterologous"
amino acid at a location distinct from the covalent attachment of
the sensing moiety or moieties. Such heterologous amino acids may
themselves impart a sensing function, so long as such a function is
in addition to the sensing function provided by the covalently
attached sensing moiety or moieties of the invention.
[0015] Accordingly, in certain embodiments, the invention provides
a modified pore-forming or pore-subunit polypeptide other than
wherein the modification of the modified pore-subunit polypeptide
exists only in that the polypeptide contains a heterologous
analyte-binding amino acid. In further embodiments, this invention
provides oligomeric and polymeric pore assemblies and biosensors
comprising at least a first modified pore-subunit polypeptide other
than wherein the modification of the modified pore-subunit
polypeptide exists only in that the polypeptide contains a
heterologous analyte-binding amino acid. In yet further
embodiments, the invention provides methods of detecting analytes,
including changes in the type and/or amount of biological and
chemical constituents in samples, and methods of detecting changes
in the physical environment, using oligomeric and polymeric pore
assemblies and biosensors that comprise at least a first modified
pore-subunit polypeptide other than wherein the modification of the
modified pore-subunit polypeptide exists only in that the
polypeptide contains a heterologous analyte-binding amino acid.
[0016] In other embodiments, the invention provides modified
pore-forming or pore-subunit polypeptides, oligomeric and polymeric
pore assemblies and biosensors thereof, and methods of using such
polypeptides, pore assemblies and biosensors, other than wherein
the modified pore-subunit polypeptide is a mutant staphylococcal
alpha hemolysin polypeptide and wherein the modification exists
only in that the polypeptide comprises a heterologous
analyte-binding amino acid, which polypeptide assembles into an
analyte-responsive heteroheptameric pore assembly in the presence
of a wild type staphylococcal alpha hemolysin polypeptides.
[0017] In yet other embodiments, the invention provides modified
pore-forming or pore-subunit polypeptides, oligomeric and polymeric
pore assemblies and biosensors thereof, and methods of using such
polypeptides, pore assemblies and biosensors, other than wherein
the modified pore-subunit polypeptide is a pore-subunit
polypeptide, such as a staphylococcal alpha hemolysin polypeptide,
wherein the modification exists only in that the polypeptide is
attached or covalently attached to a chelating molecule for metal
detection.
[0018] The modified, covalently-linked, sensing pore-forming or
pore-subunit polypeptides of the invention are capable of
assembling into pores, or into oligomeric and/or polymeric pore
assemblies in the presence of a plurality of pore-forming or
pore-subunit polypeptides.
[0019] All such pores and pore assemblies are herein termed "pore
assemblies" for simplicity, irrespective of whether the pore is
formed by a single polypeptide or two or more such polypeptides.
The formation of the pore assemblies can take place in any suitable
environment, such as any suitable lipid environment, e.g., a
bilayer, cell membrane, liposome and the like.
[0020] In certain preferred embodiments, the parent "pore-subunit
polypeptides" and modified versions of the invention are capable of
assembling into oligomeric and/or polymeric pore assemblies in the
presence of a plurality of "like" pore-subunit polypeptides.
[0021] This includes assemble with a plurality of unmodified and
modified versions of the same pore-subunit polypeptides. Equally,
the use of "pore-subunit polypeptides" capable of forming
oligomeric and/or polymeric pore assemblies in the presence of
distinct pore-subunit polypeptides is included within the
invention.
[0022] The invention thus provides homomeric pore assemblies, in
which all the pore-subunit polypeptides are modified pore-subunit
polypeptides of the invention. The invention further provides a
range of heteromeric pore assemblies, in which at least one of the
pore-subunit polypeptides is a modified pore-subunit polypeptide of
the invention, but in which the overall pore assembly includes at
least one distinct type of pore-subunit polypeptide. The
heteromeric pore assemblies may be further sub-divided into those
heteromeric pore assemblies in which the pore-subunit polypeptides
are modified and unmodified versions of the same polypeptide; and
those heteromeric pore assemblies that comprise at least two
pore-subunit polypeptides or different origins, whether in modified
or unmodified form.
[0023] Any polypeptide, whether of natural or totally synthetic
origin, may be used in the invention so long as it can be
effectively covalently attached to one or more sensing moieties and
so long as it meets the pore-forming criteria described herein and
known those of ordinary skill in the art. Exemplary pore-forming
polypeptides are the pore-subunit polypeptides known in nature,
such as bacterial pore-subunit polypeptides.
[0024] For example, certain preferred pore-forming and pore-subunit
polypeptides for use in the invention include, but are not limited
to, porins, complement pore polypeptides, hemolysin C polypeptides,
streptolysin O polypeptides and membrane channel polypeptides, such
as potassium channel polypeptides. In certain preferred embodiments
of the invention, the pore-subunit polypeptide is a staphylococcal
hemolysin polypeptide, with staphylococcal alpha hemolysin
polypeptides being particularly preferred.
[0025] As described above, the invention contemplates the use of
engineered, mutant and variant pore-forming or pore-subunit
polypeptides, including those with heterologous amino acid(s), so
long as an exogenous sensing moiety is covalently attached. As
such, in certain preferred embodiments, the pore-subunit
polypeptides of the invention are mutant staphylococcal alpha
hemolysin polypeptides that comprise at least a first heterologous
amino acid. For example, such as wherein the mutant staphylococcal
alpha hemolysin polypeptide comprises a cysteine residue in place
of serine at position 106 of the wild-type staphylococcal alpha
hemolysin polypeptide; or wherein the mutant staphylococcal alpha
hemolysin polypeptide comprises a cysteine residue in place of
lysine at position 8 of the wild-type staphylococcal alpha
hemolysin polypeptide.
[0026] The modified pore-forming or pore-subunit polypeptides are
"covalently linked or attached" to at least a first sensing moiety
in any manner that substantially preserves the ability of the
polypeptide to assemble into oligomeric and/or polymeric pore
assemblies and that substantially preserves the ability of the
sensing moiety to provide a useful sensing function.
[0027] The sensing moiety or moieties may be covalently linked to
the pore-forming or pore-subunit polypeptide so that they occupy a
position in a transmembrane channel, project into the lumen of, or
occupy a position in a stem domain of the resultant oligomeric
and/or polymeric pore assembly. The sensing moiety or moieties may
also be covalently linked to a surface position on the pore-forming
or pore-subunit polypeptide, so that they occupy a position close
to the entrance to the channel or pore, as exemplified by the
attached oligonucleotides disclosed herein.
[0028] The "covalent linkage" can be formed by one or more covalent
bonds between the pore-forming or pore-subunit polypeptide and the
sensing moiety or moieties. Direct covalent attachment is preferred
in various aspects. However, "covalent linkage" also includes other
functional chemical attachments, and does not exclude the use of
linkers, such as short chains of chemical groups or peptides, which
covalently link the two components without being an integral part
of either component in its natural form. Synthetic linking
methodology is well known in the art and can be readily adapted for
use herewith in light of the inventive teaching of the present
disclosure.
[0029] In certain aspects of the invention, the sensing moiety is a
functional group. The term "functional group", as employed herein,
is used for convenience to mean a functional sensing moiety "other
than a polymer", wherein the sensing moiety provides a useful
sensing function. In preferred aspects, the functional group is an
"analyte-binding" functional group. In certain embodiments, the
functional group binds to one or more analytes, while in other
embodiments, the analyte binds to the functional group. Other
functional groups are those that sense changes in the physical
environment, such as changes in pH, light, voltage, temperature and
the like. Although the present invention may be used in combination
with radiolabels, an advantage of the invention is that radioactive
substances are by no means necessary to practice the invention.
[0030] The functional group can be a naturally occurring molecule,
a synthetic molecule or a combination thereof. Functional groups
that are naturally occurring molecules contemplated for use in the
present invention include, but are not limited to, enzyme
inhibitors, haptens, nucleotides, amino acids, lipids, toxins,
saccharides, chelators and/or cyclodextrins. Synthetic molecules
contemplated for use in the present invention include, but are not
limited to, calixarenes and/or crown ethers.
[0031] In other aspects of the present invention, the sensing
moiety is a polymer. Polymers contemplated for use in the present
invention can be homopolymers, heteropolymers and functionalized
polymers. The polymers can also be naturally occurring molecules or
synthetic molecules. In certain aspects of the invention, the
polymer is polyethylene glycol (PEG) or polyethylene glycol
(PEG)-biotin. In preferred aspects of the invention, the polymer is
an analyte-binding polymer, including, but not limited to,
oligonucleotides, polynucleotides, oligosaccharides,
polysaccharides, lipopolysaccharides, proteins, glycoproteins,
polypeptides and/or peptides. In particularly preferred
embodiments, the attached polymer is a single-stranded
oligonucleotide or polynucleotide, such as DNA or RNA.
[0032] In various embodiments of the invention, the covalently
attached sensing moiety responds to a change in the type,
concentration and/or amount of a biological or chemical constituent
in the environment of the oligomeric pore assembly. The constituent
may be an organic molecule or even a microorganism, such as a
bacterium, fungi or virus. The organic molecules may be biological,
physiological and/or pharmacological molecules, or may be
byproducts, pollutants, environmental toxins, explosives, or such
like. As such, the sensing moiety may bind to a metal or a metal
ion (e.g., zinc, cobalt, copper, nickel and cadmium), a toxin, an
enzyme, a nucleotide, an oligonucleotide, an amino acid, a peptide,
a polypeptide, a saccharide, an oligosaccharide, a hapten, a lipid,
an antibody or antigen-binding fragment thereof, or any one or more
a range of organic molecules.
[0033] In still further embodiments, the sensing moiety responds to
a change in the physical environment of the oligomeric pore
assembly, including, but not limited to, changes in pH, light,
voltage, current, resistance and/or temperature.
[0034] In additional aspects of the present invention, the
pore-forming or pore-subunit polypeptide is covalently linked to at
least a first and at least a second, third, fourth, etc. sensing
moiety. In certain aspects, the first sensing moiety is distinct
from the second, third, fourth, etc. sensing moieties. In other
aspects, the first sensing moiety is the same as at least one of
the second, third, fourth, etc. sensing moieties, or is the same as
each of the second, third, fourth, etc. sensing moieties. In
further aspects, the pore-subunit polypeptide is covalently linked
to a plurality of sensing moieties, which may all be the same, all
be different, or combinations of sensing moieties may be used.
Exemplary combinations include the attachment of various
oligosaccharides and/or oligonucleotides. Therefore, more than one
analyte or physical parameter can be assayed at the same time.
[0035] Thus, where the pore-forming or pore-subunit polypeptide is
P and certain distinct sensing moieties are S.sub.1, S.sub.2,
S.sub.3, etc., the invention includes modified polypeptides, pore
assemblies, biosensors, arrays, kits and methods wherein the
modified polypeptide is P--S.sub.1, P--S.sub.2, P--S.sub.3,
P--S.sub.1S.sub.1, P--S.sub.2S.sub.2, P--S.sub.3S.sub.3,
P--S.sub.1S.sub.1S.sub.1, P--S.sub.2S.sub.2S.sub.2,
P--S.sub.3S.sub.3S.sub.3, P--S.sub.1S.sub.2, P--S.sub.2S.sub.1,
P--S.sub.1S.sub.3, P--S.sub.3S.sub.1, P--S.sub.2S.sub.3,
P--S.sub.3S.sub.2, P--S.sub.1S.sub.2,S.sub.3,
P--S.sub.3S.sub.2,S.sub.1, and such like, with the same or
different sensing moieties attached, and where different sensing
moieties are used, in various orders of attachment. Those of
ordinary skill in the art will understand that the same individual
combinations are possible where the pore-forming or pore-subunit
polypeptides are different, e.g., P.sub.1, P.sub.2, P.sub.3,
P.sub.4, P.sub.5, P.sub.6 and P.sub.7 and such like, and that any
one or more of the same or different polypeptides may be combined
with any one or more of the same or different attached sensing
moieties, and that the modified polypeptides may be combined into
pores in any operative combination.
[0036] Further aspects of the invention are kits, which comprise,
generally in one or more suitable containers, a plurality of
pore-forming or pore-subunit polypeptides sufficient to form a
pore, wherein at least one of the pore-subunit polypeptides is a
modified pore-subunit polypeptide of the invention, or a precursor
thereof, i.e., a modified pore-subunit polypeptide comprising a
pore-subunit polypeptide covalently linked to a sensing moiety, or
a precursor thereof. Where the modified pore-subunit polypeptides
are supplied as precursors, the materials for converting the
precursor into a modified pore-subunit polypeptide of the invention
are included in the kit, such as one or more sensing moieties and,
optionally, one or more components for covalently linking the one
or more sensing moieties to the pore-subunit polypeptides.
[0037] The present invention also provides oligomeric and polymeric
pore assemblies comprising a number of pore-subunit polypeptides
sufficient to form a pore, wherein at least one of the pore-subunit
polypeptides is a modified pore-subunit polypeptide of the
invention, i.e., a modified pore-subunit polypeptide comprising a
pore-subunit polypeptide covalently linked to a sensing moiety.
[0038] In certain aspects of the invention, the pore assembly
comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified
pore-forming or pore-subunit polypeptides. Thus, in certain
aspects, the pore assembly comprises a plurality of modified
pore-subunit polypeptides. In yet other aspects, the pore assembly
is comprised completely of modified pore-subunit polypeptides.
[0039] Irrespective of the number of modified pore-subunit
polypeptides within the pore assembly, so long as there is at least
one, the pore assemblies of the invention may comprise between
about 1 and about 100 pore-subunit polypeptides. The use of one
pore-subunit polypeptide requires that the pore-subunit polypeptide
form a pore by itself and be a modified pore-subunit polypeptide of
the invention.
[0040] In certain aspects, the pore assemblies of the invention
comprise 1, 2, about 3, about 4, about 5, about 6, about 7, about
8, about 9, about 10, about 11, about 12, about 13, about 14, about
15, about 16, about 17, about 18, about 19, about 20, about 21,
about 22, about 23, about 24, about 25, about 26, about 27, about
28, about 29, about 30, about 31, about 32, about 33, about 34,
about 35, about 36, about 37, about 38, about 39, about 40, about
41, about 42, about 43, about 44, about 45, about 46, about 47,
about 48, about 49, about 50, about 51, about 52, about 53, about
54, about 55, about 56, about 57, about 58, about 59, about 60,
about 61, about 62, about 63, about 64, about 65, about 66, about
67, about 68, about 69, about 70, about 71, about 72, about 73,
about 74, about 75, about 76, about 77, about 78, about 79, about
80, about 81, about 82, about 83, about 84, about 85, about 86,
about 87, about 88, about 89, about 90, about 91, about 92, about
93, about 94, about 95, about 96, about 97, about 98, about 99,
about 101 or about 102 or more pore-subunit polypeptides.
[0041] In such pore assemblies, all, or substantially all, of the
pore-subunit polypeptides may be modified pore-subunit polypeptides
of the present invention. Alternatively, the modified pore-subunit
polypeptides of the present invention may make up about 5%, about
10%, about 15%, about 20%, about 30%, about 40%, about 50%, about
60%, about 70%, about 80%, about 90% or about 95% or so of the
pore-subunit polypeptides within the pore assembly.
[0042] Where the pore assemblies of the invention comprise two or
more, or a plurality of the modified pore-subunit polypeptides of
the invention, the modified pore-subunit polypeptides may each be
linked to the same sensing moiety, or each be linked to distinct
sensing moieties. The modified pore-subunit polypeptides may be
linked to at least two distinct sensing moieties, up to and
including being linked to as many distinct sensing moieties as
modified pore-subunit polypeptides within the pore assembly.
[0043] In preferred embodiments, the pore assemblies comprise the
amount of pore-subunit polypeptides that form in the natural
environment. For example, the pore assemblies may comprise 3, 4, 5,
6, 7, 8, 9 or 10 or so pore-subunit polypeptides. Where the
pore-subunit polypeptides and modified pore-subunit polypeptides
are substantially all staphylococcal hemolysin polypeptides, such
as staphylococcal alpha hemolysin polypeptides, the pore assemblies
may preferably comprise 7 pore-subunit polypeptides, which may be
include any number from 1 to 7 modified pore-subunit polypeptides
of the invention. In other instances, the use of 3 polypeptides is
particularly preferred, such as in certain porins that form pores
as trimers.
[0044] Thus, the invention provides pore assemblies of the formula
WTx-nMODn, wherein WT is an unmodified pore-subunit polypeptide,
MOD is a modified pore-subunit polypeptide of the invention, n is
an integer of 1 or greater than 1 and n is less than or equal to x,
with x being an integer. That is, n is an integer of I or greater
than 1 and x is an integer that is greater than or equal to n.
Preferably, n and x are each integers from 1 to 100. Where x is
equal to n, the pore assembly comprises only modified pore-subunit
polypeptides of the invention.
