U.S. patent application number 11/576723 was filed with the patent office on 2009-02-26 for channel current cheminformatics and bioengineering methods for immunological screening, single-molecule analysis, and single-molecular-interaction analysis.
This patent application is currently assigned to Board of Supervisors of Louisiana State University And Agricultural And Mechanical College Acting For The Louisiana State Univer. Invention is credited to Seth Pincus, Stephen Winters-Hilt.
Application Number | 20090054919 11/576723 |
Document ID | / |
Family ID | 35056663 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090054919 |
Kind Code |
A2 |
Winters-Hilt; Stephen ; et
al. |
February 26, 2009 |
Channel Current Cheminformatics And Bioengineering Methods For
Immunological Screening, Single-Molecule Analysis, And
Single-Molecular-Interaction Analysis
Abstract
Analytical tools and methods employing processing of channel
current blockade measurements to detect or assess changes in
environmental conditions generally, and more specifically, to
detect and assess interactions between a blockade-producing
auxiliary molecule in the channel and either the surrounding
conductive medium generally or molecules in that conductive medium.
The auxiliary molecule is disposed within the channel in order to
generate a highly structured channel current blockade signal. The
blockade signal resulting from inducing ionic current flow through
the channel with auxiliary molecule present, is based on an
auxiliary molecule chosen to be highly susceptible to modulation
when the auxiliary molecule interacts with other molecules in the
surrounding conductive medium, or when it otherwise is modified or
affected at the molecular level by changes in the physical or
chemical conditions of that surrounding medium. The tools and
methods enable, amongst other things, candidate antibody screening,
molecular affinity and bond strength analysis and detection of a
variety of changes in test media, even at the molecular level.
Inventors: |
Winters-Hilt; Stephen;
(Mandeville, LA) ; Pincus; Seth; (New Orleans,
LA) |
Correspondence
Address: |
McGLINCHEY STAFFORD, PLLC
4703 BLUEBONNET BLVD
BATON ROUGE
LA
70809
UNITED STATES
2252914600
225-291-4606
|
Assignee: |
Board of Supervisors of Louisiana
State University And Agricultural And Mechanical College Acting For
The Louisiana State University Health Sciences Center in New
Orleans
433 Bolivar Street Room 824
New Orleans
LA
70112
Board of Supervisors of Louisiana State University And
Agricultural And Mechanical College Acting For The University of
New Orleans
2045 Lakeshore Drive Suite 526
New Orleans
LA
70122
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20080097502 A1 |
April 24, 2008 |
|
|
Family ID: |
35056663 |
Appl. No.: |
11/576723 |
Filed: |
April 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60/616274 |
Oct 6, 2004 |
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60/616275 |
Oct 6, 2004 |
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60/616276 |
Oct 6, 2004 |
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60/616277 |
Oct 6, 2004 |
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Current U.S.
Class: |
606/172 ;
606/167 |
Current CPC
Class: |
G01N 2500/00 20130101;
A61B 5/15144 20130101; A61B 5/15117 20130101; A61B 2090/034
20160201; A61B 5/7267 20130101; A61B 5/150465 20130101; A61B
5/15111 20130101; G16B 40/00 20190201; A61B 2090/0814 20160201;
G16C 99/00 20190201; A61B 5/15128 20130101; A61B 5/150022 20130101;
G01N 33/6872 20130101; A61B 5/150916 20130101; A61B 17/32093
20130101; A61B 5/150259 20130101 |
Class at
Publication: |
606/172 ;
606/167 |
International
Class: |
A61B 17/3209 20060101
A61B017/3209 |
Claims
1. A method comprising receiving a blockade channel current signal
carried by an ionic current flowing through a partially-blockaded
nano-scale membrane channel defined by a membrane which partitions
a conductive medium, exposing the channel to a test condition
during ionic current flow while extracting from the blockade
channel current signal, a set of one or more pattern features to
establish over a period of time either a blockade channel current
signal pattern or a change in the blockade channel current signal
pattern.
2. A method according to claim 2, further comprising comparing the
blockade channel current signal pattern, or the change in the
blockade channel current signal pattern, to at least one blockade
channel current signal pattern, or at least one change in the
blockade channel current signal pattern, associated with at least
one known condition, to thereby correlate the test condition with
the known condition.
3. A method according to claim 2, wherein the known condition is
selected from the group consisting of (i) the presence of a known
molecule, (ii) a known molecular characteristic, and (iii) a known
environmental condition.
4. A method according to claim 3, wherein the nano-scale membrane
channel is partially blockaded by the presence of an auxiliary
molecule in the membrane channel.
5. A method according to claim 4, wherein the test condition is
comprised of the introduction of a test molecule to the conductive
medium.
6. A method according to claim 5, wherein the auxiliary molecule is
at least a portion of a single antibody, wherein the test molecule
is an antigen, and wherein the known condition is one or more
antigen-antibody binding events.
7. A method according to claim 6, wherein the known condition is a
plurality of discrete antigen-antibody binding events, and wherein
the process further comprises measuring the average length of time
during which the one or more discrete antigen-antibody binding
events takes place during the period of time.
8. A method according to claim 5, wherein (a) further comprising
correlating the blockade channel current signal pattern or the
change in the blockade channel current signal pattern with a
characterization of interaction between the test molecule and the
auxiliary molecule.
9. A method according to claim 5, wherein the auxiliary molecule is
an aptamer, wherein the test molecule is a biomolecule, and wherein
the known condition is binding affinity.
10. A method for screening candidate agents for preventing pore
formation, comprising in a conductive medium, contacting a membrane
with one of the candidate agents and then placing a
membrane-permeabilizing agent in the presence of the membrane and
the candidate agent under conditions which would permit pore
formation in the membrane in the absence of the candidate agent,
and introducing an ionic charge to the membrane in the conductive
medium and measuring the time which passes from the moment the
membrane-permeabilizing agent is placed in the presence of the
membrane until pore formation in the membrane permits an ionic
current to flow through the pore to be detected.
11. A method for identifying an effective cytosolic antigen
delivery mechanism for use in evoking a cytotoxic T Lymphocyte
response in an organism challenged by one or more cytosolic
virulence factors, comprising introducing a single pore-forming
toxin to a membrane so as to form at least one pore through which
an ionic current may flow, and causing the current to flow through
the pore to generate one or more signals indicative of at least one
characteristic of antigen transmembrane transport.
12. Apparatus comprising a membrane which partitions a conductive
medium, the membrane defining at least one nano-scale channel
through which an ionic current may flow, the channel having
disposed therein an auxiliary molecule which causes modulations in
the ionic current when the ionic current flows through the channel,
an ionic current source, a sensor for measuring the ionic current
flowing through the channel and generating a blockade channel
current signal, a computer system programmed and configured for
receiving and processing the blockade channel current signal from
the sensor to enable the extraction of a set of one or more pattern
features.
13. Apparatus according to claim 12, wherein the computer system is
programmed and configured to further enable recognition of a
pattern of the blockade channel current signal, or a change in the
pattern of the blockade channel current signal, over time.
14. Apparatus according to claim 13, wherein the membrane is a
solid-state membrane.
15. Apparatus according to claim 13, wherein the membrane is a
lipid bi-layer.
16. Apparatus according to claim 13 wherein the auxiliary molecule
is covalently bound to a first study molecule to produce a first
study molecule/auxiliary molecule blockade channel current signal
carried by the ionic current flowing through the channel, and the
computer system is programmed and configured to detect a change in
the pattern of the first study molecule/auxiliary molecule blockade
channel current signal upon interaction of the first study molecule
with a second study molecule over time, and to correlate the change
in the pattern with at least one previously measured pattern, or at
least one previously measured change in pattern, associated with a
known molecule or a known molecular characteristic, respectively.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] Claim is hereby made to the benefits of prior, co-pending
U.S. Provisional Patent Applications 60/616,274, 60/616,275,
60/616,276 and 60/616,277, all filed on Oct. 6, 2004. The
disclosures of each of the foregoing are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention pertains to the field of molecular
chemistry, and in particular, to methods of molecular
characterization and analysis using electrophoretically driven ion
flow through one or more channels in a suitable substrate.
SEQUENCE LISTING
[0003] A Sequence Listing, which lists the sequences identified by
Sequence ID Number, corresponding to the Sequence ID Number used
herein, accompanies this disclosure and is incorporated herein by
reference.
[0004] When used herein, unless the context demands otherwise, the
term "channel" is synonymous with the term "pore." A "conductive
medium" means a medium capable of conducting an ionic flow.
BACKGROUND
[0005] The notion of using channels as detection devices dates back
to the Coulter counter, where pulses in channel flow were measured
in order to count bacterial cells. Cell transport through the
Coulter counter is driven by hydrostatic pressure--and interactions
between the cells and the walls of the channel are ignored. Since
its original formulation, channel sizes have reduced from
millimeter scale to nanometer scale, and the detection mechanism
has shifted from measurements of hydrostatically driven fluid flow
to measurements of electrophoretically driven ion flow. Analytes
observed via channel measurements are likewise reduced in scale,
and are now at the scale of single biomolecules such as DNA and
polypeptides. To a limited extent, some intramolecular,
Angstrom-level, features are beginning to be resolved as well.
[0006] For nanoscopic channels, interactions between channel wall
and translocating biomolecules can't, usually, be ignored. On the
one hand this complicates analysis of channel blockade signals, on
the other hand, tell-tale on-off kinetics are revealed for binding
between analyte and channel, and this is what has allowed the
probing of intramolecular structure on single DNA molecules.
[0007] Biophysicists and medical researchers have performed
measurements of ion flow through single nanopores since the 1970's.
It was they who first designed the sensitive patch clamp amplifiers
needed for the picoampere ionic current measurements. Typically,
these single channel techniques have been applied to low
conductance, ion selective channels, such as gated potassium
channels, but they have also been applied to larger channels
involved in metabolite and macromolecule transport. The use of
large biological pores as polymer sensors is a relatively new
possibility that dates from the pioneering experiments of Bezrukov
et al., circa 1994, and Kasianowicz et al., circa 1996. Bezrukov,
S. M., I. Vodyanoy, V. A. Parsegian. 1994, Counting polymers moving
through a single ion channel, Nature 370 (6457), pgs 279-281;
Kasianowicz, J. J., E. Brandin, D. Branton, and D. W. Deamer, 1996,
Characterization of Individual Polynucleotide Molecules Using a
Membrane Channel, Proc. Natl. Acad. Sci. USA 93(24), 13770-73. This
work proved that resistive pulse measurements, familiar from cell
counting with the Coulter counter, could be reduced to the
molecular scale and applied to polymers in solution. A seminal
paper by Kasianowicz et al., circa 1996, then showed that
individual DNA and RNA polymers could be detected via their
translocation blockade of a nanoscale pore formed by
.alpha.-hemolysin toxin. In such prior nanopore detection work, the
data analysis problems were of a familiar "Coulter event"
form--where the event was associated with a current blockade at a
certain, fixed level. For this type of blockade signal, information
on transitions between sub-blockades is often negligible.
[0008] Until now, there has been very little, if any, recognition
of the wealth of information which might be obtained from current
channel blockades with frequent transitions between distinct
sub-blockades. The present invention involves not only the
recognition of this information, but also techniques for obtaining
the information and applying it in very useful ways.
SUMMARY OF THE INVENTION
[0009] The present invention brings forth a novel modification of a
channel current-based detection system, using what are herein
termed auxiliary molecules and artificial intelligence (e.g.,
pattern recognition) software systems for the analysis of the data
obtained. The auxiliary molecule employed produces a "toggling"
blockade between several different levels (with two usually
dominating). The resulting blockade signal for the auxiliary
molecule by itself is no longer at approximately a fixed blockade
level, but now consists of a telegraph-like blockade signal with
stationary statistics. Upon binding of analyte to the auxiliary
molecule the toggling signal is greatly altered, to one with
different transition timing and different blockade residence
levels. Building on this as a biosensing foundation requires
sophisticated computational tools, such as Hidden Markov Models and
Support Vector Machines (SVMs), but offers at least a hundred-fold
improvement to the sensitivity of the device. This is because the
events of analyte bound vs non-bound (detected or not) can now be
discerned with the much greater information in the multiple-level
residencies and transition timings. The sparse information provided
by previous systems, with non-bound having one blockade level and
bound having another, single, blockade level, presents a minimal
amount of information when to compared to the present invention.
Given the noise in the prior systems and the limited dynamic range
for blockades of the open channel current, the systems devoid of an
auxiliary molecule component are greatly restricted because they
are not endowed with this sensitive timing information.
[0010] Thus, the present invention provides in one embodiment a
method comprising receiving a blockade channel current signal
carried by an ionic current flowing through a partially-blockaded
nano-scale membrane channel defined by a membrane which partitions
a conductive medium, and exposing the channel to a test condition
during ionic current flow while extracting from the blockade
channel current signal, a set of one or more pattern features to
establish over a period of time either a blockade channel current
signal pattern or a change in the blockade channel current signal
pattern. In another embodiment of the invention, this method
further comprises comparing the blockade channel current signal
pattern, or the change in the blockade channel current signal
pattern, to at least one blockade channel current signal pattern,
or at least one change in the blockade channel current signal
pattern, associated with at least one known condition, to thereby
correlate the test condition with the known condition.
[0011] The present invention also provides, in another embodiment,
a method for detecting antigens using at least one nanometer-scale
channel through which an ionic current may flow, which channel is
coupled to an antibody to form a blockade sufficient to modulate
ionic current passing through the channel so as to provide a signal
indicative of the blockade in the channel, thereby providing a
signal which, when processed in accordance with the present
invention, enables bio-sensing. Another embodiment of the invention
involves utilization of pattern recognition software specifically
designed to analyze channel currents.
[0012] In another embodiment, the method comprises drawing part of
a single antibody into a single nanometer-scale channel, where only
a single channel is established to conduct current through a
membrane (e.g., an organic or solid-state membrane). A
single-antibody blockade event forms the background channel current
signal that is monitored for antigen binding events. An
antigen-binding event results in a perturbation of the channel
current signal of the antibody channel blockade, and is the basis
of the detection observation.