[0045] Additionally, the present invention provides biosensor
devices and arrays, digital biosensor devices, and arrays and
integrated circuits thereof, comprising one or more of the modified
pore-subunit polypeptides of the invention, preferably, in the form
of one or more of the oligomeric and/or polymeric pore assemblies
of the invention. That is, the biosensor devices comprise a number
of pore-subunit polypeptides sufficient to form a pore, wherein at
least one of the pore-subunit polypeptides is a modified
pore-subunit polypeptide comprising a pore-subunit polypeptide
covalently linked to at least a first sensing moiety.
[0046] In certain preferred embodiments, the biosensors, devices
and arrays of the invention are fabricated to detect electrical
current. The biosensor devices may detect a single channel current
or may detect a current through two, more than two or a plurality
of channels. In detecting electrical current, the devices of the
invention are able to detect changes in ionic current flowing
through a pore so that they are able, for example, to detect,
quantitate and/or discriminate between components driven through
the pore by an applied potential.
[0047] Biosensor arrays preferably comprise two or more of the
oligomeric and/or polymeric pore assemblies of the invention, most
preferably where the pore assemblies comprise modified pore-subunit
polypeptides comprising distinct sensing moieties that sense
distinct analytes or physical parameters. Such arrays provide for
the simultaneous detection of multiple analytes or physical
parameters. In the stochastic sensing of the present invention,
each element does not need to be entirely specific for a given
analyte, as a signature or "profile" is still generated that allows
the detection, and optional quantification, of the given or more
then one analyte(s).
[0048] Biosensors generally have three elements: a sensing moiety
that either binds to or is bound by one or more target analytes, or
responds to one or more physical parameters or properties; a
transduction mechanism that signals the binding of the analyte(s)
or the alteration of the sensing moiety in response to the physical
parameter(s); and a means for, or a method of, measuring, and
preferably processing, the transduction signal. Operative aspects
of biosensors that may be combined for use in the present invention
are described in published PCT patent application WO 99/05167,
filed Jul. 24, 1998; U.S. provisional patent application
60/053,737, filed Jul. 25, 1997; U.S. patent application Ser. No.
09/122,583, filed Jul. 24, 1998; and in U.S. Pat. Nos. 5,777,078,
5,817,771 and 5,824,776, each of which are specifically
incorporated herein by reference in their entirety.
[0049] The sensing moieties of the invention are covalently linked
to pore-forming polypeptides to give modified pore-forming
polypeptides that self-assemble into pores. The transduction
mechanisms that signal the binding of analytes and/or the
alteration of the sensing moieties in response to physical
parameters are adapted for use with the particular sensing moieties
and detection means. For example, transduction mechanisms that
signal the fractional occupancy of the sensing moiety by analytes
and/or the physical and/or chemical state of sensing moiety under
different conditions. Exemplary transduction mechanisms include
materials flowing through the pore assemblies.
[0050] The biosensors of the present invention are thus useful in
the detection of any analyte, component or physical parameter that
contacts or impacts the measurable channel of the pore assembly.
For use in single channel mode, an individual analyte is detected
as it randomly, i.e., stochastically, binds to and releases from a
single binding site. These events are detectable as modification or
perturbations of the ion conductance in the single channel.
[0051] Preferably, the biosensor devices of the invention comprise
means to detect the signal sensed by the at least a first sensing
moiety of the modified pore-subunit polypeptide, oligomeric and/or
polymeric pore assembly or assemblies. In certain preferred
embodiments, the means is means to detect an electrical current or
ion flux. In other embodiments, the detection means is means to
detect signals based upon fluorescence or phosphorescence, or means
to detect signals based upon atomic force microscopy. Means to
detect an electromagnetic signal, such as a visible, ultraviolet,
infrared, near-infrared or x-ray signal are also possible.
[0052] The biosensor devices of the invention may also comprise
additional components, such as one, two or a plurality of signal
amplification means and processing means, such as microprocessors
and amplifiers. Sampling means may also be provided.
[0053] The present invention also provides various detection
methods using one or more of the modified pore-subunit
polypeptides, preferably, in the form of one or more of the
oligomeric and/or polymeric pore assemblies or one or more of the
biosensor devices of the invention. That is, the invention provides
detection methods using at least a first of the oligomeric or
polymeric pore assemblies or biosensor devices of the invention in
which a signal, such as an electrical current, is detected through
at least a first channel, a single channel or two or more
channels.
[0054] Preferably, the signal, whether an electrical current or
other signal, and whether detected through a single channel or two
or more channels, is compared to a "control" signal measurement,
such as a control current measurement; wherein a modulation or
perturbation in signal, e.g., current, compared to an equivalent
measurement in the control indicates the presence of the substance,
event or change to be detected. In certain embodiments, the
"control" signal measurement is actually measured in a "control
sample", but it need not be. An advantageous feature of the
invention is that parallel controls do not need to be run, i.e.,
the invention is self-calibrating.
[0055] The invention thus provides methods of detecting, and
optionally quantifying, the presence of an analyte in a sample,
comprising contacting the sample with one or more oligomeric and/or
polymeric pore assemblies or biosensor devices of the invention,
and detecting a signal or electrical current through at least a
first channel, wherein a modulation in signal or current compared
to a signal or current measurement in a control sample lacking the
analyte indicates the presence of the analyte in the sample. The
amount of the analyte in the sample may be readily quantitated by
quantifying the signal or electrical current detected.
[0056] These methods comprise contacting the sample with one or
more oligomeric and/or polymeric pore assemblies or biosensor
devices of the invention, i.e., comprising a number of pore-subunit
polypeptides sufficient to form a pore, wherein at least one of the
pore-subunit polypeptides is a modified pore-subunit polypeptide
comprising a pore-subunit polypeptide covalently linked to a
sensing moiety, and detecting an electrical current through at
least a first channel, wherein a modulation in current compared to
a current measurement in a control sample lacking the analyte
indicates the presence of the analyte in the sample.
[0057] An electrical current may be detected through a single
channel. Such single channel detection in the digital mode provides
a signature of the analyte, providing information on both the
concentration of the analyte, as well as the identity of the
analyte. In certain aspects of the invention, once the pore
assembly has been validated, there is no need to run a control to
determine the analyte signature, thus creating a "self-calibrating"
sensor. As such, the "comparison" step can be an inherent feature
that is not re-executed in real time alongside every analyte
measurement. Such digital monitoring can also be used in single
molecule detection.
[0058] An electrical current may also be detected through at least
two channels, wherein a modulation in current compared to a current
measurement in a control sample lacking the analyte indicates the
presence of the analyte in the sample. In general, using two or
more, or macroscopic, channels provides information in the change
of the pore environment, without providing a specific analyte
signature.
[0059] In addition to single and multiple channel detection, a
number of other detection methods are contemplated for use in the
present invention, including, but not limited to, fluorescence,
phosphorescence, and atomic force microscopy. Such signals may be
detected by the detection means exemplified above and known those
of ordinary skill in the art in light of the present
disclosure.
[0060] The present invention also provides methods of detecting the
presence of an unknown analyte in a sample, comprising contacting
the sample with one or more oligomeric and/or polymeric pore
assemblies or biosensor devices of the invention, and detecting a
signal or electrical current through at least a first channel to
determine a sample current signature, and comparing the sample
current signature to a standard current signature of a known
analyte, wherein a concurrence of the sample current signature and
the standard current signature indicates the identity of the
unknown analyte in the sample.
[0061] Such methods comprise contacting the sample with one or more
oligomeric pore assemblies comprising a number of pore-subunit
polypeptides sufficient to form a pore, wherein at least one of the
pore-subunit polypeptides is a modified pore-subunit polypeptide
comprising a pore-subunit polypeptide covalently linked to a
sensing moiety, detecting an electrical current through a single
channel to determine a sample current signature, and comparing the
sample current signature to a standard current signature of a known
analyte, wherein a concurrence of the sample current signature and
the standard current signature indicates the identity of the
unknown analyte in the sample.
[0062] These methods further contacting the sample with one or more
oligomeric pore assemblies comprising a number of pore-subunit
polypeptides sufficient to form a pore, wherein at least one of the
pore-subunit polypeptides is a modified pore-subunit polypeptide
comprising a pore-subunit polypeptide covalently linked to a
sensing moiety, detecting an electrical current through at least
two channels to determine a sample current signature, and comparing
the sample current signature to a standard current signature of a
known analyte, wherein a concurrence of the sample current
signature and the standard current signature indicates the identity
of the unknown analyte in the sample.
[0063] Furthermore, the present invention provides methods of
detecting a change in the type or amount of a biological or
chemical constituent in a sample, comprising the steps of
contacting the sample with one or more oligomeric and/or polymeric
pore assemblies or biosensor devices of the invention at a first
time point; determining a first sample current signature by
detection of an electrical current through at least a first
channel, a single channel or through two channels; contacting the
sample with one or more oligomeric and/or polymeric pore assemblies
or biosensor devices of the invention at a second time point;
determining a second sample current signature by detection of an
electrical current through at least a first channel, a single
channel or through two channels; and comparing the first sample
current signature to the second sample current signature, wherein a
difference between the first sample current signature and the
second sample current signature is indicative of a change in the
type or amount of a biological or chemical constituent in the
sample.
[0064] In all methods where measurements are made at least at a
first and second time point, the time points may be two or more
time points at any instance of operation in a continuous flow
mode.
[0065] Such methods comprise contacting the sample with one or more
oligomeric pore assemblies comprising a number of pore-subunit
polypeptides sufficient to form a pore, wherein at least one of the
pore-subunit polypeptides is a modified pore-subunit polypeptide
comprising a pore-subunit polypeptide covalently linked to a
sensing moiety at a first time point; determining a first sample
current signature by detection of an electrical current through at
least a first channel, a single channel or through two channels;
contacting the sample with one or more oligomeric pore assemblies
comprising a number of pore-subunit polypeptides sufficient to form
a pore, wherein at least one of the pore-subunit polypeptides is a
modified pore-subunit polypeptide comprising a pore-subunit
polypeptide covalently linked to a sensing moiety at a second time
point; determining a second sample current signature by detection
of an electrical current through at least a first channel, a single
channel or through two channels; and comparing the first sample
current signature to the second sample current signature, wherein a
difference between the first sample current signature and the
second sample current signature is indicative of a change in the
type or amount of a biological or chemical constituent in the
sample.
[0066] In embodiments where the attached polymer is an
oligonucleotide or polynucleotide, such as single-stranded DNA or
RNA, the invention further provides methods of nucleic acid
detection and analysis. For example, the invention provides methods
of detecting defined nucleic acid sequences using one or more
oligomeric and/or polymeric pore assemblies or biosensor devices of
the invention in which the attached sensing moiety is itself a
nucleic acid. A range of such sequence detection methods is
possible. These include, but are not limited to, methods of
detecting the presence of a nucleic acid of unknown sequence in a
sample.
[0067] Such methods generally comprise contacting the sample with
one or more oligomeric and/or polymeric pore assemblies or
biosensor devices of the invention, in which at least a first
pore-forming or pore-subunit polypeptide is covalently linked to at
least a first nucleic acid that acts as a sensing moiety,
preferably, at least a first nucleic acid of known sequence that
acts as a sensing moiety; detecting a signal or electrical current
through at least a first channel, a single channel or two or more
channels, to determine a sample current signature; and comparing
the sample current signature to a standard current signature of a
nucleic acid of known sequence, wherein a concurrence of the sample
current signature and the standard current signature indicates the
identity of the nucleic acid of unknown sequence in the sample.
[0068] Preferably, the at least a first nucleic acid that acts as a
sensing moiety has a known sequence; and the methods are used to
discriminate between nucleic acids in the sample of exactly the
complementary sequence, substantially the complementary sequence
and those nucleic acids that do not have exactly or substantially
the complementary sequence. The nucleic acids are preferably on the
order of between about 6 and about 50 nucleotides in length. The
sequences to be detected are limitless, as exemplified by detecting
sequence variations of diagnostic and/or prognostic significance in
human, veterinary, agricultural, environmental and/or
microbiological significance.
[0069] Further embodiments of using pores, pore assemblies and
biosensors with covalently attached nucleic acid elements are in
sequencing nucleic acids. In these aspects of the invention, the at
least a first pore-forming or pore-subunit polypeptide of the
invention may be covalently linked to at least a first nucleic acid
of known or unknown sequence. Those with known attached sequences
may be used as described above to specifically detect, and thus
sequence, complementary nucleic acids.
[0070] Accordingly, multiple copies of nucleic acids with sequences
from a given molecule may be arrayed in a pore assembly or
biosensor. The plurality of oligonucleotides arrayed in the pore
assembly or biosensor may each have a substantially distinct
sequence from a given molecule, such as, e.g., a pathogen or
oncogene, thus arrowing detection of hybridizing sequences. The
plurality of oligonucleotides arrayed as such may also have
sequences from a parent molecule that overlap by one nucleotide
residue per oligonucleotide, such that an overlapping array of
pathogen- or oncogene-derived sequences are presented.
[0071] Pores, pore assemblies and biosensors in which the
polypeptides of the invention are covalently linked to at least a
first nucleic acid of unknown or partially unknown sequence can
also be readily used in sequencing. In such embodiments, the pores
are interrogated in sequence with candidate oligonucleotides,
allowing those that hybridize to be identified in sequential
format.
[0072] Arrays of pores, pore assemblies and biosensors with
covalently attached nucleic acids of known, unknown or partially
known and unknown sequences may thus be used in essentially the
same manner as the sequence detection chips with immobilized
nucleic acids available in the art. Although the biosensors of the
present invention provide the various improved features described
herein and apparent in the practice of the invention, the execution
of the nucleic acid binding steps and assimilation and analysis of
the information generated, preferably using computer-based
algorithms, has parallels in the "sequencing chip" technology. The
following patents and patent applications are each incorporated
herein by reference for purposes of even further exemplifying the
use of immobilized nucleic acids in detection and sequencing: WO
95/09248: U.S. Pat. Nos. 5,202,231; 5,695,940; 5,525,464;
5,667,972; 5,202,231; 5,492,806; WO 99/09217; and WO 98/31836.
[0073] Additionally, the present invention provides methods of
detecting, and optionally quantifying, a change in the physical
environment of a sample, comprising contacting the sample with one
or more oligomeric and/or polymeric pore assemblies or biosensor
devices of the invention at a first time point; determining a first
sample current signature by detection of an electrical current
through at least a first channel, a single channel or through two
channels; contacting the sample with one or more oligomeric and/or
polymeric pore assemblies or biosensor devices of the invention at
a second time point; determining a second sample current signature
by detection of an electrical current through at least a first
channel, a single channel or through two channels; and comparing
the first sample current signature to the second sample current
signature, wherein a difference between the first sample current
signature and the second sample current signature is indicative of
a change in the physical environment of the sample.
[0074] The change in the physical environment may be readily
quantitated by quantifying the signal or electrical current
detected. The change(s) in the physical environment may also be
determined as an ongoing process, i.e., in a continuous flow mode.
That is, the first and second samples, pore assemblies and current
signatures do not need to be physically separate, only temporally
distinct.
[0075] These methods of the invention comprise the steps of
contacting the sample with one or more oligomeric pore assemblies
comprising a number of pore-subunit polypeptides sufficient to form
a pore, wherein at least one of the pore-subunit polypeptides is a
modified pore-subunit polypeptide comprising a pore-subunit
polypeptide covalently linked to a sensing moiety at a first time
point; determining a first sample current signature by detection of
an electrical current through at least a first channel, a single
channel or through two channels, contacting the sample with one or
more oligomeric pore assemblies comprising a number of pore-subunit
polypeptides sufficient to form a pore, wherein at least one of the
pore-subunit polypeptides is a modified pore-subunit polypeptide
comprising a pore-subunit polypeptide covalently linked to a
sensing moiety at a second time point; determining a second sample
current signature by detection of an electrical current through at
least a first channel, a single channel or through two channels;
and comparing the first sample current signature to the second
sample current signature, wherein a difference between the first
sample current signature and the second sample current signature is
indicative of a change in the physical environment of the
sample.
BRIEF DESCRIPTION OF THE DRAWING
[0076] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of illustrative
embodiments presented herein. All drawings in U.S. provisional
application Ser. No.60/182,097, filed Feb. 11, 2000, are
specifically incorporated herein by reference without
disclaimer.
[0077] FIG. 1. Schematics of H.sub.6S106C-PEG5K.sub.1 and
H.sub.6K8C-PEG5K.sub.1, shown as sagittal sections. In each
engineered pore, only one of the seven subunits is modified. In
this work, the mutant K8A was used as the unmodified .alpha.HL
subunit (H), so that the net charge at the cis channel entrance
would not be altered in heteromers containing K8C-PEG5K.
[0078] FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. FIG. 2A and FIG. 2B.