[0013] The methods of this invention also can be applicable in a
variety of ways where many channels are present, where each channel
offers parallel conductance paths for the ionic current, and where
each channel is exposed to antibody to establish a background
collection of channel/antibody signals that is modifiable in the
presence of antigen. Multiple antibody species can be present in
this multi-channel embodiment. Anything that can evoke an antibody
response can be taken as the antigen or collection of antigens for
which the bio-sensing is designed.
[0014] One application of the present invention is in the field of
bio-defense. Thus, in accordance with one embodiment of this
invention, a fractionated bio-terror organism, such as, e.g.,
fractionated anthrax spores (i.e., pieces of protein, etc.), may be
injected into a mouse to evoke a broad-spectrum antibody response.
A collection of nanopore/antibody channel currents could then be
prepared with those antibodies for detection of fractionated
anthrax spores obtained via an air-concentrator with the same
protein fractioning processes. Introduction of the fractionated
air-concentrate when spores are present to the nanopore/antibody
detector would then evoke a discernible indication of the presence
of that microorganism.
[0015] Yet another embodiment of the present invention provides a
method for identifying pore inhibiting agents for treatment against
pore-forming toxins associated with disease, bacterial and viral
infection, and chemical exposure. The method makes use of pattern
recognition software specialized to analysis of channel currents.
In each embodiment a pore-forming toxin, or membrane-permeabilizing
agent, is introduced into a membrane (typically a synthetic
biological membrane, such as a lipid bilayer, although hybrid
organic/solid state membranes may also be used). In another
embodiment of the invention, the lifetime of a single toxin channel
is characterized prior to its disruption by the pore inhibiting
agent being screened. The method also enables one to screen for
agents that might prevent pore formation, by examination of the
average time before channel formation under identical circumstances
(pore forming toxin w/wo pore inhibiting agents).
[0016] In another embodiment, multiple toxin channels are
introduced and disruptions to their cumulative currents are
studied, or the toxin is introduced and the timescale of
multi-channel formation is examined in the presence of pore
inhibiting agent. The multi-pore embodiments are applicable on very
fast time-scales, while the single-pore embodiments provide
single-molecule information about the action of the pore inhibiting
agents. The single-channel embodiments can also be used to inform
the design and statistical modeling of the multi-channel
embodiments in the screening process. Pore-inhibiting agents that
can be studied via these embodiments include antimicrobial
peptides, the larger family of amphipathic molecules, and
antibodies.
[0017] Medicines and vaccines that provide resistance to
pore-forming and membrane permeabilizing toxins can be rapidly
assayed using methods of this invention. Pore-forming toxins are
often virulence factors associated with bacterial and viral
infections, or with toxic chemical exposure. The medicines obtained
can either provide resistance to pore-forming toxin prior to their
pore-formation or act as treatment after exposure to pore-forming
toxins.
[0018] Still another embodiment of this invention provides a method
for identifying effective cytosolic antigen delivery mechanisms for
use in evoking cytotoxic T lymphocyte (CTL) responses in organisms
challenged by cytosolic virulence factors. In this particular
embodiment, pore-forming toxins are used that allow introduction of
peptides and other molecules into the cytosol of their host cell.
The embodiment entails establishing a single such pore-forming
toxin in a membrane followed by measurement of peptide (antigen)
transmembrane transport via channel current measurements. This
embodiment make use of established procedures for attaching target
antigen to the recognition sequence of virulence factors associated
with the pore-forming toxin in the natural setting. Via this
transmembrane transport mechanism, a number of antigen/recognition
molecules can be assayed for effective use with the chosen
pore-forming toxin, and thereby provide an embodiment of a
cytosolic antigen delivery assayer.
[0019] Medicines and vaccines that provide resistance to cytosolic
virulence factors can be rapidly assayed with this approach.
Pore-forming toxins and viruses (e.g., HIV) are often associated
with cytosolic virulence factors. Medicines and vaccines can
provide treatment or resistance to such cytosolic virulence factors
if an effective mechanism is devised for delivery of
virulence-associated antigen to the cytosol of the host cell.
Effective delivery of antigen to the cytosol is the first step in
obtaining an effective agent for evoking a CTL response against
virulence factors of the invading microorganism.
[0020] Yet another embodiment of the present invention is a method
for measuring the binding kinetics of an antibody to an associated
antigen, and more generally for measuring the affinity of an
antibody for a given antigen. The invention employs a
nanometer-scale channel that is coupled to an antibody as an
antibody characterization and antibody-antigen efficacy-screening
tool. In one embodiment, the method employs pattern recognition
software specialized to analysis of channel currents. In another
embodiment, the method comprises drawing part of a single antibody
into a single nanometer-scale channel. When only a single channel
is conducting current through a membrane (e.g., a lipid bilayer or
solid state membrane) the antibody blockade is a clearly
discernible event, and forms the background channel current signal
that is monitored (e.g., using a software-based signal processor)
for antigen binding events. An antigen binding event then perturbs
the channel current signal resulting from the antibody
channel-blocking alone, and is the basis of the detection
observation. The methods are also applicable in a variety of
embodiments when many channels are present (offering parallel
conductance paths for the ionic current), where each channel is
exposed to the same antibody to establish a background collection
of channel/antibody signals that is modifiable in the presence of
antigen that binds to that antibody. In either the single
nanopore/antibody or multiple nanopore/antibody embodiments this
provides a platform for antibody characterization. With addition of
antigen that led to the antibody obtained, the binding kinetics can
be observed, and the strength (efficacy) of the antibody for that
antigen assessed.
[0021] Through practice of the present invention, single antibody
studies can be performed in a new way via the nanopore device. Of
particular importance are studies of the antibody-antigen binding
affinities for assessment of the antibody efficacy in medical
treatments.
[0022] These and other embodiments, features and advantages of the
present invention shall now become apparent from the ensuing
detailed description of embodiments of the invention, the appended
figures and accompanying claims.
SUMMARY OF THE FIGURES
[0023] FIG. 1a. (prior art) (A) shows a nanopore device based on
the .alpha.-hemolysin channel. It has been used for analysis of
single DNA molecules, such as ssDNA, shown, and dsDNA, a nine
base-pair DNA hairpin is shown in (B) superimposed on the channel
geometry. The channel current blockade trace for the nine base-pair
DNA hairpin blockade from (B) is shown in (C).
[0024] FIG. 1b is a flow diagram illustrating the signal processing
architecture that was used to classify DNA hairpins in accordance
with one embodiment of this invention: Signal acquisition was
performed using a time-domain, thresholding, Finite State
Automaton, followed by adaptive pre-filtering using a
wavelet-domain Finite State Automaton. Hidden Markov Model
processing with Expectation-Maximization was used for feature
extraction on acquired channel blockades. Classification was then
done by Support Vector Machine on five DNA molecules: four DNA
hairpin molecules with nine base-pair stem lengths that only
differed in their blunt-ended DNA termini, and an eight base-pair
DNA hairpin. The accuracy shown is obtained upon completing the
15.sup.th single molecule sampling/classification (in approx. 6
seconds), where SVM-based rejection on noisy signals was
employed.
[0025] FIG. 2. A sketch of the hyperplane separability heuristic
for SVM binary classification. An SVM is trained to find an optimal
hyperplane that separates positive and negative instances, while
also constrained by structural risk minimization (SRM) criteria,
which here manifests as the hyperplane having a thickness, or
"margin," that is made as large as possible in seeking a separating
hyperplane. A benefit of using SRM is much less complication due to
overfitting (a problem with Neural Network discrimination
approaches).
[0026] FIG. 3. (a) The channel current blockade signals observed
when selected DNA hairpins are disposed within the channel. The
left panel shows the five DNA hairpins, with sample blockades, that
were used to test the sensitivity of the nanopore device. The top
right panel shows the power spectral density for signals obtained.
The bottom right panel shows the dominant blockades, and their
frequencies, for the different hairpin molecules. (b) A graph
showing the single-species classification prediction accuracy as
the number of signal classification attempts increases (allowing
increase in the rejection threshold). (c) A graph showing the
prediction accuracy on 3:1 mixture of 9TA to 9GC DNA hairpins. 8GC
has SEQ ID NO: 1. 9TA has SEQ ID NO: 2. 9GC has SEQ ID NO 3. 9CG
has SEQ ID NO: 4. 9AT has SEQ ID NO 5.
[0027] FIG. 4. Signal analysis from an experiment carried out in
accordance with an embodiment of this invention, involving a single
antibody but different blockade signals. (A) The lower-level (LL)
blockade of this signal is not as deep as the others, and may
correspond to antibody capture of a free heavy chain. (B) The LL
blockade of this signal is about mid-way between those shown, so
may correspond to capture a Fab arm. (C) The LL blockade of this
signal is the deepest, and difficult to eject with voltage reversal
(requiring several attempts), so may correspond to an entire Fc arm
capture. Each graph shows the level of current in picoamps over
time in milliseconds.
[0028] FIG. 5. Signal analysis from an experiment carried out in
accordance with an embodiment of this invention, showing (A)
Blockade due to T6 antibody and (B) T6 antibody blockade one minute
after exposure to antigen. Each graph shows the level of current in
picoamps over time in milliseconds.
[0029] FIG. 6. (A) Channel current blockade signal where the
blockade is produced by 9GC DNA hairpin with 20 bp stem. (B)
Channel current blockade signal where the blockade is produced by
9GC 20 bp stem with magnetic bead attached. (C) Channel current
blockade signal where the blockade is produced by c9GC 20 bp stem
with magnetic bead attached and driven by a laser beam chopped at 4
Hz, in accordance with an embodiment of this invention. Each graph
shows the level of current in picoamps over time in
milliseconds.
[0030] FIG. 7. Shows the architecture of the FSA employed in an
embodiment of this invention (specific details on tuning parameters
can be found in Winters-Hilt, S., W. Vercoutere, V. S. DeGuzman, D.
W. Deamer, M. Akeson, and D. Haussler. 2003. Highly Accurate
Classification of Watson-Crick Basepairs on Termini of Single DNA
Molecules. Biophys. J. 84:967-976 (hereinafter referred to as
"Winter-Hilt, et al. 2003"). Tuning on FSA parameters was done
using a variety of heuristics, including tuning on statistical
phase transitions and feature duration cutoffs.
[0031] FIG. 8. The time-domain FSA shown in FIG. 7 is used to
extract fast time-domain features, such as "spike" blockade events.
Automatically generated "spike" profiles are created in this
process. One such plot is shown here for a radiated 9 base-pair
hairpin, with a fraying rate indicated by the spike events per
second (from the lower level sub-blockade). Results: the radiated
molecule has more "spikes" which are associated with more frequent
"fraying" of the hairpin terminus--the radiated molecules were
observed with 17.6 spike events per second resident in the lower
sub-level blockade, while for non-radiated there were only 3.58
such events (shown in FIG. 9).
[0032] FIG. 9. Automatically generated "spike" profile for the
non-radiated 9 base-pair hairpin. Results: the non-radiated
molecule had a much lower fraying rate, judging from its much less
frequent lower-level spike density (3.58 such events per
LLsec).
[0033] FIG. 10. This figure shows the blockade sub-level noise
reduction capabilities of an HMM/EM .times.5 filter with gaussian
parameterized emission probabilities. The sigma values indicated
are multiplicative (i.e. the 1.1 case has standard deviation
boosted to 1.1 times the original standard deviation). Sigma values
greater than one blur the gaussians for the emission probabilities
to greater and greater degree, as indicated for each resulting
filtered signal trace in the figure. The levels are not preserved
in this process, but their level transitions are highly preserved,
now permitting level-lifetime information to be extracted easily
via a simple FSA scan (that has minimal tuning, rather than the
very hands-on tuning required for solutions purely in terms of
FSAs).
[0034] FIG. 11. This figure shows results from various SVM
implementations. From the blockade data analyzed, it is typically
found that SMO .eta.-management done in the S-W way indicated, and
divergence-based kernels, allow for the best, most stable,
classifiers.
[0035] FIG. 12. The preliminary aptamer experiments performed are
based on the dsDNA molecule shown here, with 5 base overhang used
as a sensing moiety for the complimentary 5-base ssDNA molecule.
The target 5-base ssDNA is introduced subsequent to obtaining a
toggler-type capture of the aptamer molecule (properly annealed).
The aptamer experiment is referred to above as a pseudo-aptamer
experiment due to its simplification to a DNA annealing
detection.
[0036] FIG. 13. Shows a collection of blockade signal traces. The
short blockades in the upper left slide result from ssDNA
translocation by the un-annealed ssDNA components. The long
blockade in the upper left, and all right, panels is for capture
with the overhang end entering first. The panel third down on the
left shows the desired toggler-type signal. Each graph shows the
level of current in picoamps over time in milliseconds.
[0037] FIG. 14. Shows the modification to the toggler-type signal
shortly after addition of 5-base ssDNA. The observed change is
hypothesized to represent annealing by the complimentary 5-base
ssDNA component, and thus detection of the 5-base ssDNA molecule.
Each graph shows the level of current in picoamps over time in
milliseconds.
DETAILED DESCRIPTION OF EMBODIMENTS
[0038] The embodiments of the present invention described here for
biological channels are also applicable on solid-state platforms in
a variety of incarnations--the underlying biophysics remains the
same: a nanometer-scale ion channel together with ion-channel
blockade analysis that uses a highly adaptable set of
machine-learning-based signal analysis and pattern recognition
methods. Going forward, solid-state channels will allow greater
control of components of the device physics, so will play a
prominent role. However, a complication with solid-state nanoscopic
channels is that the technology for preparing them in solid-state
media is still in its infancy. Preparing a single channel in a
silicon nitride, for example, is fraught with difficulties in
maintaining a stable channel--partly because it is found that
atomic flow occurs at the surface of the substrate that leads to
closing of such small channels. Certain protein channels, on the
other hand, are known to be very stable, and benefit from the fact
that nature has designed them to self-assemble. Their biological
origin has also led to their robust properties of heat dissipation,
etc., upon polymer translocation, something that poses added
complication in the solid-state setting. For these reasons,
advances to date in nanoscopic-channel based detection have
primarily involved single nanometer-scale protein channels
("nanopores") intercalated in lipid bilayer substrates. These
biological channels provide a plethora of interesting research
milestones with the information gained about the biological systems
themselves.