A representative current trace and semi-logarithmic amplitude
histogram for a single channel current from
H.sub.6S106C-PEG5K.sub.1. The current was recorded at +100 mV under
symmetrical buffer conditions: 300 mM KCl, 5 mM Tris-HCl (pH=7.00),
100 .mu.M EDTA. The bilayer lipid was
1,2-diphytanoyl-sn-glycerophosphocholine. Protein was added to the
cis chamber, which was at ground. A positive potential indicates a
higher potential in the trans chamber and a positive current is one
in which cations flow from the trans to the cis chamber. The
current was low-pass filtered at 100 Hz and sampled at 10 kHz. An
expanded view of a high amplitude substate (the last spike in the
trace) is shown filtered at 3 kHz. d, low amplitude subconductance
state; s, short-lived spike. FIG. 2C and FIG. 2D. Signal from the
same channel after treatment with 12 mM DTT in the cis chamber,
filtered at 100 Hz.
[0079] FIG. 3A and FIG. 3B. Single channel properties of the
heteromeric pore H.sub.6106C-PEG-biotin.sub.1. FIG. 3A.
Representative single channel current trace exhibiting short-lived
high-amplitude spike-like partial closures. FIG. 3B. Threshold
histogram from an extended period (1 min) of the current trace
excerpted in FIG. 3A. The signal was filtered at 8 kHz and sampled
at 200 kHz. The threshold was set at 15 pA. Only the signal 0.5 ms
before and after each spike was recorded and used in the histogram.
Hence the peak at .about.10 pA, arising from the spikes, is
exaggerated.
[0080] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E-1, FIG. 4E-2,
FIG. 4E-3, FIG. 4F, FIG. 4G and FIG. 4H. Response of
H.sub.6106C-PEG-biotin.su- b.1 to WT and W120A streptavidins.
Single-channel current recordings. In FIG. 4A-FIG. 4D, the bars
show biotin capture events. The streptavidins were added before the
start of each trace. FIG. 4A. WT streptavidin (12 nM) added to the
cis chamber abolishes the high-amplitude spikes. FIG. 4B. WT
streptavidin (12 nM) added to the trans chamber leads to a
permanent partial channel blockade closely similar in amplitude to
the amplitude of the spikes. FIG. 4C. W120A streptavidin (7.25 nM)
added to the cis chamber leads to transient disappearances of the
high-amplitude spikes, but does not alter the underlying current.
FIG. 4D. W120A streptavidin (7.25 nM) added to the trans chamber
produces transient partial channel blockades closely similar in
amplitude to the amplitude of the spikes. FIG. 4E-1, FIG. 4E-2 and
FIG. 4E-3. The inter-event interval (.tau..sub.on) decreases with
increasing W120A streptavidin. FIG. 4F. Plot of 1/.tau..sub.on
versus W120A streptavidin concentration. Data from a single typical
study are plotted in FIG. 4E-1, FIG. 4E-2, FIG. 4E-3 and FIG. 4F as
least-squares fits. In FIG. 4G and FIG. 4H, the thick bars show
trans biotin capture events and the thin bars cis events. The
streptavidins were added before the start of each trace. FIG. 4G.
W120A streptavidin (7.25 nM) was added to the cis chamber and WT
streptavidin (71.7 nM) to the trans chamber. Cis capture of biotin
by W120A streptavidin produces transient disappearances of the
spikes. By contrast, trans capture by WT streptavidin resulted in a
permanent blockade. FIG. 4H. W120A streptavidin (7.25 nM) was added
to the cis chamber and WT120A streptavidin (29 nM) was added to the
trans chamber producing transient events by capture on both the cis
and trans sides.
[0081] FIG. 5A and FIG. 5B. Response of
H.sub.6106C-PEG-biotin.sub.1 to a mouse anti-biotin monoclonal
IgG.sub.1 (mAb). Single-channel current recordings. Thick bars show
trans biotin capture events and the thin bars cis events. The
biotin-binding proteins were added before the start of each trace.
FIG. 5A. mAb (5.8 nM) was added to the trans chamber. A single
capture event is shown. FIG. 5B. mAb (29 nM) was added to the trans
chamber with W120A streptavidin (7.25 nM) in the cis chamber. Both
cis and trans capture events are shown.
[0082] FIG. 6A and FIG. 6B. Attachment of a single DNA
oligonucleotide to the .alpha.HL pore. In the heteroheptameric
.alpha.HL pore, containing six unmodified and one DNA-modified
subunit, the 5'-end of the oligonucleotide is tethered via a
hexamethylene linker and a disulfide bond to Cys.sup.17 introduced
by mutagenesis. An applied, positive electrical potential drives
negatively charged molecules from the cis to the trans side of the
bilayer. FIG. 6A and FIG. 6B show the preparation of the .alpha.HL
pore H.sub.6(17C-oligo-A).sub.1. FIG. 6A. Autoradiogram of an
SDS-polyacrylamide gel after electrophoresis of a mixture of
unmodified .alpha.HL monomers, H, and 17C-oligo-A-D4 monomers
cross-linked to oligo-A (5'-CATTCACC-3'; SEQ ID NO:1) through a
disulfide bond, in the absence (lane 1) and presence (lane 2) of
the reducing agent DTT. The DNA-modification causes 17C-oligo-A-D4
to migrate more slowly (compare lane 1, 17C-oligo-A-D4 with lane 2,
17C-D4). 17C-D4 (lane 2) migrates more slowly than H by virtue of a
C-terminal extension of four aspartates (D4-tag). FIG. 6B.
Autoradiogram of an SDS-polyacrylamide gel containing
heteroheptamers formed by the assembly of a mixture of H and
17C-oligo-A-D4 monomers. Heptamers H.sub.7, H.sub.6(17C-oligo-A),
and H.sub.5(17C-oligo-A).sub.2 migrate in different gel bands due
to a shift caused by the D4-tag in the 17C-oligo-A-D4 subunits. The
modification with DNA does not change the electrophoretic mobility
of modified heptamers. The size of two molecular weight markers is
indicated.
[0083] FIG. 7A-1, FIG. 7A-2, FIG. 7B-1, FIG. 7B-2, FIG. 7C-1 and
FIG. 7C-2. An .alpha.HL pore modified with a single
DNA-oligonucleotide responds to individual binding events of
oligonucleotides of complementary sequence (FIG. 7A-2, FIG. 7B-2
and FIG. 7C-2). FIG. 7A-1. Representative single channel current
trace of H.sub.6(17C-oligo-A).sub.1 at a transmembrane potential of
+100 mV relative to the cis side of the bilayer in 2 M KCl, 12 mM
MgCl.sub.2, 5 mM Tris-HCl, pH 7.4. FIG. 7B-1. Representative trace
of the same channel as in FIG. 7A-1 in the presence of 67 nM
oligo-B (3'-GTAAGTGG-5'; SEQ ID NO:2) in the chamber at the cis
side of the protein. Negative current deflections (b) represent
individual binding events of oligo-B to the tethered oligo-A. The
short downward spike (s) in the trace is a translocation event of
oligo-B that did not bind to the tethered oligonucleotide. FIG.
7C-1. Trace of the same channel as in FIG. 7A-1 and FIG. 7B-1 with
67 nM oligo-B and 3.3 .mu.M oligo-A in the cis chamber. Excess
oligo-A hybridizes to oligo-B and thereby competes for the binding
of oligo-B to the tethered oligonucleotide. The short downward
spikes in the trace are translocation events of excess oligo-A
molecules through the pore.
[0084] FIG. 8A and FIG. 8B. Statistical summary of the binding
events of DNA oligonucleotides oligo-B to
H.sub.6(17C-oligo-A).sub.1. FIG. 8A. Definition of event lifetime
.tau..sub.off and event amplitude I.sub.E FIG. 8B. An event diagram
shows the event lifetime .tau..sub.off and event amplitude I.sub.E
for a single channel current recording of 3 min with 200 nM oligo-B
in the cis chamber. Each point in the diagram represents an
individual binding event of oligo-B to the tethered oligo-A in
H.sub.6(17C-oligo-A).sub.1.
[0085] FIG. 9. A DNA-nanopore detects a common mutation, which
confers resistance to the drug nevirapine in the reverse
transcriptase gene of HIV. The event diagram shows the event
lifetime, .tau..sub.off, and event amplitude, I.sub.E, for two
HIV-derived 30-nt DNA strands, oligo-181C and oligo-181Y,
interacting with H.sub.6(17C-5'-TGACAGAT-3' (SEQ ID NO:3)).sub.1.
Oligo-181C (SEQ ID NO:5) carries the drug resistance mutation and
forms a 8 bp duplex with the tethered oligonucleotide, whereas the
wild type oligo-181Y (SEQ ID NO:4) forms a duplex with a single
base mismatch. The dashed box indicates an event window populated
only by 181C binding events. The event diagram displays data from
one current recording for each of oligo-181C and oligo-181Y. The
DNA strand concentration was 670 nM and the recordings were 5 min
in duration. The study was repeated and gave the same result.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0086] Staphylococcal .alpha.-hemolysin (.alpha.HL) has been a
useful model system with which to test new approaches for
engineering membrane proteins and indeed proteins in general. The
.alpha.-Hemolysin toxin is secreted by Staphylococcus aureus as a
monomeric polypeptide of 293 amino acids. The monomer forms
heptameric, mushroom-shaped pores of known three-dimensional
structure in lipid bilayers (Song et al., 1996; Gouaux, 1998). The
opening of the channel on the cis side of the bilayer measures 29
.ANG. in diameter and broadens into a cavity .about.41 .ANG. across
(FIG. 1).
[0087] The .alpha.HL pore allows the passage of molecules of up to
.about.2000 Da across the bilayer (Fussle et al., 1981; Krasilnikov
et al., 1992; Bezrukov et al., 1996; Bezrukov et al., 1997) and is
only weakly selective for the charge of transported ions
(Menestrina, 1986). Besides being the object of a variety of
studies using mutagenesis (Walker et al., 1993; Walker and Bayley,
1995a; Braha et al., 1997; Cheley et al., 1999), .alpha.HL has been
subjected to protein engineering by targeted chemical modification.
These studies include the attachment of photocleavable protecting
groups to block assembly (Chang et al., 1995), the restoration of
activity to an inactive mutant by site-specific alkylation (Walker
and Bayley, 1995b), and the formation of channel blocker sites with
non-covalent molecular adapters (Gu et al., 1999).
[0088] The present invention describes new, targeted modifications
that are introduced, including the attachment of a synthetic
polymer chain, which may be attached at the surface or within the
lumen of the pore. Single channel electrical recording has been
used to observe current fluctuations associated with the attachment
of the polymer, a 3000 or 5000 Da polyethylene glycol (PEG)
molecule.
[0089] In addition to the protein engineering, the present
invention has applications in at least two areas: single molecule
detection and the development of biosensors. The examination and
manipulation of individual molecules is a thriving area of
research. Single molecule detection methods, which include
electrical recording (Hladky and Haydon, 1970; Sakmann and Neher,
1995), optical spectroscopy (Moerner and Orrit, 1999; Weiss, 1999)
and force measurements (Mehta et al., 1999), can provide structural
and functional information that is often difficult or impossible to
obtain by conventional techniques, which measure the properties of
large ensembles of molecules. Recent accomplishments include
observations of the movement of individual atoms and small
molecules (Gimzewski and Joachim, 1999), the movement of linear and
rotary motor proteins (Mehta et al., 1999), the turnover of
individual enzymes (Xie and Lu, 1999) and the unfolding and
refolding of proteins (Mehta et al., 1999).
[0090] In the area of biosensors, significant progress has been
made in developing protein channels and pores as sensor elements
(Ziegler and Gopel, 1998; Bayley, 1999; Hoffman, 1995; Urry, 1998;
Hubbel, 1999). According to this concept, analyte molecules
modulate the ionic current passing through the pores under a
transmembrane potential. For example, binding sites can be
engineered into pores expressly for capturing analyte molecules,
which act as partial channel blockers. Stochastic sensing, which
uses currents from single pores, is an especially attractive
prospect (Braha et al., 1997; Gu et al., 1999). The approach yields
both the concentration and identity of an analyte, the latter from
its distinctive current signature. By using .alpha.HL as a
stochastic sensing element, the inventors have succeeded in
detecting divalent metal ions (Braha et al., 1997) and a variety of
organic molecules (Gu et al., 1999). The present invention
represents a major step towards using responsive polymers for
stochastic sensing.
[0091] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention, and thus can be considered
to constitute preferred modes for its practice. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific
embodiments that are disclosed and still obtain a like or similar
result without departing from the spirit and scope of the
invention.
EXAMPLE 1
A Protein Pore with a Single Polymer Chain Tethered within the
Lumen
[0092] The present example describes a transmembrane protein pore
with a single 5000 Da polyethylene glycol (PEG) molecule attached
covalently within the channel lumen has been constructed from seven
staphylococcal .alpha.-hemolysin subunits. The modified heptamer is
stable and can be purified by electrophoresis in sodium dodecyl
sulfate, without dissociation of the subunits. The properties of
the modified pore were studied by single channel current recording.
The PEG molecule reduces the mean conductance of the pore by 18%,
as would be predicted from the effects of PEG on the conductivity
of bulk electrolytes. The recordings also reveal a variety of low
amplitude current fluctuations on a timescale of seconds, which are
tentatively ascribed to the reorganization of the PEG molecule
within the channel lumen and associated movements of the
polypeptide chain. Another class of events, comprising uniform
high-amplitude negative fluctuations in current with durations of
milliseconds, is ascribed to motions of the PEG molecule into one
of the channel entrances, thereby producing more extensive channel
block. When instead a 3000 Da PEG is attached within the channel
lumen, the single channel properties are changed in keeping with
the lower mass of the polymer. For example, the high-amplitude
fluctuations occur more frequently and are of shorter duration
suggesting that the 3000 Da PEG is more mobile than the 5000 Da
chain. The approach taken here is useful for the indirect
monitoring of polymer dynamics at the single molecule level. By
using polymers that respond to analytes, biosensors can be made
from the covalently modified pores.
[0093] A. Materials and Methods
[0094] 1. Proteins
[0095] The mutant .alpha.HL S106C gene was obtained by cassette
mutagenesis of the semisynthetic gene .alpha.HL-RL210. S106C also
contains the mutation Lys-8.fwdarw.Ala, and four conservative
replacements: Val-124.fwdarw.Leu, Gly-130.fwdarw.Ser,
Asn-139.fwdarw.Gln, and Ile-142.fwdarw.Leu. These changes, which
were introduced to prevent adventitious proteolysis (Walker and
Bayley, 1994) and to facilitate cassette mutagenesis (Cheley et
al., 1999), do not alter the electrical properties of the pore
(Cheley et al., 1999). The K8A and K8C constructs have already been
described (Walker and Bayley, 1995a; Walker and Bayley, 1994).
.sup.35S-labeled .alpha.HL polypeptides K8A, K8C and S106C were
obtained by expression in vitro (Walker et al., 1992a). To increase
the yield of protein, unlabeled methionine was included in the
translation mix (Cheley et al., 1999; Walker et al., 1992b). K8A
was used as the unmodified .alpha.HL subunit (H) in
heteroheptameric pores. Therefore, when a PEG-modified K8C subunit
is included in the heptamer, the charge at position 8 is not
altered as it would be if the wild-type protein were used.
[0096] 2. Chemical Modification of Single-Cysteine Mutants
[0097] K8C or S106C monomers were diluted 6-fold from the
translation mix into a buffer containing 10 mM MOPS-NaOH, pH 7.4,
150 mM NaCl, 0.5 mM EDTA and reduced with 0.5 mM DTT for 10 min,
before modification with 10 mM
monomethoxy-PEG5000-o-pyridyldisulfide (MePEG5K-OPSS, Shearwater
Polymers) for 20 min at 25.degree. C. Treatment with MePEG3K-OPSS
was performed in the same way.
[0098] 3. PEG-Modified Heteroheptameric Pores
[0099] The modified K8C or S106C monomers were mixed with
unmodified K8A .alpha.HL monomers (H) in various initial ratios and
the mixed subunits were allowed to assemble on rabbit erythrocyte
membranes (Walker et al., 1992a). To analyze which heteromers had
been formed with PEG5000-modified subunits, the membranes were
recovered by centrifugation, dissolved in gel loading buffer and
loaded, without heating, onto a 6% SDS-polyacrylamide gel (5-cm
long, 0.75 mm thick, Miniprotean II, Biorad). To determine subunit
ratios, the samples were heated and analyzed in a 10%
SDS-polyacrylamide gel. The dried gels were subjected to
phosphorimager or autoradiographic analysis.
[0100] To prepare the heteromers H.sub.6S 106C-PEG5K.sub.1 and
H.sub.6K8C-PEG5K.sub.1 for bilayer recording, subunits were
assembled in the ratios: H: S106C-PEG5K, 3:4; H: K8C-PEG5K, 6:1.