[0039] A need continues to exist for improved understanding of
specific and general membrane constituents that contribute to pore
formation, as mentioned above, and for a new, nanopore-based
cheminformatics characterization of the many currently known
protein channels. Areas of research accessible to nanopore-based
cheminformatics include: ion channel and pore-forming toxin
characterization; assays of molecules that confer beneficial
modification to channel function; and understanding the
pore-forming mechanism. Agents that stimulate membrane damage
include not only bacterial pore-forming toxins (PFTs), but some
enveloped viruses, animal and plant toxins, and man-made molecules
and environmental pollutants. PFTs are one of the most potent and
versatile weapons wielded by invading microbes. A number of
medically relevant pathogens are known to produce pore-forming
proteins. In many cases, PFTs are significant virulence agents:
markedly reduced virulence is observed in mutant bacterial strains
that have lost their ability to produce their PFT. Antibodies
against the alpha-hemolysin PFT, for example, have been shown to
protect against various S. aureus infections, while immunization
against the PFT of the human pathogen Aeromonas hydrophilia leads
to protection. It should also be noted that, although PFTs directly
have detrimental impact by destroying tissue cells and first-line
immune cell defenses, their most deleterious impact is often less
obvious, due to a wide spectrum of short- and long-range secondary
reactions to the intracellular signaling confusion resulting from
the lysed cells. It is hypothesized that the additional information
obtained through practice of this invention will lead to a better
understanding of these pore-forming constituents.
Membrane Environment and Alternative Channels for use in Biosensing
and Screening
[0040] The .alpha.-hemolysin channel of use in certain embodiments
of the present invention is a protein heptamer, formed by seven
identical 33 kD protein molecules secreted by Staphylococcus
aureus. The total channel length is 10 nm and is comprised of a 5
nm trans-membrane domain and a 5 nm vestibule that protrudes into
the aqueous cis compartment. The narrowest segment of the pore is a
1.5 nm-diameter aperture, see FIG. 1a. By comparison, a single
strand of DNA is about 1.3 nm in diameter. Given that water
molecules are 0.15 nm in diameter, this means that one hydration
layer separates ssDNA from the amino acids in the limiting
aperture. This places the charged phosphodiester backbone, hydrogen
bond donors and acceptors, and apolar rings of the DNA bases within
one Debye length (3 .ANG. in 1 M KCl) of the pore wall. Not
surprisingly, DNA and RNA strongly interact with the
.alpha.-hemolysin channel during translocation. Although dsDNA is
too large to translocate, about ten base-pairs at one end can still
be drawn into the large cis-side vestibule. This actually permits
the most sensitive experiments to date, as the ends of "captured"
dsDNA molecules can be observed for as long as desired to resolve
features. For ssDNA translocation under normal operating
conditions, approximately one nucleotide passes the limiting
aperture of the channel every microsecond, and a vigorous effort is
underway to find ways to slow down and control this translocation
process.
[0041] Thus, in some embodiments of the present invention there is
provided a nanopore detector, using either (i) a membrane protein
that forms an aqueous transmembrane pore, or (ii) a solid state
nanopore formed in a suitable solid state substrate. The
alpha-hemolysin nanopore detector provides an explicit description
of one of these embodiments of the channel detection invention (see
FIG. 1a). Alpha-hemolysin is a membrane protein that forms an
aqueous transmembrane pore (similar embodiments are possible with
solid-state variants). In order for such a pore to form in a lipid
bilayer it is necessary for lipid molecules to be laterally
displaced. For microbial pore-forming toxins, the energy used to
drive the pore-formation process is thought to be solely provided
by conformational changes in the toxin molecules themselves (i.e.,
the process is ATP-independent). For alpha-hemolysin, the energy
needed for the pore-formation is shown to be due to the
oligomerization of toxin monomers to form the channel heptamer
complex. Although pore-forming toxins, and membrane-permeabilizing
molecules in general, have incredibly diverse sequence and
structure, they all share in the same mechanism. They either
directly intercalate into target membranes, or bind to particular
target molecules in the membranes, and do so from a solution
soluble monomeric form. They then assemble into multimeric,
membrane-spanning pores. Attributes of the membrane, other than
specific binding molecules, are often critical to this process,
such as cholesterol-rich microdomains or lipid rafts. The role of
cholesterol as a specific binding agent for individual formation
events is well documented, and is required for pore-formation by
many toxins (in natural setting, otherwise, channel formation can
be activated by introduction of solvent, e.g., n-decane), including
the Anthrax pore-forming toxin. But cholesterol-rich microdomains
also play a role in channel formation in a non-specific manner.
This is due to the microdomains acting as concentration platforms
that can aid in the assembly of proto-channel multimers as is
described for the aerolysin heptamer in Abrami, L., M. Fivaz, F. G.
van der Goot, Surface dynamics of aerolysin on the plasma membrane
of living cells, Int. J. Med. Microbiol. 290, 363-367 (2000). Once
a proto-channel multimer has formed, such microdomains can also aid
in the last step of transmembrane channel formation. This is due to
the junctions between cholesterol-sphingolipid-rich domains and
fluid-phase phosphoglyceride domains having locally favorable
(weakened) bilayer characteristics that favor membrane penetration.
The opposite effect is also known to be medically relevant: unknown
membrane constituents, or the lack thereof, can block an
alpha-hemolysin heptamer complex from inserting a transmembrane
functional domain. This is found to be the case for human
granulocytes, where the agent preventing channel formation is
unknown.
[0042] Cholesterol not only factors into protein-channel based
detection as a specific and general pore-formation co-agent, but
also, paradoxically, as a membrane strengthening agent in that it
allows for greater vibrational shock resistance in the bilayer. For
this reason, even though cholesterol is not required for
alpha-hemolysin channel formation in bilayers, it is still
important in nanopore experiments purely as a bilayer-strengthening
agent, which also serves to reduce the membrane noise contribution
in the nanopore detector by approximately 35% (see Methods part of
the EXPERIMENTAL Section).
A New Method for Single Molecule Detection and Characterization
[0043] Angstrom precision structures for numerous DNA, RNA, and
protein molecules have been revealed by X-ray diffraction analysis
and NMR spectroscopy. These approaches rely upon average properties
of very large numbers of molecules and are often biased towards
crystallization and NMR conformer structures different from those
present in solution under physiological conditions. With the
introduction of single molecule analytical techniques in the early
1990's, however, new explorations into polymer structure and
dynamics have begun. Others have shown that atomic force microscopy
and laser tweezers, in particular, have permitted three direct
measures of the force at the single molecule level: (1) the force
required to break A.cndot.T or G.cndot.C base pairs, (2) the force
required to extend single or double stranded DNA through distinct
structural conformations, e.g., B form to S form DNA, etc., and (3)
the forces exerted by polymerases working on polynucleotides.
[0044] Channel current based nanopore cheminformatics provides a
new, incredibly powerful, method for biophysical and biochemical
analysis. Single biomolecules, and the ends of biopolymers such as
DNA, can now be examined in solution with nanometer-scale
precision. In early studies, it was found that complete base-pair
dissociations of dsDNA to ssDNA, "melting", could be observed for
sufficiently short DNA hairpins. In later work (e.g., Winters-Hilt
et al., 2003), the nanopore detector attained Angstrom resolution
and was used to "read" the ends of dsDNA molecules, and was
operated as a chemical assayer. In recent work, the nanopore
detector is being used to observe the conformational kinetics at
the termini of single DNA molecules. As part of the unique work in
connection with the present invention, the nanopore is used to
observe individual molecular interaction on-off binding events (see
Results part of EXPERIMENTAL Section).
Detection of Short Term Binding and Stationary Phase.
[0045] There are important distinctions in how a nanopore detector
can function: direct vs. indirect measurement and static or
stationary vs. dynamic (possibly modulated) or non-stationary
measurement.
[0046] A nanopore-based detector can directly measure molecular
characteristics in terms of the blockade properties of individual
molecules--this is possible due to the kinetic information that is
embedded in the blockade measurements, where the
adsorption-desorption history of the molecule (to the surrounding
channel), and the configurational changes in the molecule itself,
imprint on the ionic flow through the channel. This offers
prospects for DNA sequencing and single nucleotide polymorphism
(SNP) analysis. Nanopore methods may even have potential for
single-molecule sequencing at some point in the future. Sequencing
and SNP analysis, however, represent a small fraction of the
immense potential of such a device. This is because it has now been
discovered that a nanopore-based detector can also measure
molecular characteristics indirectly, reporting on important
binding kinetics in particular.
[0047] The nanopore-based detector works indirectly if it uses a
reporter molecule that binds to certain molecules, with subsequent
distinctive blockade by the bound-molecule complex. One example of
this, with the established DNA experimental protocols, would be
exploration of transcription factor binding sites via the different
dsDNA blockade signals that occur with and without DNA-binding of a
hypothesized transcription factor. Similarly, a channel-captured
dsDNA "gauge" that is already bound to an antibody could provide a
similar blockade shift upon antigen binding to its exposed
antibody. The latter description provides the general mechanism for
directly observing the single molecule antigen-binding affinities
of any antibody. An unexpected result has been obtained, however,
that simplifies this picture. It has been observed that antibodies
can be directly captured and can produce a toggler-type signal on
the nanopore detector, without the need for a linkage to a dsDNA
gauge that is designed for that purpose. The hypothesized shift in
blockade pattern (non-stationary) with antigen binding is also
shown to occur in those preliminary studies (see Results part of
EXPERIMENTAL Section).
[0048] The nanopore signal with the most utility and inherent
information content is not merely the channel current signal for
some static flow scenario, but one where that flow is modulated, at
least in part, by the blockade molecule itself (with dynamic or
non-stationary information, such as changing kinetic information).
The modulated ion flow due to molecular motion and transient fixed
positions (bound states) is much more sensitive to environmental
changes than a blockade molecule (or open channel flow) where the
flow is at some fixed blockade value (the rate of toggle between
blockade levels could change, for example, rather than an almost
imperceptible shift in a blockade signal residing at a single
blockade value).
[0049] Part of the present novel device and method utility is not
be based so much on the ionic flow (whether static or stationary),
as changes in the modulations of that ionic flow. A transduction of
the molecular binding and conformational kinetics to observed
current measurement, for example, can be 100 times, or more,
sensitive to alterations of that molecules environment (and thus
its kinetics) than blockade observations based on molecules that
blockade the channel at one fixed position.
[0050] In order to use nanoscopic pores to detect and identify
single strands of DNA it is necessary to have automated signal
processing. This is due to both the millisecond timescale desired
for the classifications, and due to the immense number of signals
to be analyzed. Even in careful studies of individual encoded
molecules, where the channel blockade signals are obtained with
seconds of measurement time, the presence of noise requires a
typical data set to range from several hundred to several hundred
thousand signals. With use of automated signal processing, the data
size and processing speed no longer pose a fundamental problem, but
they do pose fundamental constraints on the methods and processing
architecture that can be employed.
[0051] FIG. 1b shows the prototype signal processing architecture
developed in (Winters-Hilt et al., 2003). The processing is
designed to rapidly extract useful information from noisy blockade
signals using feature extraction protocols, wavelet analysis,
Hidden Markov Models (s) and Support Vector Machines (SVMs). For
blockade signal acquisition and simple, time-domain,
feature-extraction, a Finite State Automaton (FSA) approach is used
that is based on tuning a variety of threshold parameters. The
utility of a time-domain approach at the front-end of the signal
analysis is that it permits precision control of the acquisition as
well as extraction of fast time-scale signal characteristics. A
wavelet-domain FSA (wFSA) is then employed on some of the acquired
blockade data, in an off-line setting. The wFSA serves to establish
an optimal set of states for on-line HMM processing, and to
establish any additional low-pass filtering that may be of benefit
to speeding up the HMM processing. HMMs can characterize current
blockades by identifying a sequence of sub-blockades as a sequence
of state emissions. See, e.g., Chung, S. H., J. B. Moore, L. Xia,
L. S. Premkumar, and P. W. Gage, 1990, Characterization of single
channel currents using digital signal processing techniques based
on Hidden Markov models. Phil. Trans. R. Soc. Lond. B 329. 265-285;
Chung, S-H., and P. W. Gage, 1998, Signal processing techniques for
channel current analysis based on hidden Markov models, in Methods
in Enzymology; Ion channels, Part B. P. M. Conn editior. Academic
Press, Inc., San Diego, 420-437; Colquhoun, D., and F. J. Sigworth,
1995, Fitting and statistical analysis of single-channel products,
in Single-channel recording, B. Sakmann and E. Neher editors,
Second edition, Plenum Publishing Corp., New York, 483-587. The
parameters of an HMM can then be estimated using a method called
Expectation/Maximization. Although HMMs can be used to discriminate
among several classes of input, multi-class computational
scalability tends to favor their use as feature extractors (see
Experimental section for further details). HMMs are well suited to
extraction of aperiodic information embedded in stochastic
sequential data such as in channel current blockades (or genomic
sequences). Classification of feature vectors obtained by the HMM
(for each individual blockade event) is then done using SVMs, an
approach which automatically provides a confidence measure on each
classification. SVMs are fast, easily trained, discriminators, for
which strong discrimination is possible without the over-fitting
complications common to neural net discriminators. SVMs strongly
draw upon variational methods in their construction and are
designed to yield the best estimate of the optimal separating
hyperplane (for classifier, see FIG. 2) with confidence parameter
information included (via hyperplane with margin optimization used
in structural risk minimization). The SVM approach also
encapsulates a significant amount of discriminatory information in
the choice of kernel in the SVM, and a number of novel kernels have
been developed (see the Experimental section for further details).
In previous work (Winters-Hilt et al., 2003) novel,
information-theoretic, kernels were successfully employed for
notably better performance over standard kernels.
[0052] Different tools are employed at each stage of the signal
analysis (as shown in FIG. 1b) in order to realize the most robust
(and noise resistant) tools for knowledge discovery, information
extraction, and classification. Statistical methods for signal
rejection using SVMs are also be employed in order to reject
extremely noisy signals (FIG. 3). Since the automated signal
processing is based on a variety of machine-learning methods, it is
highly adaptable to any type of channel blockade signal. This
enables a new type of informatics (cheminformatics) based on
channel current measurements, regardless of whether those
measurements derive from biologically based or a semiconductor
based channels.