Samples corresponding to a total of 12 .mu.l of translation mix per
lane were loaded, without heating, onto 6% SDS-polyacrylamide gels
(35 cm long, 1.5 mm thick), which were run for 18 h at 150 V. The
unfixed gels were vacuum dried at 50.degree. C. onto Whatman 3 MM
paper and the protein bands located by autoradiography. The desired
bands were cut from the gel and rehydrated in water (300 .mu.l per
lane). After removal of the paper, the gel was crushed in the water
and the mixture was left to stand for 10 h at 4.degree. C. A
solution of PEG-modified heptamers was then obtained by removal of
the acrylamide with a cellulose acetate filter (0.2 .mu.m diameter,
Rainin, Woburn, Mass.). PEG3000 produced no gel shift in
heteroheptamers modified at position 106. Therefore, to make
H.sub.6S106C-PEG3K.sub.1, unmodified subunits (H) and S 106-PEG3K
were assembled together in a ratio of 4:3. After preparative
SDS-polyacrylamide gel electrophoresis, as described above, the
major band contained .about.40% H.sub.6S106C-PEG3K.sub.1 and
.about.60% H.sub.7, as deduced from the subunit ratio determined by
analytical SDS-polyacrylamide gel electrophoresis and by the
results of single channel recording.
[0101] 4. Planar Bilayer Recordings
[0102] Planar lipid membrane recordings were carried out at
24.+-.1.degree. C. (Braha et al., 1997; Gu et al., 1999; Montal and
Mueller, 1972). The cis and trans chambers, each of 2 ml, were
separated by a 25-.mu.m-thick Teflon septum (Goodfellow
Corporation, Malvern, Pa.). An aperture in the septum
(.about.150-.mu.m diameter) was pretreated with 10% (v/v)
hexadecane (Aldrich Chemical Co., Milwaukee, Wis.) in n-pentane
(Burdick & Jackson, Muskegon, Mich.). The electrolyte in both
chambers was 300 mM KCl, 5 mM Tris-HCl, pH 7.0, containing 100
.mu.M EDTA. A bilayer membrane was formed (Montal and Mueller,
1972) with 1,2-diphytanoyl-sn-glycerophosphocholine (Avanti Polar
Lipids, Alabaster, Ala.). .alpha.HL pores were introduced by adding
gel-purified heptamers (1 to 6 .mu.l) to the cis chamber, to give a
final protein concentration of 0.05-0.3 ng/ml. The cis solution was
stirred for 5-30 min until a single pore inserted (8-10 pA step at
-40 mV).
[0103] Currents were recorded by using a patch clamp amplifier
(Axopatch 200B, Axon Instruments, Foster City, Calif.) connected to
the chambers by Ag/AgCl electrodes, and monitored with an
oscilloscope (Model TAS250, Tektronix, Heenveen, Netherlands). The
cis chamber was grounded and a negative current (downward
deflection) represents positive charge moving from the cis to trans
side. A Pentium PC equipped with a DigiData 1200 A/D converter
(Axon Instruments, Foster City, Calif.) and a strip chart recorder
(BD112, Kipp & Zonen, Bohemia, N.Y.) were used for data
acquisition. For the most of the studies, the current traces were
low-pass filtered with a built-in 4-pole Bessel filter at a
frequency of 5 kHz and stored by using a digital audio tape
recorder (DAS-75, Dagan Corporation, Minneapolis, Minn.). For
computer analysis, the data were further filtered with a 8-pole
Bessel filter at frequencies in the range 100-3000 Hz and sampled
at 10 kHz. For display and statistical analysis, the FETCHAN and
pSTAT programs were used, both from the software package pCLAMP7
(Axon Instruments) and Origin (Microcal Software, Northampton,
Mass.). In the case of H.sub.6S106C-PEG3000.sub.1, a different
protocol was used to allow the examination of rapid events. The
signal, filtered at 10 kHz, was recorded on digital audio tape. For
analysis, the signal was filtered at 7 kHz with a low-pass Bessel
filter and sampled at 333 kHz for computer acquisition using a
threshold protocol in the CLAMPEX program from pCLAMP7.
[0104] Current amplitudes and life-times of the various conductance
states are given as mean values (.+-.s.d.). The value of "n"
denotes the number of studies analyzed or, when indicated, the
number of events examined.
[0105] B. Results
[0106] 1. Engineering an .alpha.HL Pore with a Single PEG Molecule
Tethered within the Central Cavity
[0107] In previous work, the inventors modified the lumen of the
heptameric .alpha.HL pore by direct genetic engineering (Braha et
al., 1997) and by non-covalent modification with molecular adapters
(Gu et al., 1999). A principal goal has been to create protein
pores that respond to various analytes and can thus be employed as
components of biosensors, especially stochastic sensors in which
single molecule detection is used. The inventors reasoned that an
additional way to modify the interior of the protein would be by
the covalent attachment of responsive molecules. Responsive
polymers attached at specific sites in proteins have demonstrated
potential. They have, for example, been used to modulate the
affinity of streptavidin for biotin (Stayton et al., 1995; Ding et
al., 1999).
[0108] To assemble an exemplary structure with an internal polymer,
a 5000 Da PEG molecule was placed within (or largely within) the
central cavity of the cap domain of the .alpha.HL pore. The
calculated volume of PEG5000 based on the experimental hydrated
radius (Krasilnikov et al., 1992; Scherrer and Gerhardt, 1971) is
comparable to the volume of the cavity, which is 36,000
.ANG..sup.3, assuming a sphere of diameter 41 .ANG.. The Flory
dimension (RF) of PEG5000 of 60 .ANG. (RF=aN.sup.0.6, where N,
number of polymer repeat units; a, effective repeat length (3.5
.ANG.)) (Doi, 1996), which has been variously interpreted as a
radius or diameter (Kenworthy et al., 1995; Rex et al., 1998),
gives a larger volume for PEG5000, but this may be unrealistic (Rex
et al., 1998).
[0109] 2. Heptamers Containing up to Seven PEG Molecules Can be
Made by Derivatization at an External Site on the .alpha.HL
Pore
[0110] The PEG conjugation chemistry and a means to analyze the
assembly of derivatized subunits were tested with the .alpha.HL
single-cysteine mutant K8C. In this case, the PEG chains would end
up located near the surface of the heptamer, at the cis mouth, and
therefore be unlikely to interfere with assembly (FIG. 1) (Walker
et al., 1995; Olson et al., 1999). .sup.35S-labeled K8C
polypeptides were obtained by expression in vitro (Walker et al.,
1992a) and modified with monomethoxy-PEG5000-o-pyri- dyldisulfide
(MePEG5K-OPSS). The PEG is attached to the protein through a
disulfide bond that is readily cleaved with dithiothreitol (DTT).
This was shown by extracting protein from an SDS-polyacrylamide gel
band generated with a low fraction of S106C-PEG5K subunits (band
a), heating to 95.degree. C. for 5 min and separating in a second
SDS-polyacrylamide gel. The bands were quantified by phosphorimager
analysis. Analyzing lanes of heated band a (H.sub.6S
106C-PEG5K.sub.1); heated and DTT-treated band a
(H.sub.6S106C-PEG5K.sub.1); PEG-modified S106C monomer before
heptamerization; and PEG-modified S106C monomer after DTT treatment
gave rise to the present finding.
[0111] Analysis of H/K8C-PEG5K heteroheptamers by
SDS-polyacrylamide gel electrophoresis and autoradiography revealed
seven bands, a-g. The subunits were .sup.35S-labeled during in
vitro expression. K8C and S106C were reduced with 0.5 mM DTT and
reacted with 10 mM MePEG5K-OPSS for 20 min at 25.degree. C. The
modified K8C polypeptides were mixed with unmodified
.sup.35S-labeled .alpha.HL monomers in all initial ratios between
0:7 and 7:0 the mixed subunits were allowed to assemble on rabbit
erythrocyte membranes (Walker et al., 1992a). Following
heptamerization on the erythrocyte membranes, autoradiograms were
obtained. The number of PEG-modified subunits present in the
heptamers in each band are: K8C a, 1;b,0and2; c, 3; d, 4; e, 5; f,
6; g, 7; S106C a, 1 and 2; b, 0.
[0112] The protein in each band was eluted and heated to dissociate
the subunits. Further electrophoresis revealed the ratio of
subunits in each band, which showed that all eight possible
combinations (Braha et al., 1997) of unmodified .alpha.HL (H) and
K8C-PEG5K subunits had been formed (both H.sub.7 and
H.sub.5K8C-PEG5K.sub.2 are in band b). Interestingly, the
electrophoretic mobility of heptamers containing a single PEG5000
molecule was increased (band a), while the mobilities of heptamers
containing three or more attached PEG5000s (bands c-g) were
decreased. Heptamers with two to five PEGs exist in more than one
form: the outcome of permutation about the central seven-fold axis
(Braha et al., 1997). Bands c and d were distinctly broadened, most
likely reflecting an incomplete separation of the five permutations
each of H.sub.4K8C-PEG5K.sub.3 and H.sub.3K8C-PEG5K.sub.4. These
studies show that heptamers containing PEG5000 in an external
location can be assembled and all eight combinations of subunits
can be identified by analytical SDS-polyacrylamide gel
electrophoresis.
[0113] 3. A Single PEG Chain can be Attached at a Point within the
Central Cavity of the .alpha.HL Pore
[0114] The same approach was used to make heptamers containing
PEG5000 attached covalently to a cysteine residue within the large
central cavity (position 106 in the polypeptide chain). Modified
S106C monomers were mixed with unmodified .alpha.HL monomers (H)
and allowed to assemble on rabbit erythrocyte membranes (Walker et
al., 1992a). By contrast with the results with K8C-PEG5K, the
analysis of H/S106C-PEG5K heteroheptamers revealed only two major
bands, suggesting that the formation of SDS-resistant heptamers
containing several modified S106C subunits is disfavored due to
crowding of the PEG chains within the central cavity. The measured
ratio of unaltered .alpha.HL (H) to S106C-PEG5K subunits in band a
was 5.9:1, and therefore the oligomer in the band must contain six
unmodified subunits (H) and one S106-PEG5K subunit, namely
H.sub.6S106C-PEG5K.sub.1. At high S106C-PEG5K:H ratios in the
assembly mix, a faint band was seen above band a and may represent
H.sub.5S106C-PEG5K.sub.2.
[0115] This study shows that H.sub.6S106C-PEG5K.sub.1 can be formed
and that it is stable as a heptamer at room temperature in the
denaturing detergent SDS. Therefore, the cavity might be large
enough to contain a PEG5000 molecule. Any hydration pressure that
develops in packing the PEG internally would have to be
insufficient to dissociate the heptamer. Alternatively, unfavorable
interactions would be reduced if part of the PEG chain were
extruded through the cis or trans entrance. Studies with PEG3000
support the latter interpretation. The electrophoretic mobility of
H.sub.6S106C-PEG3K.sub.1 is the same as that of the unmodified
heptamer (H.sub.7), suggesting that the hydrodynamic properties of
the heptamer are unaltered and the PEG3000 chain is largely
contained within the cavity. By contrast, the altered
electrophoretic mobility of H.sub.6S106C-PEG5K.sub.1 suggests that
part of the PEG5000 chain is exposed to solvent.
[0116] 4. Ionic Current Through Individual PEG-Modified .alpha.HL
Pores
[0117] The cavity in the .alpha.HL pore lies on the conductive
pathway and so the incorporation of a PEG molecule at position 106
would be expected to alter the current that flows through the pore
in response to an applied potential. This was tested by performing
single channel current measurements on H.sub.6S 106C-PEG5K.sub.1
eluted from preparative gels (Braha et al., 1997). The control
homoheptamer (H.sub.7) exhibits a uniform unitary conductance state
(Table 1; Braha et al., 1997; Cheley et al., 1999; Gu et al.,
1999).
[0118] By contrast, the PEG-modified .alpha.HL pore showed dynamic
gating behaviors centered around a main conductance state of
diminished amplitude compared with H.sub.7 (FIG. 2A and FIG. 2B,
Table 1). The mean of the main peaks in the conductance histograms
for H.sub.6S 106C-PEG5K.sub.1 was 221.+-.9 pS (n=7) at +100 mV, in
symmetric 300 mM KCl, 5 mM Tris-HCl (pH 7.00), 100 .mu.M EDTA, a
reduction of 18% over the value for H.sub.7 (Table 1). Two distinct
subconductance behaviors were observed: relatively long-lived low
amplitude fluctuations (mean life-time, 14.5.+-.1.7 s, n=27 events)
and short-lived higher amplitude negative spikes (mean life-time,
13.7.+-.2.2 ms, n=87 events; frequency of occurrence 0.20.+-.0.02
s.sup.-1). Typically there were three to five low amplitude states
separated by .DELTA.g=10.+-.1 pS. The excess current noise of the
low amplitude states over H.sub.7 single channel noise was modest,
<5% broadening at half-width of the individual peaks in current
histograms (filtered at 5 kHz), denoting an absence of unusual
higher frequency events within these states. In two cases (out of
seven that were analyzed), the typical low amplitude behavior (FIG.
2A and FIG. 2B) was preceded by two-state behavior with faster
kinetics (.DELTA.g=7.+-.1 pS; mean life-time of lower conductance
state 709.+-.81 ms, n=19 events; frequency of occurrence 0.32
s.sup.-1). The faster transitions lasted for five and eight minutes
before irreversible (>15 min) conversion to the typical
behavior.
[0119] When the PEG was cleaved from the pore, by reduction of the
disulfide bond with DTT, the current increased to a value similar
to that observed with H.sub.7 (FIG. 2C and FIG. 2D), after a lag
period of 18-25 minutes (n=4). Long-lived low amplitude
fluctuations were also observed with H.sub.6K8C-PEG5K.sub.1,
centered around a mean conductance of 244.+-.19 pS (n=8) (Table 1),
which is higher than the value for H.sub.6S106C-PEG5K.sub.1. There
were typically three to five substates with life-times ranging from
a few tens of milliseconds to hundreds of milliseconds. .DELTA.g
values (8 pS to 50 pS) were often larger than those of the
substates of H.sub.6S 106C-PEG5K.sub.1. Strikingly, the short-lived
high amplitude spikes were completely absent.
1TABLE 1 Conduction properties of unaltered (H.sub.7) and
PEG-modified .alpha.HL pores.sup.a After PEG Mean Conductance
Substates Cleavage Channel (pS).sup.b (pS).sup.c (pS).sup.d H.sub.7
268 .+-. 5 (11) none n.a. H.sub.6K8C-PEG5K.sub.1 244 .+-. 19 (8) 17
.+-. 4, d (8) 270 .+-. 5 (8) H.sub.6S106C-PEG5K.sub.1 221 .+-. 9
(7) 10 .+-. 1, d; 267 .+-. 7 (4) 120 .+-. 7, s (7)
H.sub.6S106C-PEG3K.sub.1 237 .+-. 4 (5) 128 .+-. 3, s (5).sup.e 272
.+-. 3 (5) .sup.aStudies were performed at a transmembrane
potential of +100 mV with 300 mM KCl, 5 mM Tris-HCl (pH = 7.00),
100 .mu.M EDTA in both chambers. The number of studies analyzed is
shown in parentheses. .sup.bThe mean (.+-. s.d.) of the mean
conductance values from the major peaks of all-points histograms
(e.g., FIG. 2A and FIG. 2B) was calculated. .sup.cThe mean change
in conductance (.DELTA.g .+-. s.d.) between the most common
substates. d, discrete low amplitude events; s, negative current
spikes. .sup.dThe conductance was determined after treatment with
DTT as described in the text. When a step to an increased steady
current was observed, the PEG was assumed to have left the cavity.
In the case of H.sub.6S106C-PEG5K.sub.1, this took 18-25 min with
10-15 mM DTT. .sup.eIn the case of H.sub.6S106C-PEG3K.sub.1 low
amplitude events were seen on one occasion in six studies and are
not recorded in Table 1.
[0120] Single channel current measurements were also performed on
H.sub.6S106C-PEG3K.sub.1. The preparation was contaminated with
H.sub.7 channels and bilayers containing them were disregarded. The
mean unitary conductance of H.sub.6S106C-PEG3K.sub.1 was 237.+-.4
pS (n=5), somewhat higher than that of H.sub.6S106C-PEG5K.sub.1.
Low amplitude events were seen in only one of the six single
channels that were observed. The short-lived higher amplitude
negative spikes (mean life-time, 132.+-.10 .mu.s, n=5) were shorter
than those seen with H.sub.6S106C-PEG5K.sub.1, were of a similar
amplitude (128.+-.3 pS, n=5) and occurred more often (26.+-.10 s
n=5). After treatment with 10 mM DTT, the PEG3000 molecule exited
the cavity after 15 sec to 4 min (n=5), far more rapidly than
PEG5000.
[0121] 5. Interpretation of Current Fluctuations in PEG-Modified
.alpha.HL Pores
[0122] The current fluctuations observed when a PEG molecule of
5000 Da is anchored within the central cavity of .alpha.HL are
remarkable, compared for example with the single invariant
conductance state observed when a more rigid cyclodextrin is bound
non-covalently within the channel lumen (Gu et al., 1999). While
switching between defined conductance states, rather than a
continuum of states, was surprising, the following explanations
account for the four main behaviors observed with
H.sub.6S106C-PEG5K.sub.1.