Software Refinements are Partly Guided by the Bioengineering Device
Optimization.
[0053] The technical difficulty is to find molecules whose
blockades interact with the channel environment, via short
time-scale binding to the channel, or via inherent conformational
changes in its high force environment, and that do so at timescales
observable given the bandwidth limitations of the device. The 9
base-pair DNA hairpins previously studied have this property, so
they can form the basis of a critical enhancement to the device,
with one end performing the critical modulated blockade of the
channel, and the other end possibly bound to any sensing moiety,
such as an antibody. In the invention described here, the sensing
moieties are bound to something that is producing a very sensitive,
rapidly changing, blockade signal due to its interaction kinetics
with the channel environment (the DNA hairpin gauges, for
example).
[0054] One of the features of the present invention is the
indication that binding of antibody to the DNA hairpin gauges as
the sensing moiety appears to be unnecessary, at least for some
antibodies, as the antibodies themselves can enter the channel and
provide the sensitive "toggling blockade" signal needed (see FIG.
4). We then see that binding of antigen can be observed as a change
in that "toggling," as hypothesized (see FIG. 5).
[0055] A practical limitation of the nanopore device is that
molecules to be detected, or specially designed blockade gauges
(for indirect attachment) can blockade the channel and get "stuck"
in one blockade state. This occurs for a 20 base-pair DNA hairpin
that has the same 9 base-pair terminus as the "sensitive" DNA gauge
described in the following: Winters-Hilt, S., "Nanopore detection
using channel current cheminformatics," SPIE Second International
Symposium on Fluctuations and Noise, 25-28 May, 2004; Winters-Hilt,
S, and M. Akeson, "Nanopore cheminformatics," DNA and Cell Biology,
October 2004; Winters-Hilt et al., 2003; Winters-Hilt, S. 2003,
Highly Accurate Real-Time Classification of Channel-Captured DNA
Termini, Third International Conference on Unsolved Problems of
Noise and Fluctuations in Physics, Biology, and High Technology, pg
355-368; and Vercoutere, W., S. Winters-Hilt, V. S. DeGuzman, D.
Deamer, S. Ridino, J. T. Rogers, H. E. Olsen, A. Marziali, and M.
Akeson, 2003, Discrimination Among Individual Watson-Crick
Base-Pairs at the Termini of Single DNA Hairpin Molecules, Nucl.
Acids Res. 31, pg 1311-1318). It no longer modulates the channel
flow, being stuck in just one blockade state. But it has been
discovered that the molecule can be re-excited to its telegraph
signaling via a bead or some other form of attachment which is
periodically tugged by a laser beam (or magnet). In this way, the
larger DNA hairpin has it's blockade signal "re-awakened" to reveal
binding kinetic information at its channel-captured end.
Modulations to the channel environment appear to push an analyte
into a highly sensitive interaction (and detection) regime
vis-a-vis the channel detector (see FIG. 6).
Using CCC Signal Analysis to Advance Understanding of the Mechanism
of Drug Interaction.
[0056] The channel current cheminformatics (CCC) software needed
for the present channel-based detection methodology furthers many
objectives insofar as channel-based research is concerned,
including high throughput screening assays for ion-channel
modulatory agents. The drugs sought in high-throughput screening
assays are meant to modify channel function in the case of ion
channels with deleterious mutations, modify or disrupt channel
function in the case of PFTs, or prevent channel-formation by PFTs
in the first-place. There can be many subtleties to this search
process. It is known that the antiviral drug amantadine, for
example, inhibits the M2 pore function of type A influenza. The
drug has a slow onset of action, however, which some hypothesize
indicates its action may be due to allosteric influences on the
channel. Such subtleties of drug action are another area where
significant advances in understanding can be made via single
channel experiments coupled with the CCC tools.
Antibody Detection Interfaces for the Nanopore Detector.
[0057] In what follows, the properties of antibodies are given in
detail to convey the subtle issues nanopore detection may help to
resolve, and thereby provide improved opportunities for drug
discovery. Other proteins, or biomolecules in general, will rarely
have the same complexity as an antibody (or antibody-antigen
interaction). Thus, in many respects, the nanopore detector
analysis of antibody and antibody-antigen blockade observations are
probing some of the most complex of protein-protein
interactions.
[0058] The basic domain structure of antibody is an elongated
globular domain consisting of beta-pleated sheets. Antigen binding
occurs in the amino-terminal domain, while the effector functions
are localized to the C-terminal domains. A flexible hinge joins the
amino-terminal domains of the molecule (Fab consisting of two
domains of the heavy chain and the entire light chain) with the
carboxy-terminal portion (Fc). In the Experimental section we
demonstrate that antibodies can be captured by the nanopore, most
likely at their amino or carboxy terminus. Moreover, perturbations
of the current blockade initiated by the capture are observed upon
introduction of antigen. Presumably these are the result of
antibody binding to the antigen. This interaction could affect the
current blockade in two ways: 1. the antibody molecule may alter
its conformation, or 2. the mass added by the (probably
asymmetrical) addition of the antigen molecule, and consequent
hinge flexibility, may affect the binding kinetics of the antibody
in the pore. Both probably occur. The following describes the
conformational perturbations in the antibody that occur on binding
antigen, and how this may play a role in the initiation of antibody
effector functions.
[0059] Immunoglobulin (Ig) is a bifunctional molecule. Antigen is
bound by the Fab portion. Upon binding to antigen, a series of
events are initiated by the interaction of the antibody Fc region
with serum proteins and cellular receptors. Biological effects
resulting from Fc interactions include activation of the complement
cascade, binding of immune complexes by Fc receptors on various
cells, and the induction of inflammation. Although effector
molecules may bind to Fc of free antibody, binding is greatly
enhanced if the antibody has bound antigen. Two different models
have been proposed to explain this: associative and allosteric. The
associative model explains enhanced binding as a result of
multivalent interactions between the effector molecules and
multiple Fc regions brought into close proximity by multiple
antibodies binding to an antigen. The allosteric model proposes
that there is a conformational change induced when antibody binds
to antigen that makes the sites on Ig more accessible to the
effector molecules. These models are not mutually exclusive and
both mechanisms may play a role. There is strong evidence showing
conformational changes within the Fab when antibody binds to
antigen (reviewed below). Although it has not been shown that this
conformational change is transmitted to the Fc region, flexibility
of the hinge and the CH.sub.2--CH.sub.3 domain interface has
clearly been demonstrated and associated with effector
function.
[0060] The Ig molecule has structural flexibility that allows
accommodation to the requirements of antigen binding and effector
function. Changes in the structure of the Fab have been
demonstrated on antigen binding. Due to difficulties in performing
structural analyses on intact Ig molecules, it has only recently
been possible to show whether conformational changes induced by
antigen binding in IgG are transmitted to the Fc and effector
molecule binding sites.