[0123] First, the reduction in current carried by the main
conductance states (FIG. 2A and FIG. 2B) most likely arises from
changes in the properties of the electrolyte in the cavity caused
by the presence of the PEG molecule. The unaltered H.sub.7 pore is
ohmic and only weakly ion selective, suggesting that ion transport
is through a channel filled with electrolyte with properties close
to that of bulk solution. The volume of the cavity is .about.36,000
.ANG..sup.3. Were the entire PEG5000 molecule within the cavity,
its "concentration" would be .about.23%. At this concentration, the
conductivity of a solution of 100 mM KCl would be reduced by 48%
(Krasilnikov et al., 1992; Bezrukov and Vodyanoy, 1993), far
greater than the 18% decrease in single channel conductance
observed. Nevertheless, the result is reasonable given that a
hydrated PEG molecule cannot occlude the entire conductive pathway,
from one entrance to another, and that the PEG chain may lie partly
outside the lumen.
[0124] Second, the slow low-amplitude fluctuations in current can
be ascribed to rearrangements of the PEG5000 molecule within the
cavity correlated with associated movements of the protein (the
fluctuations do not occur with unmodified H.sub.7). Protein motions
can occur over a wide range of time scales (Kay, 1998) and recently
they have been observed at the single molecule level. For example,
substrate fluorescence revealed fluctuations in a rate constant of
cholesterol oxidase with a correlation time of about one second
(Xie and Lu, 1999; Lu et al., 1998) and FRET measurements revealed
fluctuations in the conformation of staphylococcal nuclease with an
average time constant of 41 ms, which was increased to 133 ms with
substrate bound (Ha et al., 1999).
[0125] The third phenomenon, the very slow (minutes)
interconversion between related states, is also likely to be
related to rearrangement of the PEG and an associated adjustment of
the protein. Long-lived conformational states in proteins have been
encountered previously (Xie and Lu, 1999; Xue and Yeung, 1995; Tan
and Yeung, 1997). Alternative explanations are that the current
fluctuations arise entirely from movements of the PEG chain or, at
the other extreme, that the fluctuations can be ascribed solely to
movements of the protein destabilized by the presence of the PEG.
It may be possible to distinguish these possibilities
experimentally. For example, if the motion of the PEG were
uncoupled from the motion of the protein, the frequency and
duration of the fluctuations would be independent of the point of
attachment of the PEG within the central cavity.
[0126] The fourth phenomenon, the short-lived, high amplitude,
negative current spikes, may represent the partial looping of the
PEG5000 chain into the transmembrane barrel or into the cis
opening. The millisecond duration of the states is far longer than
the dwell time of free PEG molecules within the .alpha.HL pore
(Bezrukov et al., 1996; Bezrukov and Kasianowicz, 1997; Bezrukov et
al., 1994), but of the same magnitude as relaxation times of PEGs
tethered to supported bilayers (Wong et al., 1997; Sheth and
Leckband, 1997). The uniform amplitude of these events (FIG. 2A and
FIG. 2B, histogram peak "s") suggests that one or the other of the
two possible looping events predominates. The pore always returns
to the conductance state from which it undergoes a high amplitude
excursion (n=55 events), further suggesting that the low amplitude
events involve protein conformational changes as well as PEG
reorganization. If instead the low amplitude events purely
represented states of the PEG molecule, the PEG would have to
retain "memory" of them during the larger excursions.
[0127] The results obtained with PEG3000 are consistent with the
interpretation of the behavior of H.sub.6S 106C-PEG5K.sub.1 as
outlined above. The mean conductance of H.sub.6S106C-PEG3K.sub.1 is
only 12% lower than the unmodified pore (Table 1), in keeping with
the lower mass of PEG3000 compared with PEG5000. The lower
"concentration" of PEG within the H.sub.6S106C-PEG3K.sub.1 pore
might also explain the faster release of the PEG chain by DTT. The
high amplitude spikes occur about 100 times more often with
H.sub.6S106C-PEG3K.sub.1, compared to H.sub.6S106C-PEG5K.sub.1, and
are about 100 times shorter in duration, suggesting that PEG3000 is
more mobile than PEG5000 within the cavity. Finally, although the
interpretation of the low amplitude events is tentative, their
rarity in the case of H.sub.6S106C-PEG3K.sub.1 suggests that
polymer motion is less readily coupled to protein movement than it
is in H.sub.6S106C-PEG5K.sub.1.
[0128] In summary, this Example shows that a multisubunit protein,
a heptameric transmembrane pore, can be constructed with a
synthetic polymer tethered within an internal cavity. It is not
currently known whether the entire polymer chain is encapsulated.
Certainly, the fluctuations of current passing through a single
pore in a transmembrane potential suggest that the PEG chain is
flexible and may therefore sample the external solvent. This
suggests that current recording is a useful tool for monitoring the
dynamic properties of PEG and other polymers, including
oligopeptides and oligonucleotides, at the single molecule level.
Further, by using polymers that respond to analytes, it is possible
to make biosensors (Braha et al., 1997; Gu et al., 1999) based on
this new class of engineered pores. This does not depend on a
detailed interpretation of the current fluctuations, only that they
are modulated by analytes in a concentration dependent manner and
at the same time provide analyte-specific signatures (Braha et al.,
1997; Gu et al., 1999).
EXAMPLE 2
Transmembrane Movement of a Single Polymer Chain Tethered within a
Protein Pore
[0129] In this Example, a protein-based structure is described in
which a single functionalized polymer chain is attached at a
defined site within the central cavity of a transmembrane pore
built by the self-assembly of staphylococcal .alpha.-hemolysin
subunits. The untethered end of the chain is capable of
translocation across the membrane, from one entrance of the pore to
the other, a distance of at least 10 nm. Hence, the engineered pore
comprises an unusual nanostructure with a moveable part. In
addition, the pore can be used to examine polymer motions on the
microsecond timescale. Furthermore, it is demonstrated that the
pore acts as a new type of biosensor element in which
polymer-ligand conjugates are covalently attached to protein pores.
A change in the ionic current carried by the pore occurs when a
protein analyte binds to the functionalized polymer.
[0130] A. Materials and Methods
[0131] 1. Formation of Heteromeric .alpha.HL Pores Containing
Covalently-Attached PEG-Biotin
[0132] The mutant .alpha.HL genes, S106C, K8C and K8A, have been
described previously (Walker and Bayley, 1994; Walker and Bayler,
1995a; Cheley et al., 1999). .sup.35S-labeled S106C, K8C and K8A
polypeptides were obtained by in vitro transcription and
translation (Walker et al., 1992). To obtain a higher yield of
protein, unlabeled methionine was included in the expression mix
(Cheley et al., 1999; Walker et al., 1992). .alpha.HL monomers
S106C and K8C were covalently modified by five-fold dilution of the
translation mix into 10 mM MOPS, pH 7.0 (NaOH), containing 150 mM
NaCl, 0.5 mM EDTA, reduction with 0.5 mM DTT for 5 min and reaction
with 10 mM with biotin-PEG3400-maleimide (Shearwater Polymers,
Huntsville, Ala., USA) for 10 min and room temperature. Modified
subunits were mixed in various ratios with unmodified K8A .alpha.HL
monomers (H) and allowed to assemble into heteroheptamers on rabbit
erythrocyte membranes (Cheley et al., 1999; Walker et al., 1992).
.alpha.HL heptamers are stable in SDS unless heated (Walker and
Bayley, 1995b) and were analyzed by SDS-PAGE and autoradiography.
Where indicated a large excess (8.5 mg/ml) of streptavidin (S-4762,
Sigma) was added prior to analysis. Heptameric pores for electrical
recording were obtained from preparative gels (Braha, 1997). The
ratios of unmodified to modified subunits in these purified
proteins were determined by heating the proteins to 95.degree. C.
and separating the dissociated subunits in a second, analytical gel
(Example 1).
[0133] 2. Bilayer Recording
[0134] The formation of bilayers of
1,2-diphytanoyl-sn-glycerophosphocholi- ne (Avanti Polar Lipids),
the insertion of heptameric .alpha.HL pores into them, and
single-channel recording have been described (Braha, 1997; Montal
and Mueller, 1972). Both the cis and trans chambers of the
apparatus contained 300 mM KCl, 5 mM Tris-HCl, pH 7.00, with 100
.mu.M EDTA. .alpha.HL pores were added to the cis chamber, at a
concentration of 0.05-0.3 ng/ml. The solution was stirred for
.about.15 minutes until a single channel inserted into the bilayer.
Currents were recorded by using a patch clamp amplifier (Axopatch
200B, Axon Instruments) at a holding potential of +100 mV (with the
cis side grounded). The signals were low-passed filtered with a
built-in 4-pole Bessel filter at a frequency of 10 kHz and recorded
on digital audio tape recorder. For computer analysis, the signals
were further filtered with an 8-pole Bessel filter at frequencies
in the range 1-4 kHz and sampled at 20 kHz, unless otherwise
specified.
[0135] Statistical analysis was carried out by using the FETCHAN
and pSTAT programs, both from the software package pCLAMP7 (Axon
Instruments), and Origin (Microcal Software). Measurements are
given as mean.+-.s.d. k.sub.off.sup.cis values were obtained from
1/.tau..sub.off, determined from dwell-time histograms.
k'.sub.on.sup.cis values were determined from the concentration
dependence of 1/.tau..sub.on. Because relatively few events were
recorded, k.sub.off.sup.trans and k'.sub.on.sup.trans values were
determined from mean dwell times and mean inter-event intervals.
For cis events, `n` refers to the number of studies. For trans
events, `n` refers to the number of events.
[0136] 3. Molecular Graphics
[0137] The molecular models of streptavidin (1swd.pdb) and
.alpha.-hemolysin (7ahl.pdb) were generated with SPOCK 6.3 software
(Christopher, 1998).
[0138] B. Results
[0139] For applications in biotechnology, engineered versions of
.alpha.HL have been prepared that contain built-in triggers and
switches actuated by physical, chemical and biochemical stimuli
(Chang et al., 1995; Panchal et al., 1996; Russo et al., 1977). In
addition, genetically engineered .alpha.HL and .alpha.HL in
combination with non-covalent molecular adapters have been used as
stochastic sensor elements to monitor individual metal ions (Braha
et al., 1997) and small organic molecules (Gu et al., 1999).
[0140] The interactions of polymers with various pores including
.alpha.HL have been studied extensively (Bezrukov et al., 1994;
Bezrukov et al., 1996; Merzlyak et al., 1999). Especially appealing
is the use of electrical recording to count polyanionic DNA and RNA
strands as they move through the .alpha.HL pore in a transmembrane
potential (Kasianowicz et al, 1996; Akeson et al., 1999).
Information about the length and base composition of the
polynucleotides is obtained by monitoring the electrical current
while the polymers are in the channel. Single neutral polyethylene
glycol molecules of 3400 Da have now been observed, by tethering
them within the lumen of the .alpha.HL pore. By measuring the
current passing through the pore, the structural dynamics of the
polymer chain can be monitored. The polymer contains a biotinyl
group at the untethered end and by using genetically engineered
streptavidin mutants with a weakened binding affinity (Sano and
Cantor, 1995; Chilkoti et al., 1995a), the appearance of the biotin
on both the cis and trans side of the membrane can be monitored
during a single study.
[0141] A preparation of heptameric .alpha.HL pores was made, which
was enriched in molecules containing six unmodified subunits and
one subunit covalently modified within the central cavity with
PEG-biotin. .sup.35S-labeled PEG-biotin-modified heteroheptameric
.alpha.HL pores were analyzed by SDS-PAGE and autoradiography.
Where required, the samples were treated with excess
WT-streptavidin before electrophoresis. Ratios of unmodified and
modified subunits in the initial assembly mix included 6:1, 1:6 and
4:3. The components of each band could be inferred from band shifts
after streptavidin treatments and dissociation of the subunits by
heating followed by additional electrophoresis.
[0142] Heptamers obtained by co-assembly (Braha et al., 1997;
Example 1) of unmodified .alpha.HL and the mutant S106C, which had
been reacted with biotin-PEG3400-maleimide, co-migrated with
unmodified heptamers upon SDS-polyacrylamide gel electrophoresis.
The addition of streptavidin (60 kDa) before electrophoresis caused
.about.75% of the material to migrate more slowly. This more slowly
migrating material contained heteroheptamers with six unmodified
and one modified subunit (H.sub.6 106C-PEG-biotin.sub.1), according
to a second analysis by SDS-PAGE performed after heating the sample
to dissociate the subunits. No band corresponding to
heteroheptamers with two modified subunits was detected in the
streptavidin-treated assembly products, but such a band was present
in a preparation of heteroheptamers derived from the mutant K8C
modified with PEG-biotin.
[0143] Position 8 is near the cis entrance to the channel lumen and
PEG molecules on all seven subunits can be tolerated at this
position (Example 1). Because H.sub.6 106C-PEG-biotin.sub.1
co-migrates with unmodified heptamers (H.sub.7), it is inferred
that the bulk of the PEG chain remains within the central cavity of
the pore where it has no appreciable effect on electrophoretic
mobility. By contrast, a single PEGS000 chain attached at position
106 increases the electrophoretic mobility of the heptamer and must
protrude into the extralumenal solvent (Example 1).
[0144] The single channel properties of H.sub.6
106C-PEG-biotin.sub.1 were examined by planar bilayer recording.
Currents arising from the contaminating H.sub.7 pores, which had
the same conductance as control H.sub.7 pores (271.+-.3 pS, n=14),
were disregarded. The results of the bilayer studies are shown in
Table 2, Table 3, Table 4 and Table 5 below.
2TABLE 2 Conduction Properties of Unaltered (H.sub.7) and
PEG-modified .alpha.HL Pores.sup.a Short-Lived and High-Amplitude
Closures Mean Frequency of Conductance Amplitude Occurrence Channel
(pS).sup.b Life Time (.mu.s) (pS) (s.sup.-1) H.sub.7 271 .+-. 3
(14) NA NA NA H.sub.6S106C- 229 .+-. 4 (17) 130 .+-. 7 (17) 121
.+-. 4 (17) 37 .+-. 6 (17) PEG-biotin.sub.1 H.sub.6S106C- 237 .+-.
4 (5) 132 .+-. 10 (5) 128 .+-. 3 (5) 26 .+-. 10 (5) PEG3K.sub.1
.sup.aStudies were carried out with symmetrical electrolyte
solutions (300 mM KCl, 5 mM Tris-HCl, 100 .mu.M EDTA, pH = 7.00
.+-. 0.01) at an applied transmembrane potential of +100 mV. The
number of studies analyzed is indicated in parentheses. .sup.bThe
mean (.+-. s.d.) of the mean conductance values were derived from
the major peaks of all-point single-channel current histograms of
individual studies.
[0145]
3TABLE 3 Conduction Properties of H.sub.6S106C-PEG-biotin.sub.1
Upon Addition of Wild-Type Streptavidin to the cis or trans
Chamber.sup.a Mean Conductance Relative Chamber Before Addition of
After Addition of Channel Added.sup.b Streptavidin (pS)
Streptavidin (pS) Block.sup.c cis 221 .+-. 12 (4) 224 .+-. 12 (4)
0% trans 230 .+-. 4 (5) 110 .+-. 9(5) 51.3 .+-. 3.3% .sup.aStudies
were carried out with symmetrical electrolyte solutions (300 mM
KCl, 5 mM Tris-HCl, 100 .mu.M EDTA, pH = 7.00 .+-. 0.01) at an
applied transmembrane potential of +100 mV. All numbers from the
table represent .+-. s.d. For cis events, the number in parentheses
refers to the number of studies. For trans events, number in
parentheses refers to the number of events. .sup.bThe final
concentration of wild-type streptavidin in the cis or trans chamber
covered the range of 12-72 nM. .sup.cRelative channel block is the
channel block accompanied by the binding of streptavidin relative
to the mean conductance before addition of streptavidin.
[0146]
4TABLE 4 Conduction Properties of H.sub.6S106C-PEG-biotin.sub.1
Upon Addition of Streptavidin Mutant W120A to the cis or trans
Chamber.sup.a Chamber Relative Channel Added.sup.b t.sub.on (ms)
t.sub.off (ms) Block.sup.c cis 4750 .+-. 230 (6) 3180 .+-. 140 (6)
0% trans 318800 .+-. 54000 (5) 667 .+-. 476 (5) 52.4 .+-. 4.1%
.sup.aStudies were carried out with symmetrical electrolyte
solutions (300 mM KCl, 5 mM Tris-HCl, 100 .mu.M EDTA, pH = 7.00
.+-. 0.01) at an applied transmembrane potential of +100 mV. All
numbers from the table represent .+-. s.d. For cis events, the
number in parentheses refers to the number of studies. For trans
events, number in parentheses refers to the number of events.
.sup.bThe protein concentration of W120A streptavidin in the cis or
trans chamber was 7.25 nM. .sup.cRelative channel block is the
channel block accompanied by the binding of streptavidin relative
to the mean conductance before addition of streptavidin.
[0147]
5TABLE 5 Conduction Properties of H.sub.6S106C-PEG-biotin.sub.1
Upon Addition of Anti-Biotin mAb to the cis or trans Chamber.sup.a
Chamber Relative Channel Added.sup.b t.sub.on (ms) t.sub.off (ms)
Block.sup.c cis 4750 .+-. 230 (6) 3180 .+-. 140 (6) 0% trans 318800
.+-. 54000 (5) 677 .+-. 476 (5) 52.4 .+-. 4.1% .sup.aStudies were
carried out with symmetrical electrolyte solutions (300 mM KCl, 5
mM Tris-HCl, 100 .mu.M EDTA, pH = 7.00 .+-. 0.01) at an applied
transmembrane potential of +100 mV. All numbers from the table
represent .+-. s.d. For cis events, the number in parentheses
refers to the number of studies. For trans events, number in
parentheses refers to the number of events. .sup.bThe protein
concentration of anti-biotin mAb in the cis chamber was 1.8 nM, the
concentration in the trans chamber was 5.8 nM. .sup.cRelative
channel block is the channel block accompanied by the binding of
streptavidin relative to the mean conductance before addition of
streptavidin.
[0148] The H.sub.6106C-PEG-biotin.sub.1 channels exhibited a
reduced unitary conductance state (229.+-.4 pS, n=17) decorated
with short-lived high-amplitude negative spikes (mean life time,
130.+-.7 .mu.s, amplitude, 121.+-.4 pS, n=17), which occurred at a
high frequency (37.+-.6 s.sup.-1, n=17) (FIG. 3A and FIG. 3B). Both
the reduced conductance and the spikes were associated with the PEG
chain (rather than the biotin), as H.sub.6106C-PEG3K.sub.1
channels, which contain a PEG of 3000 Da without the biotinyl
group, showed very similar characteristics (unitary conductance
237.+-.4 pS; spike life time, 132.+-.10 .mu.s; amplitude, 128.+-.3
pS; frequency, 26.+-.10 s.sup.-1, n=5). When 12 nM wild-type (WT)
streptavidin was added to the cis side of a bilayer containing a
H.sub.6106C-PEG-biotin.sub.1 pore, the spikes disappeared
completely after a short lag period (117.+-.11 s, n=4), leaving the
mean conductance unchanged (FIG. 4A). By contrast, the addition of
12 nM WT streptavidin to the trans side of the bilayer caused a
permanent partial channel block of 120.+-.9 pS (n=5) (FIG. 4B). The
extent of the block (51.+-.3%, n=5) was closely similar to the
average amplitude of the short-lived spikes (121.+-.4 pS, 53.+-.2%,
n=17) and occurred after a lag period of 158.+-.29 s (n=5). The
above results are interpreted as the essentially permanent capture
of the PEG-biotin chain by WT streptavidin
(K.sub.d=4.times.10.sup.-14 M in solution (Chilkoti et al., 1995a))
at the trans or cis side of the bilayer.
[0149] When W120A streptavidin, a mutant with considerably lower
affinity for biotin (K.sub.d=1.1.times.10.sup.-7 M (Chilkoti et
al., 1995a; Chilkoti et al., 1995b; Perez-Luna et al., 1999)) was
added to the trans or cis side of the H.sub.6106C-PEG-biotin.sub.1
pore, transient instead of permanent disappearances of the spikes
were observed (FIG. 4C and FIG. 4D). However, in terms of the
extent of channel block, the transient binding events brought about
by W120A were closely similar to those seen with WT streptavidin
(FIG. 3A and FIG. 3B): no measurable block on the cis side, and a
52.+-.4% block on the trans side. As expected for a bimolecular
interaction, the frequency of occurrence of blocking events
(1/.tau..sub.on=k.sub.on[W120A]) was proportional to the
concentration of W120A (FIG. 4E-1, FIG. 4E-2, FIG. 4E-3 and FIG.
4F). Remarkably, sequential binding events of W120A streptavidin
from both sides of the bilayer were able to be monitored in a
single study, due to the different signatures of the trans and cis
events (FIG. 4G and FIG. 4H).
[0150] At identical protein concentrations, the reversible binding
events of W120A streptavidin occurred more than 50 times less
frequently at the trans side than at the cis side. In addition, the
trans events exhibited a shorter dwell time. Through the analysis
of inter-event intervals (.tau..sub.on) and event lifetimes
(.tau..sub.off) apparent kinetic constants (k') for the association
and true kinetic constants (k) for the dissociation of the W120A
streptavidin.biotin complex were obtained for each side of the
lipid bilayer in 300 mM KCl, 5 mM Tris-HCl, pH 7.00, containing 100
.mu.M EDTA at +100 mV: k'.sub.on.sup.cis=0.38.+-.0.03.time-
s.10.sup.7 M.sup.-1 s.sup.-1 (all k'.sub.on values for streptavidin
are corrected for the presence of four biotin binding sites on each
protein); k.sub.off.sup.cis=0.31.+-.0.01 s.sup.-1;
K'.sub.d.sup.cis=0.82.+-.0.02.ti- mes.10.sup.-7 M;
k'.sub.on.sup.trans=1.08.+-.0.15.times.10.sup.5 M.sup.-1 s.sup.-1;
k.sub.off.sup.trans=1.48.+-.0.61 s.sup.-1;
K'.sub.d.sup.trans=1.37.+-.0.56.times.10.sup.-5 M. The value of
K'.sub.d.sup.cis is closely similar to the reported K.sub.d value
(1.1.times.10.sup.-7 M) (Chilkoti et al., 1995a).
[0151] Similar findings were obtained with a mouse anti-biotin
monoclonal IgG.sub.1 (mAb). For example, application of the mAb to
the cis side of a bilayer containing H.sub.6106C-PEG-biotin.sub.1
was accompanied by the transient disappearance of the spikes but
did not alter the amplitude of the main conductance state. Because
all three biotin-binding proteins fail to alter the unitary
conductance when they bind on the cis side of the bilayer, it is
likely that a major fraction of the PEG chain remains within the
.alpha.HL cavity during cis captures. By contrast, biotin capture
by the mAb at the trans side of the bilayer led to a drop in the
mean conductance of 55.+-.2% (FIG. 5A), close to the value for
W120A the streptavidins (W120A, 52.+-.4%; WT, 51.+-.3%). Because
both the streptavidins and the mAb produce a similar block, it is
likely that the physical origin of the block derives from the PEG
chain passing through the inner restriction, rather than from the
binding protein itself.
[0152] Kinetic constants for the association and dissociation of
the mAb were obtained for both sides of the lipid bilayer in 300 mM
KCl, 5 mM Tris-HCl, pH 7.00, containing 100 .mu.M EDTA at +100 mV:
k'.sub.on.sup.cis=4.86.+-.1.02.times.10.sup.7 M.sup.-1 s.sup.-1
(all k'.sub.on values for the mAb are corrected for the presence of
two biotin binding sites on each protein);
k.sub.off.sup.cis=0.019.+-.0.003 s.sup.-1;
K'.sub.d.sup.cis=0.39.+-.0.06.times.10.sup.-9 M;
k'.sub.on.sup.trans=4.32.+-.1.01.times.10.sup.5 M.sup.-1 s.sup.-1;
k.sub.off.sup.trans2.88.+-.0.74.times.10.sup.-2
s.sup.-1K'.sub.d.sup.tran- s=0.66.+-.0.16.times.10.sup.-7 M. Again,
biotin-binding events could be observed on both sides of the
bilayer in a single study. Indeed, captures by two different
biotin-binding proteins were recorded, e.g., cis: W120A
streptavidin, trans: mAb (FIG. 5B).
[0153] The results with both W120A streptavidin and the monoclonal
antibody are consistent with a simple kinetic model. In such a
kinetic model of the interactions between the .alpha.HL pore
H.sub.6106C-PEG-biotin.sub.1 and streptavidin at the cis and trans
sides of the bilayer, let p.sub.cis and p.sub.trans be the
probabilities that the biotinyl group is on the cis and trans sides
of the bilayer. p.sub.inside is the probability that it is in the
lumen of the .alpha.HL pore. Assuming that the equilibria are not
disturbed by biotin capture,
p.sub.cis/p.sub.inside=k.sub.+.sup.cis/k.sub.-.sup.cis,
p.sub.trans/p.sub.inside=k.sub.+.sup.trans/k.sub.-.sup.trans, and
p.sub.cis+p.sub.trans+p.sub.inside=1. Therefore,
p.sub.inside(1+k.sub.+.s-
up.cis/k.sub.-.sup.cis+k.sub.+.sup.trans/k.sub.-.sup.trans)=1,
p.sub.cis=k.sub.+.sup.cis/K.sub.-.sup.cis(1+k.sub.+.sup.cis/k.sub.-.sup.c-
is+k.sub.+.sup.trans/k.sub.-.sup.trans), and
p.sub.trans=k.sub.+.sup.trans-
/k.sub.-.sup.trans(1+k.sub.+.sup.cis/k.sub.-.sup.cis+k.sub.+.sup.trans/k.s-
ub.-.sup.trans). Let k.sub.+.sup.trans/k.sub.-.sup.trans be small
based on the finding that appearances on the trans side of the
bilayer are infrequent. Then
p.sub.cis=k.sub.+.sup.cis/(k.sub.-.sup.cis+k.sub.+.sup.c- is),
p.sub.trans=(k.sub.+.sup.trans/k.sub.-.sup.trans)(k.sub.-.sup.cis/(k.-
sub.-.sup.cis+k.sub.+.sup.cis)), and
p.sub.cis/p.sub.trans=(k.sub.+.sup.ci-
s/k.sub.-.sup.cis)(k.sub.+.sup.trans/k.sub.-.sup.trans). For
capture, k.sub.off/k'.sub.on=K'd, kinetic constants as measured
herein, k.sub.off/k.sub.on=K.sub.d, actual kinetic constants,
k'.sub.on.sup.cis=p.sub.cisk.sub.on.sup.cis,
k'.sub.on.sup.trans=p.sub.tr- ansk.sub.on.sup.trans,
K.sub.d.sup.cis=p.sub.cisK'.sub.d.sup.cis,
K.sub.d.sup.trans=p.sub.transK'.sub.d.sup.trans, and
p.sub.cis/p.sub.trans=(K.sub.d.sup.cis/K.sub.d.sup.trans)(K'.sub.d.sup.tr-
ans/K'.sub.d.sup.cis). In the cases examined,
K'.sub.d.sup.cis.about.K.sub- .d.sup.cis, p.sub.cis.about.1,
K'.sub.d.sup.trans>>K.sub.d.sup.trans- ,
p.sub.trans<<1, and p.sub.cis/p.sub.trans.about.150.
[0154] As the biotinyl group is rarely captured on the trans side
of the bilayer, k.sub.+.sup.trans is likely relatively small.
Therefore, the fraction of time spent by the biotinyl group on the
cis side is given by
p.sub.cis=k.sub.+.sup.cis/(k.sub.-.sup.cis+k.sub.+.sup.cis) and the
fraction of time spent on the trans side by
p.sub.trans=(k.sub.+.sup.tran-
s/k.sub.-.sup.trans).times.(k.sub.-.sup.cis/(k.sub.-.sup.cis+k.sub.+.sup.c-
is)). In terms of measurable dissociation constants
K.sub.d.sup.cis=p.sub.cisK'.sub.d.sup.cis and
K.sub.d.sup.trans=p.sub.tra- ns K'.sub.d.sup.trans. Therefore:
p.sub.cis/p.sub.trans=(K.sub.d.sup.cis/K.sub.d.sup.trans).times.(K'.sub.d.-
sup.trans/K'.sub.d.sup.cis) (1)
[0155] Assuming that the dissociation constants for the
biotin-streptavidin interaction are the same as those determined
under other circumstances (Chilkoti et al., 1995a) and the same on
both sides of the bilayer (K.sub.d.sup.cis=K.sub.d.sup.trans), then
p.sub.cis/p.sub.trans=167.+-.68. The latter assumption is not
strictly true; for example, for streptavidin W120A,
k.sub.off.sup.trans is about five times larger than
k.sub.off.sup.cis; perhaps elongation of the PEG chain lowers the
activation barrier for dissociation. While the value of
p.sub.cis/p.sub.trans is approximate, it does provide a qualitative
picture of biotin localization with respect to the bilayer.
Gratifyingly, equation 1 yields a similar value of
p.sub.cis/p.sub.trans=169.+-.41 for studies with the mAb.
[0156] When the biotinyl group is captured on the cis side of the
bilayer by the streptavidins or the mAb, there is no detectable
change in channel conductance suggesting that the PEG chain is
still largely contained within the central cavity, where it reduces
the flow of ions by about 15.5% (Example 1), and that the
streptavidin molecule does not itself perturb current flow. By
contrast, capture on the trans side is accompanied by a dramatic
reduction in single channel conductance that is strikingly similar
in amplitude to the current reduction seen during the transient
current spikes that occur in the absence of the biotin-binding
proteins or between captures in their presence. This similarity
indicates that the spikes represent excursions of the biotinyl
group towards the trans entrance into the transmembrane
.beta.-barrel. In accordance with this interpretation, there is a
complete absence of spikes during the cis capture events when the
end of the polymer is unavailable for threading into the barrel.
The spikes occupy about 0.48% of the current trace in the absence
of streptavidin, which is roughly in accord with the value of
p.sub.cis/p.sub.trans, therefore, the frequency of occurrence of
the spikes of 37.+-.6 s.sup.-1 is likely to be the upper limit for
the rate of appearance at the trans entrance and for transmembrane
movement (appearances at the cis entrance being yet more frequent
but electrically silent).
[0157] In summary, a nanoscale protein pore was assembled with a
covalently-attached and functionalized moving arm. The untethered
end of the arm is free to move across the bilayer from one mouth of
the pore to the other, a distance of more than 10 nm. Despite the
great interest in nanostructures, few assemblies with moving parts
have been made; one recent achievement is a nanomechanical device
based on the B-Z transition of DNA (Mao et al., 1999). The
functionalized pore of the present invention can also include the
ability to control the position of the arm, for example with the
transmembrane potential, which can be used to drive transmembrane
transport. This system is also applicable to examining the dynamics
of polymers other than PEG at the single molecule level, including
biological molecules such as polynucleotides, oligosaccharides and
peptides.
[0158] The characterization of single polymer molecules is active
area of research, which is usually limited to optical microscopy or
force measurements (Weiss, 1999; Mehta et al., 1999; Xie and Lu,
1999; Marszalek et al., 1999). Finkestein and colleagues have
examined the transmembrane movement of biotinylated toxins by
capture with streptavidin (Slatin et al., 1994), and the inventors
contemplate that biotin-binding proteins with reduced affinity,
might have advantages in such studies, as demonstrated herein.
Finally, these results show how engineered protein pores can be
used, at the single-molecule level, as stochastic sensor elements
for protein analytes. The present invention thus shows that
proteins such as antibodies can be detected at low nanomolar
concentrations (e.g., FIG. 4F) by using a chemically modified pore.
Stimulus-responsive polymers (Stayton et al., 1995) can also be
attached in the channel lumen to yield another class of sensor
elements.
EXAMPLE 3
[0159] Sequence-Specific Detection of DNA Using Engineered Protein
Pores
[0160] The present example shows various means of applying the
present invention to the field of DNA biochemistry. A
single-stranded DNA (ssDNA) molecule was covalently attached to the
a-hemolysin pore of Staphylococcus aureus. Changes in the current
flowing through an engineered pore revealed the sequence-specific
binding of individual ssDNA molecules to the tethered DNA strand.
The DNA-nanopore was able to discriminate, at the single molecule
level, between DNA strands up to 30 nucleotides in length differing
by a single base substitution. The use of the nanopore as a
biosensor element was exemplified by the detection of a drug
resistance-conferring mutation in the reverse transcriptase gene of
HIV. In addition, the present example demonstrates the use of such
DNA-nanopore compositions to sequence codons in tethered
DNA-strands. This example therefore shows the application of the
covalently modified nanopore technology of the invention in the
generation of nanopores modified with ssDNA or RNA and the use of
such oligonucleotide-nanopores to sense and sequencing DNA
molecules by single molecule detection.
[0161] The examination of individual RNA or DNA molecules is a
thriving area of research. Individual polynucleotide molecules can
be studied by fluorescence correlation spectroscopy (Eigen and
Rigler, 1994; Kinjo et al., 1998), force measurements (Strunz et
al., 1999; Baumann et al, 2000; Smith et al., 1996), and electrical
recordings (Andersen, 1999; Deamer and Akeson, 2000; Henrickson et
al., 2000). In electrical recordings, a single strand of RNA or DNA
is driven by an applied potential through a single nanopore, which
leads to a detectable change in the ionic current flowing through
the pore (Kasianowicz et al., 1996). This approach has been
employed to discriminate between RNA and DNA homo- or block
polymers with different base compositions (Akeson et al., 1999;
Meller et al., 2000). However, the single base resolution required
to sequence individual strands of DNA has been so far elusive. In
these aspects of the invention, the sensitivity of single channel
current recording is surprisingly combined with the selectivity of
nucleic acid hybridization (Taton et al., 2000; Lipshutz et al.,
1999) to sense the binding of individual DNA molecules to a DNA
strand tethered to a nanopore.
[0162] In heptameric pores of .alpha.HL, the 293 amino acid
monomeric polypeptide assembles to form a known structure (Song et
al., 1996) resembling a mushroom of 10 nm in height and up to 10 nm
in width. The lumen of .alpha.HL measures 3 nm at the cis entrance,
widens to 4.1 nm in the internal cavity and narrows at the inner
constriction to a diameter of 1.6 nm. In the transmembrane barrel,
the lumen has an average diameter of 2 nm. Because of the narrow
inner constriction, ssDNA but not double stranded DNA (dsDNA) can
pass through the pore (Kasianowicz et al., 1996). Molecular
graphics simulations reveal, however, that the internal cavity is
big enough to accommodate a DNA duplex 10 base pairs (bp) in
length.
[0163] A. Materials and Methods
[0164] 1. Formation of Heteromeric .alpha.HL Pores Containing
Covalently-Attached Oligonucleotides
[0165] An .alpha.HL pore carrying a single DNA oligonucleotide
attached to a site located at the cis entrance of the lumen was
generated. The DNA-nanopore was composed of six unmodified subunits
and one subunit covalently modified with the oligonucleotide.
Heptamers with this composition were obtained by assembly of
unmodified .alpha.HL (H) and the cysteine mutant 17C-D4, which had
been coupled through a disulfide linkage to oligo-A
(5'-CATTCACC-3'; SEQ ID NO:1), 8 nucleotides (nt) in length.
[0166] To achieve this, oligonucleotides were first activated and
then reacted with the single cysteine residue of .alpha.HL-17C-D4.
5' thiol-modified DNA oligonucleotides with a hexamethylene linker
were purchased from Research Genetics (Huntsville, Ala.) and
activated with 2,2'-dipyridyl disulfide to yield 5'-S-thiopyridyl
oligonucleotide (Corey et al., 1995) for coupling to the protein.
The mutant .alpha.HL-17C-D4 was generated by site-directed
mutagenesis (Howorka and Bayley, 1998) of the engineered gene
.alpha.HL-WT-RL-D4, which encodes the wild-type .alpha.HL protein
and a C-terminal polypeptide extension of four aspartate residues.
.sup.35S-labeled .alpha.HL polypeptides H (wild type) and 17C-D4
were generated by expression in vitro (Cheley et al., 1999).
[0167] For the coupling to oligonucleotides, translation mixes of
17C-D4 (3 .mu.l, 300 ng .alpha.HL protein) and of H (15 .mu.l, 1.5
.mu.g) were combined and separated from excess
.beta.-mercaptoethanol by using spin filter columns with a
molecular weight cut off of 10 kDa (#42407, Millipore). For this
treatment, the combined mixes were diluted into 0.1 mM DTT (0.5 ml)
and concentrated by centrifugation to a volume of 30 .mu.l. The
procedure was repeated two times. The retentate (30 .mu.l) was then
diluted 2-fold into a buffer containing 10 mM MOPS-NaOH, pH 7.4,
150 mM NaCl, 0.5 mM EDTA and reacted with 50 nmol 5'-S-thiopyridyl
oligonucleotide for 10 min at 25.degree. C. The monomeric subunits
were then co-assembled on rabbit erythrocyte membranes (Walker et
al., 1992a) and the resultant heptamers were purified by
SDS-polyacrylamide gel electrophoresis (Howorka et al., 2000; FIG.
6A).
[0168] Heteroheptamer H.sub.6(17C-oligo-A).sub.1 was purified from
heptamers H.sub.7 and H.sub.5(17C-oligo-A).sub.2, which also formed
during the assembly process, by SDS-PAGE (FIG. 6B). In this, the
various heptamers migrated in separate bands by virtue of a gel
shift caused by a C-terminal polypeptide extension of four
aspartates (D4), present only in 17-oligo-A-D4 but not in the H
subunits. Interestingly, the modification of C-D4 with DNA caused
the monomer to migrate more slowly (FIG. 6A, compare lanes 1 and
2), but did not alter the electrophoretic mobility of the
heptamer.). The subunit ratio in heteroheptamer
H.sub.6(17C-oligo-A).sub.1 was confirmed by additional SDS-PAGE
after the protein had been extracted from the first gel and heated
to dissociate the subunits.
[0169] 2. Bilayer Recordings
[0170] Planar lipid bilayer recordings were used to examine the
single-channel properties of H.sub.6(17C-oligo-A).sub.1 and its
interaction with oligonucleotides of complementary sequence added
to the cis chamber. These recordings were carried out at
22.+-.1.degree. C. (Braha et al., 1997). Briefly, a bilayer of
1,2-diphytanoyl-sn-glyceropho- sphocholine (Avanti Polar Lipids,
Alabaster, Ala.) was formed on an aperture (140 .mu.m in diameter)
in a Teflon septum (Goodfellow Corporation, Malvern, Pa.), which
separated the cis and trans chambers (1.5 ml each) of a planar
bilayer apparatus. The electrolyte in both chambers was 2 M KCl, 12
mM MgCl.sub.2 and 5 mM Tris-HCl, pH 7.4. Heptameric a.alpha.HL
protein was added to the cis chamber, at a concentration of 0.01 to
0.1 ng/ml, and the electrolyte in the cis chamber stirred until a
single channel inserted into the bilayer. Electrical recordings
were performed at a holding potential of +100 mV (with the cis side
grounded) by using a patch clamp amplifier (Axopatch 200B, Axon
Instruments, Foster City, Calif.). Currents were low-pass filtered
with a built-in 4-pole Bessel filter at 10 kHz and sampled at 50
kHz by computer with a Digidata 1200 A/D converter (Axon
Instruments) and analyzed (Movileanu et al., 2000). Traces shown in
the figures were filtered at 1 kHz and sampled at 5 kHz. Unless
otherwise stated, DNA oligonucleotides were purchased from
Integrated DNA Technologies (Coralville, Iowa) and used without
further purification.
[0171] The channels were analyzed under an electric field of +100
mV, which drives negatively charged molecules such as DNA from the
cis to the trans side of the bilayer. In 2 M KCl, 12 mM MgCl.sub.2
and 5 mM Tris-HCl, pH 7.4, the unitary conductance was 1750.+-.140
pS (n=4). The single channel currents were decorated with brief
current fluctuations (mean lifetime, 0.15.+-.0.07 ms; amplitude,
140.+-.42 pS; frequency of occurrence, 8.6.+-.1.5 s.sup.-1, n=3).
Due to their short lifetimes, some current spikes were not
completely resolved at the filter frequency of 10 kHz. As this
study focuses on the sequence-specific binding events, the current
spikes, which presumably represent translocation events were not
investigated further; FIG. 7A-1).
[0172] B. Results
[0173] The conductance of H.sub.6(17C-oligo-A), is lower than the
value for H.sub.7 channels (1950.+-.100 pS, n=3) or
H.sub.6(17C-oligo-A), channels, which had been treated with DTT to
cleave the disulfide bond between the oligonucleotide and .alpha.HL
(1900.+-.110 pS, n=3). The reduced conductance of
H.sub.6(17C-oligo-A), indicates that the tethered
DNA-oligonucleotide partly blocks the current flowing through the
nanopore (FIG. 7A-2).
[0174] When 67 nM oligo-B (3'-GTAAGTGG-5'; SEQ ID NO:2), with a
sequence fully complementary to the tethered oligo-A
(5'-CATTCACC-3'; SEQ ID NO:1), was added to the cis side of the
bilayer two type of events occurred: negative current deflections
(FIG. 7B-1, symbol b) characterized by a duration of hundreds of
milliseconds, a mean amplitude of 605.+-.31 pS and a frequency of
occurrence of 0.48.+-.0.08 s.sup.-1 (n=4); and spike-like events
(FIG. 7B-1, symbol s) with a mean lifetime of 0.3.+-.0.1 ms, a mean
amplitude of 590.+-.120 pS and a frequency of occurrence of
0.13.+-.0.02 s.sup.-1 (n=4).
[0175] The current deflections (b) most likely represent single
oligo-B molecules, which enter the DNA-nanopore 5'-end first and
form a duplex with the tethered, complementary oligo-A. The spike
at the end of the binding event (FIG. 7B-1) indicates that after
dissociation oligo-A passes the inner constriction to exit on the
trans side of the pore (FIG. 7B-2). The spikes (s) probably arise
from oligo-B strands, which enter the DNA-nanopore with the 3'-end
first, leaving them unable to form a duplex with the tethered
oligonucleotide. Alternatively, spikes (s) could also stem from
oligo-B strands, which enter the pore with the 5'-end first but do
not bind.
[0176] To prove that the current deflections (b) represented
oligo-B binding to the tethered oligo-A, excess free oligo-A was
added on the cis side. If the binding were specific, excess oligo-A
would compete for the binding of oligo-B to tethered oligo-A (FIG.
7C-2). Indeed, the frequency of occurrence of the proposed binding
events was reduced 21-fold (0.02 s.sup.-1), while spikes, now
presumably stemming from oligo-B transiting the lumen without
binding, appeared with a frequency of occurrence 5.2 s.sup.-1 (FIG.
7C-1).
[0177] Single channel current recording was used to derive the
kinetic constants for the association and the dissociation of
individual DNA strands. Each binding event, oligo-B to
H.sub.6(17C-oligo-A), (FIG. 7B-1), was characterized by its event
amplitude I.sub.E and its event lifetime .tau..sub.off (FIG. 8A).
The two characteristic parameters for hundreds of individual events
from one recording were plotted onto an event diagram, in which
each point represents one event (FIG. 8B).
[0178] While the event amplitudes were narrowly distributed
(597.+-.20 pS), the event lifetimes were scattered between 50 and
2000 ms with a mean value of 470.+-.400 ms. Lifetime histogram
analysis of four recordings with a total number of 4000 events
revealed that the event population was composed of two different
event types with .tau..sub.off values of 119.+-.23 ms (20.+-.3% of
the events) and 620.+-.63 ms (80.+-.3%). Fitting the kinetic
parameters to different kinetic schemes revealed, that the two
event types represent two classes of binding events with different
stability constants: K.sub.d-1=9.3.times.10.sup.-6,
K.sub.d-2=4.5.times.10.sup.-7.
[0179] In more detail, the kinetic scheme for DNA duplex formation
and dissociation in the internal cavity of .alpha.HL is as
follows.
[0180] Single channel current analysis revealed two .tau..sub.off
and proportion (P) values for the dissociation of oligo-B
(5'-GGTGAATG-3'; SEQ ID NO:2) from the tethered DNA strand oligo-A
(5'-CATTCACC-3'): .tau..sub.off-1=119.+-.23 ms, P.sub.1=20.+-.3% of
the events; .tau..sub.off-2=620.+-.63 ms, P.sub.2=80.+-.3%.
[0181] To account for the values, two simple kinetic models can be
envisioned. 1
[0182] In this model, DNA duplex AB forms by the association of DNA
strands A and B and is assumed to dissociate along two kinetically
different routes characterized by the rate constants k.sub.off-1
and k.sub.off-2. In accordance, the overall rate of duplex
dissociation is:
v.sub.off=(k.sub.off-1+k.sub.off-2).multidot.[AB]
[0183] The probability for duplex AB to dissociate along route 1
would is given by:
P.sub.1=k.sub.off-1/(k.sub.off-1+k.sub.off-2)=.tau..sub.off-2/(.tau..sub.o-
ff-1+.tau..sub.off-2)
[0184] The experimentally derived values of .tau..sub.off-1=119 ms
and .tau..sub.off-2=620 ms, yields
.tau..sub.off-2/(.tau..sub.off-1+.tau..sub- .off-2)=0.84. But, the
experimental value for P.sub.1 is 0.2. Therefore, the observed
kinetic parameters can-not be explained by the kinetic model I.
More likely, hybridization follows kinetic model II, characterized
by two completely separate binding events: 2
[0185] The total rate of strand association in this model is:
v.sub.on=k.sub.on.multidot.[A].multidot.[B]=(k.sub.on-1+k.sub.on-2).multid-
ot.[A].multidot.[B]
[0186] The individual rate constants for duplex formation and
dissociation and the stability constants are:
6 k.sub.on-1 = P.sub.1 .multidot. k.sub.on k.sub.on-2 = P.sub.2
.multidot. k.sub.on K.sub.off-1 = 1/.tau..sub.off-1 K.sub.off-2 =
1/.tau..sub.off-2 K.sub.d-1 = k.sub.off-1/k.sub.on-1 K.sub.d-2 =
k.sub.off-2/k.sub.on-2
[0187] Inserting the values for P.sub.1=0.2, P.sub.2=0.8,
k.sub.on=4.5.times.10.sup.6M.sup.-1 s.sup.-1, .tau..sub.off-1=119
ms and .tau..sub.off-2=620 ms gives
7 K.sub.on-1 = 9 .times. 10.sup.5 M.sup.-1 s.sup.-1 K.sub.on-2 =
3.6 .times. 10.sup.6 M.sup.-1 s.sup.-1 K.sub.off-1 = 8.4 s.sup.-1
K.sub.off-2 = 1.6 s.sup.-1 K.sub.d-1 = 9.3 .times. 10.sup.-6
K.sub.d-2 = 4.5 .times. 10.sup.-7
[0188] While it would be interesting to further investigate the
nature of the two different binding events, it is clear that
K.sub.d-2 dominates the composite K.sub.d obtained from the mean
lifetime, .tau..sub.off. Therefore, to simplify the analysis,
composite kinetic constants are used here. The inter-event
intervals (.tau..sub.on) showed a linear dependence on the
concentration of oligo-B in the examined range (5 to 400 nM).
Through the analysis of the inter-event intervals (.tau..sub.on)
and the event lifetimes (.tau..sub.off ) of three independent
single channel current recordings, it was possible to obtain the
kinetic constants for strand association (k.sub.on) and strand
dissociation (k.sub.off).
[0189] The association constant (k.sub.on) for duplex formation was
calculated from k.sub.on=1/(c.times..tau..sub.on), where
.tau..sub.on is the inter-event interval and c the concentration of
oligo-B in the cis chamber. The strand dissociation constant
(k.sub.off) was derived from the event lifetime (.tau..sub.off ):
k.sub.off=1/.tau..sub.off (Moczydlowski, 1986).
[0190] The value of k.sub.on was 4.5.times.10.sup.6
M.sup.-1s.sup.-1 and k.sub.off was 1.9 s.sup.-1. The value of
k.sub.on falls within the range of values usually observed for
strand association in homogeneous solution (Braunlin and
Bloomfield, 1991; Porschke and Eigen, 1971; Cantor and Schimmel,
1980; Riesner and Romer, 1973); and k.sub.off is slightly higher
than the calculated value for dissociation in solution (0.6
s.sup.-1). The rate constant for duplex dissociation, k.sub.off, in
homogeneous solution was calculated using the relation
k.sub.off=k.sub.on.times.K.sub.d. Given that the association rate
constant k.sub.on is usually not strongly dependent on the length
and type of oligonucleotide (Cantor and Schimmel, 1980), a value of
10.sup.6 M.sup.-1s.sup.-1 was assumed for k.sub.on. The
dissociation equilibrium constant K.sub.d was derived from
thermodynamic data (Martin et al., 1971).
[0191] It might be concluded that the kinetics of duplex formation
in a nanopore are very similar to duplex formation in homogeneous
solution. Alternatively, the kinetics might be affected by opposing
but compensating factors; for example, sterical constraints or
effects of the applied potential (Gilles et al., 1999). Hence,
single channel current studies with a DNA-nanopore give kinetic
data consistent with established literature values and, in
addition, offer the ability to detect properties (e.g., complex
kinetics) often difficult to investigate by conventional methods
which measure bulk properties.
[0192] As mismatched bases are known to weaken duplex formation
between DNA strands (Aboul-ela et al., 1985), the inventors tested
whether a DNA-nanopore could discriminate between DNA molecules
differing by a single base. A common point mutation in the reverse
transcriptase gene of HIV was examined, which confers resistance to
the widely used antiviral drug nevirapine (Hanna et al., 2000;
Richman et al., 1994).
[0193] The tethered oligonucleotide with a length of 8 nt
(5'-TGACAGAT-3'; SEQ ID NO:3) was fully complementary to an 8 nt
portion of a 30 nt coding fragment from the drug-resistant virus
strain (FIG. 9, oligo-181C; SEQ ID NO:5), while the wild type (wt)
virus (FIG. 9, oligo-181Y; SEQ ID NO:4) included a single mismatch.
DNA strands oligo-181C and oligo-Y can be derived from HIV RNA by
RT-PCR.TM. of the reverse transcriptase gene, followed by digestion
with restriction enzyme NlaIII (with the recognition sequence and
cleavage site CATG.vertline.) and linear PCR.TM. with a primer of
the sequence 5'-ACAAAATCCAGA-3' (nucleotides 1 through 12 of SEQ ID
NO:4 and SEQ ID NO:5).
[0194] When wt oligo-181Y was added to the cis chamber, five events
with an event lifetime longer than the cut-off of 5 ms were
recorded in 300 s (FIG. 9; open circles). By contrast, in the same
time period, mutant oligo-181C gave rise to 70 events with event
amplitudes (I.sub.E) greater than those found for the five events
with oligo-181Y (FIG. 9, filled squares). This indicates that the
oligo-181C strands, when bound to the tethered oligonucleotide,
thread into the transmembrane barrel through the central
constriction producing a strong block (Movileanu et al., 2000;
compare with the block caused by shorter oligos in FIG. 7A-1, FIG.
7B-1 and FIG. 7C-1 with FIG. 8A and FIG. 8B).
[0195] Because 181C events populated a distinct area in the event
diagram, a 181C-specific event window (FIG. 9, box) useful for the
assignment of new events was defined. Any new event falling into
the window can be identified as stemming from a single strand of
181C (no 181Y events, but one hundred and fifty 181C events, fell
in the box during two recordings). This study shows that the
DNA-nanopore was able to discriminate, on the single molecule
level, between two 30 nt-long ssDNA strands differing only by a
single base. Hence, DNA-nanopores represent novel biosensor
elements for the ultrasensitive detection of DNA from medically or
environmentally important samples.
[0196] A DNA-nanopore was also used to sequence a codon on a single
strand of DNA. The sequencing principle was based on the
match/mismatch-dependen- t binding time of hybridized
oligonucleotides (Table 6A). In the following description, the
unknown nucleotides (indicated by X and Z of defined designations
in the described oligonucleotides) are also represented as "N" in
the appended sequence listing.
[0197] A ssDNA oligonucleotide (5'-GCATTCX.sub.1X.sub.2X.sub.3-3';
SEQ ID NO:6) with three unknown bases (X.sub.1, X.sub.2, X.sub.3)
was tethered to Cys.sup.17 of .alpha.HL. To identify the first base
X.sub.1, four oligonucleotides with sequences 3'-CGTAAGZ.sub.1-5'
(SEQ ID NO:7; Z.sub.1=A, C, G, T) were used and their interactions
with the tethered DNA strand analyzed by single channel current
recording. Of the four oligonucleotides, one was characterized by a
higher average event lifetime compared with the other three
oligonucleotides (Table 6A). Therefore, this oligonucleotide,
carrying a T in position Z.sub.1 was fully complementary to the
tethered DNA strand, and hence, the base X.sub.1 was defined to be
A. To identify the other two bases (X.sub.2, X.sub.3), two
additional rounds were performed, each with a different set of
oligonucleotides (3'-GTAAGTZ.sub.2-5', SEQ ID NO:8 and
3'-TAAGTGZ.sub.3-5', SEQ ID NO:9; Table 6A). Sequence information
obtained in one round was used to design the oligonucleotides for
the next round. In this way, the codon X.sub.1X.sub.2X.sub.3 was
unambiguously deduced to be ACC (Table 6A).
[0198] The success of this method of sequencing depends on the
differences in the match/mismatch-dependent event lifetimes.
Therefore, the influence of the position of the mismatch on the
lifetime was analyzed using the oligonucleotides: 5'-ATTCACC-3'
(SEQ ID NO:10); 3'-TAAZ.sub.4TGG-5' (SEQ ID NO:11) and
3'-TAZ.sub.5GTGG-5' (SEQ ID NO:12). It was found that the mismatch
had the most dramatic effect when it was positioned in the middle
of the oligonucleotide (Table 6B). For two different internal
positions, the event lifetimes for mismatched oligonucleotides were
8- and 60-times shorter than those of the completely complementary
oligonucleotide (Table 6B). By contrast, the event lifetimes for
three oligonucleotides with different terminal mismatches were 2.3,
5.9 and 4.7 shorter than the lifetimes of the corresponding
complementary oligonucleotides (Table 6A).
[0199] The sequencing of tethered DNA using the present
hybridization-based method, would require molecular biological and
chemical manipulations such as linear PCR.TM. with
5'-thiol-modified primers and chemical attachment of the DNA-strand
to the nanopore. Clearly, time-consuming manipulations could be
greatly reduced if copies of a non-tethered DNA strand were
sequenced with an array of DNA-nanopores modified with
oligonucleotides of known sequence. The viability of this approach
was proven for the determination of a single base (Table 6C).
[0200] Oligonucleotide 6 (3'-GTAAGTX.sub.6G-5'; SEQ ID NO:14) with
the unknown base X.sub.6 was added to the cis side of four
different .alpha.HL pores, which had been modified with
5'-CATTCAZ.sub.6-3' (SEQ ID NO:13; Z.sub.6=A, C, G, T), and
analyzed by single channel current recording. The average lifetime
of binding events with .alpha.HL-oligonucleotides wherein Z.sub.6=G
was longer than the values for the other three DNA-nanopores (Table
6C), and the unknown base X.sub.6 was deduced to be C.
8TABLE 6A event lifetimes .tau..sub.off [ms] for oligos 1, 2 and 3
interacting with .alpha.HL-SS-5'-GCATTCX.sub.1X- .sub.2X.sub.3-3'
(SEQ ID NO:6) .alpha.HL-SS-5'- GCATTCX.sub.1X.sub.2X.sub.3-3' (SEQ
ID NO:6) Z.sub.n Interacting with A C G T Z.sub.n X.sub.n oligo-1
3'-CGTAAGZ.sub.1-5' .fwdarw. 6.7 8.2 7.9 19 T .fwdarw. A (SEQ ID
NO:7) oligo-2 3'-GTAAGTZ.sub.2-5' .fwdarw. 1.7 1.5 10 1.6 G
.fwdarw. C (SEQ ID NO:8) oligo-3 3'-TAAGTGZ.sub.3-5' .fwdarw. 5.3
3.9 25 4.0 G .fwdarw. C (SEQ ID NO:9) codon ACC
[0201]
9TABLE 6B event lifetimes .tau..sub.off [ms] for oligos 4 and 5
interacting with .alpha.HL-SS-5'-ATTCACC-3' (SEQ ID NO:10)
.alpha.HL-SS-5'-ATTCACC-3' (SEQ ID NO:10) Z.sub.n Interacting with
A C G T oligo-4 3'-TAAZ.sub.4TGG-5' .fwdarw. <0.5 <0.5 29
<0.5 (SEQ ID NO:11) oligo-5 3'-TAZ.sub.5GTGG-5' .fwdarw. 29
<0.5 3.5 1.6 (SEQ ID NO:12)
[0202]
10TABLE 6C event lifetimes .tau..sub.off [ms] for oligo 6
interacting with .alpha.HL-SS-5'-CATTCAZ6-3' (SEQ ID NO:13) oligo 6
3'-GTAAGTX.sub.6G-5' (SEQ ID NO:14) Z.sub.6 Interacting with A C G
T Z.sub.6 X.sub.6 .alpha.HL-SS-5'-CATTCAZ.sub.6-3' .fwdarw. 1.1 13
2.0 1.6 .fwdarw. C .fwdarw. G (SEQ ID NO:13)
[0203] Table 6A, Table 6B and Table 6C: Single base mismatches
influence the binding time of individual DNA strands to a
DNA-nanopore. Table 6A. Sequencing of a codon in an individual
ssDNA molecule tethered to the .alpha.HL pore. The sequence was
determined by the match/mismatch-dependent binding time of
hybridizing oligonucleotides. Table 6B. The position of a single
base mismatch in an oligonucleotide interacting with a DNA-nanopore
strongly influences the event lifetime .tau..sub.off Table 6C.
Hybridization-based determination of an unknown base in
non-tethered ssDNA by using an "array" of four DNA-nanopores with
known sequence. The values in A, B, C are the arithmetic means of
the lifetimes .tau..sub.off of one single channel current
recording. All studies were repeated and gave similar values. The
concentration of oligonucleotides in the cis chamber was 200 nM,
and events were counted if 400 pS.ltoreq.I.sub.E.ltoreq.700 pS and
.tau..sub.off.gtoreq.0.5 ms. Each recording had a duration of 4 min
and the number of events in a recording ranged from 400 to
1600.
[0204] The use of this strategy to sequence a target gene would
require multiple ssDNA copies with lengths between seven and 20 nt.
In one approach, these fragments could be obtained by PCR.TM.
amplification, followed by selective enzymatic degradation of the
template strands and fragmentation of the product strands. The
protocol includes pre-amplification of the desired PCR.TM.-product
using the nucleotide UTP instead of TTP, followed by linear PCR.TM.
in the presence of TTP and UTP to yield product strands containing
TTP and UTP at random positions. The degradation of the template
strands containing UTP and the fragmentation of the product strands
to ssDNA oligonucleotides is accomplished by the enzymes uracil
DNA-glycosylase and E. coli endonuclease IV. If necessary, the DNA
fragments can be partially purified.
[0205] Alternatively, short fragments can be generated by using
restriction endonucleases with a 2 bp recognition sequence.
Chlorella virus-encoded restriction endonucleases have short (2 to
4 bp) recognition sites. For example, the enzyme CviTI cuts at the
site (NG.vertline.CN). Additional information is available at
www.cvienzymes.com.
[0206] In summary, .alpha.HL pores modified with a single DNA
oligonucleotide have been used to study duplex formation by
individual DNA molecules, thus extending the proof of principle for
the present invention. The DNA-nanopores can be used in at least
two different modes. In the first mode, a single DNA strand is
tethered to the pore and analyzed by the binding of partly or
completely complementary oligonucleotides. In the second mode, a
solution of free analyte DNA is added to the cis chamber and
analyzed with DNA-nanopores of known sequence.
[0207] Using DNA-nanopores operating in the first mode, the
kinetics of DNA duplex formation was studied at the single molecule
level, thereby avoiding problems of conventional techniques such as
surface plasmon resonance (SPR). In SPR, the transport of the
analyte to the sensor surface can be impeded by slow diffusion
through the immobilization matrix (Schuck, 1997). Indeed, k.sub.on
values derived by SPR are reported to be one to two orders of
magnitude lower (Jensen et al., 1997; Gotoh et al., 1995) than the
values for duplex formation in solution (Braunlin and Bloomfield,
1991; Porschke and Eigen, 1971; Cantor and Schimmel, 1980; Riesner
and Romer, 1973). In comparison, the present invention yields
kinetic constants in excellent agreement with
experimentally-derived data for duplex formation in solution.
Furthermore, the use of DNA-nanopores provides kinetic parameters
not readily obtained by conventional techniques, which measure bulk
properties. For example, two different binding events were observed
characterized by their k.sub.on, k.sub.off and K.sub.d values.
[0208] DNA-nanopores operating in the first mode can also be used
to sequence an individual tethered DNA strand as shown for a
complete codon in the present study. In the current configuration,
at least one codon can be determined per attached DNA strand;
hence, sequencing of a 1000 bp gene would require at least 334
chemically modified .alpha.HL pores. Therefore, the utility of
DNA-nanopores for sequencing will be improved by miniaturization of
single channel current recordings to allow the simultaneous and
automated analysis of hundreds of different channels.
[0209] To produce microfabricated chip-based channel arrays, the
single channels should be maintained in stabilized membranes and
individual channels should be electronically addressed within an
array of hundreds of channels. Improving the stability of membranes
is achieved using supported bilayers (Cornell et al., 1997; Sotra
et al., 1999) and nanoscale apertures in a variety of materials
(Hulteen et al., 1998). Electronically addressing individual
channels within an array of hundreds of channels is achieved using
microfabrication expertise (Quake and Scherer, 2000) and the
production of chip-based circuits. Sequencing of single DNA strands
using the first mode would require fragments of each target DNA
strand to be covalently attached to a nanopore. Under optimized
conditions for chemical tethering of the DNA strands, the detection
limit would lie at a few copies of a target strand.
[0210] Using the second mode, non-tethered target DNA strands could
be sequenced by arrays of nanopores modified with known DNA
sequences. In contrast to the first mode, this configuration would
allow re-use of the arrays. The viability of this strategy was
shown for the identification of a base with an "array" of four
DNA-pores. A particularly preferred application of arrays of
DNA-pores operating in the second mode lies in the sequencing of
variants of a known gene, such as the protease gene of
drug-resistant HIV strains, or the diagnostic screening of single
nucleotide polymorphisms (SNPs) in human genes. In order to
identify single point mutations in a 400 bp-gene, 400.times.4=1600
DNA-pores with different DNA 7 mers would be sufficient. The use of
arrays of DNA-pores will likely also offer advantages.
[0211] The current detection limit of this system in terms of final
target DNA concentration is 1 nM. To decrease the detection limit
and fully capitalize on the high sensitivity of DNA-nanopores, the
sample volume can be reduced by the miniaturization of the chamber
reservoir. Assuming a sample volume of one nL, a detection limit of
one attomole can be achieved. The transport of a sample volume of a
few nL is readily done with state-of-the-art fluidic systems (Quake
and Scherer, 2000). To further reduce the detection limit, the
target DNA can be electrophoretically transported and concentrated
on the biosensor surface (Gilles et al., 1999). Furthermore, the
recording time for DNA-nanopores is a few minutes, while the time
required for the hybridization and read out of DNA-chips is 45 to
60 minutes (Hegde et al., 2000).
[0212] DNA-nanopores can also improve the prospects for DNA
sequencing by translocation. This approach assumes that individual
bases can be identified by their characteristic channel blockades
and/or dwell times as a single DNA strand moves through a nanopore
(Akeson et al., 1999; Meller et al., 2000). DNA homo- or block
polymers 100 nt in length and with different base compositions have
been identified by their characteristic signatures (Akeson et al.,
1999; Meller et al., 2000). Single base resolution has been
elusive, because the DNA strands translocate too quickly through
the nanopore to allow the identification of single bases (for
example 190 .mu.s for (dA).sub.100=1.9 .mu.s per nt). To improve
the resolution, the translocating DNA strand should be slowed down.
The DNA-nanopores of the present invention represent an important
advance here, because the tethered DNA strand provides a physical
constriction and the chemical "stickyness" necessary to retard the
translocating DNA strand. In addition, the bases of the tethered
DNA can selectively interact with bases of the translocating DNA
and thereby cause base-specific differences in the dwell-times
and/or current blockades.
[0213] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods, and
in the steps or in the sequence of steps of the methods described
herein, without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
14 1 8 DNA UNKNOWN misc_feature ()..() SYNTHETIC OLIGONUCLEOTIDE 1
cattcacc 8 2 8 DNA UNKNOWN misc_feature ()..() SYNTHETIC
OLIGONUCLEOTIDE 2 ggtgaatg 8 3 8 DNA UNKNOWN misc_feature ()..()
SYNTHETIC OLIGONUCLEOTIDE 3 tgacagat 8 4 30 DNA UNKNOWN
misc_feature ()..() SYNTHETIC OLIGONUCLEOTIDE 4 acaaaatcca
gacatagtta tctatcaata 30 5 30 DNA UNKNOWN misc_feature ()..()
SYNTHETIC OLIGONUCLEOTIDE 5 acaaaatcca gacatagtta tctgtcaata 30 6 9
DNA UNKNOWN misc_feature ()..() SYNTHETIC OLIGONUCLEOTIDE 6
gcattcnnn 9 7 7 DNA UNKNOWN misc_feature ()..() SYNTHETIC
OLIGONUCLEOTIDE 7 ngaatgc 7 8 7 DNA UNKNOWN misc_feature ()..()
SYNTHETIC OLIGONUCLEOTIDE 8 ntgaatg 7 9 7 DNA UNKNOWN misc_feature
()..() SYNTHETIC OLIGONUCLEOTIDE 9 ngtgaat 7 10 7 DNA UNKNOWN
misc_feature ()..() SYNTHETIC OLIGONUCLEOTIDE 10 attcacc 7 11 7 DNA
UNKNOWN misc_feature ()..() SYNTHETIC OLIGONUCLEOTIDE 11 ggtnaat 7
12 7 DNA UNKNOWN misc_feature ()..() SYNTHETIC OLIGONUCLEOTIDE 12
ggtgnat 7 13 7 DNA UNKNOWN misc_feature ()..() SYNTHETIC
OLIGONUCLEOTIDE 13 cattcan 7 14 8 DNA UNKNOWN misc_feature ()..()
SYNTHETIC OLIGONUCLEOTIDE 14 gntgaatg 8
* * * * *
References