[0061] There is clear evidence of a conformational change on
antigen binding by pentameric IgM. In the absence of antigen, IgM
exists as a multimer yet binds to C1q with an affinity of only
10.sup.4 to 10.sup.5 M.sup.-1. Complexed with antigen, the affinity
increases to greater than 10.sup.7 M.sup.-1, and a single IgM
complexed to antigen can activate complement. Electron microscopy
has been performed on IgM, both free and bound to antigen. In its
uncomplexed form, the pentameric IgM assumes a star-shaped, planar
conformation. When bound to antigen, the molecule folds, with the
Fab regions dislocated out of the plane of the formed by the Fc's.
This change in conformation either results in the exposure of
additional complement-binding sites (associative model) or a change
in the site itself that is more favorable to complement binding
(allosteric model). In this case, the allosteric model is best
supported by the data. Pentameric Fc isolated by the proteolytic
removal of Fab binds C1q with the same affinity as uncomplexed
intact IgM. Thus the effect of antigen binding cannot simply be the
exposure of additional C1q-binding sites by movement of Fab arms,
since the physical removal of these arms does not increase
affinity.
[0062] There is structural flexibility inherent in the domain
structure of the Ig molecule. Defined motions include: 1. Fc
wagging, 2. Fab arm rotation, relative to the Pc, 3. Fab arm
waving, alters the angle formed between the two Fab's relative to
the Fc (T-shape vs Y-shape), and 4. Fab elbow bend, changing the
angle between the Fv and the second domain of Fab (formed by CH1
and CL). All of these motions occur between different domains of
the antibody; none involve changes in conformation within a domain.
The greatest flexibility, and the major difference between Ig
isotypes, is in the hinge region. Although it was originally
thought, primarily on the basis of hinge-deletion mutants, that the
structure of the hinge region was a primary influence on antibody
effector function, more recent data suggests that hinge may have
less of an effect on Fc-mediated functions than was initially
assumed.
[0063] Interdomain flexibility of antibody probably serves several
functions. Fab motions allow for bivalent antigen binding when
individual epitopes are separated by variable distances and may
even be necessary for optimal binding of monovalent antigen. Fc
wagging probably increases accessibility of complement and Fc
receptor binding motifs to their receptors, accomplished by
minimizing steric hinderance that may occur on antigen binding.
Finally, it is possible that interdomain flexibility transmits a
conformational signal on antigen binding from the Fab to the
Fc.
[0064] High resolution X-ray crystallographic studies have been
performed with Fab and Fv fragments in the presence and absence of
antigen. These studies have shown that the antibody combining site
changes conformation to accommodate the antigen and bring
hypervariable residues into contact with antigenic structures. For
some antibodies these changes were minor, but in others major
conformational changes were observed which not only affected the
Fv, but were transmitted into the constant domains. Induced fit was
also demonstrated in the structure of the antigens.
[0065] (i) The Fab fragment of the monoclonal anti-sweetener
antibody NC6.8 was studied by others at 1.4 angstrom resolution. On
complexation with antigen there were local changes affecting the
positioning of both heavy and light chain CDRs. One of the
consequences of this was to disrupt a hydrogen bond between Tyr H96
and Ser H98, causing CDR3 to expand and move 4.5 angstroms and a
new hydrogen bond to form between Tyr H96 and the hapten. This
caused V.sub.H to flex and shorten. There was a concomitant
lengthening of V.sub.L by 10 angstroms, resulting in a decrease of
31.degree. in the elbow bend between Fv and the first constant
domain. The first constant domain was shifted as a unit so that the
carboxyl terminus had moved 19 angstroms toward the Fv. The authors
hypothesize that in an intact antibody, this allosteric change
could be "relayed as tug (by tensile forces) on the segment
connecting the Fab to the Fc region, perhaps altering the
orientations of the constituents responsible for such effector
functions as complement activation."
[0066] (ii) An antibody to the V3-loop of the HIV-envelope protein
has been crystallized by others in the presence and absence of the
synthetic peptide to which it binds. The presence of antigen
induced a large rotation of V.sub.H relative to V.sub.L and
influenced the orientation of the first constant domains as
well.
[0067] (iii) X-ray crystal studies by others on intact
immunoglobulins have been limited because of the difficulty in
forming crystals caused by the inherent flexibility of the antibody
molecule and because when crystals have formed the Fc units are
disordered. Several intact antibodies have yielded a structure for
the entire molecule including Fc. Interesting findings include: 1.
an asymmetric configuration particularly within the CH.sub.2
domain, resulting in somewhat independent conformations of the two
CH.sub.2 domains, and 2. flexibility of the CH.sub.2--CH.sub.3
junction a site of many effector molecule interactions. On the
basis of these 3-D studies it has been concluded by others that the
structure illustrated the dynamic range inherent in the antibody
and that the antibody is an assembly of units possessing a high
degree of flexibility.
[0068] (iv) Electron microscopy and 2-D crystallography have
demonstrated to others that both IgG and IgM are relatively planar
structures in the absence of antigen, but that on antigen binding
the Fab is dislocated from this plane. It has been proposed that
this dislocation allows greater access of C1 q and Fc receptors to
the Fc portion of the antibody once it has bound antigen.
[0069] The flexibility of the antibody molecule is well
established. There is also considerable data indicating that there
are conformational changes induced by antigen binding.
Aptamer Detection Interfaces for the Nanopore Detector.
[0070] Unlike the previous section on antibody biophysics, in this
section details on the DNA (aptamer) biophysics are omitted for
brevity.
[0071] An aptamer is a nucleic acid (DNA or RNA) that has strong
binding affinity for a target protein, DNA/RNA, or other
biomolecule. The binding affinity is selected for by PCR
amplification of the most fit molecules, with fitness given by
binding strength. Since the DNA hairpins with the desired toggle
blockade properties can be viewed as "dumb aptamers," i.e.,
aptamers with no binding target, selection of DNA-hairpin variants
with the desired binding properties is a logical starting point for
the search process (constrained in both "toggler" captured-end
parameters and sensing moiety parameters). Further detail is in the
Results section of the EXPERIMENTAL Section below.
A Novel Nanopore Device Implementation.
[0072] The relevant prior published literature has relied upon
biosensing mechanisms that involve a nanometer-scale channel and
blockades of that channel. Some of the literature has described
modifications to the channel geometry and its binding properties
(thus sensing properties) by introduction of auxiliary molecules.
The auxiliary molecules that modify the channel sensing environment
can be covalently bound (main case) or non-covalently bound to the
channel (e.g., cyclodextrins non-covalently bound, or short ssDNA
oligomers covalently bound). The design of these modifications is
focused on providing alternative binding sites with no or scant
attention paid to the role of the auxiliary molecules vis-a-vis
their on-off binding with the channel itself. In the case of
auxiliary molecules covalently bound to the channel, the sensitive
on-off binding kinetics to the channel is eliminated entirely. In
the case of the non-covalently bound (such as, e.g., the
cyclodextrins), the information pertaining to the on-off binding of
the auxiliary molecule is not pursued in its own right as the
critically sensitive reporting mechanism that it entails. Instead,
lengthy phases of fixed cyclodextrin conformation are sought. In
essence, the channel blockade states associated with analyte
detection, or non-detection, can all be described in the prior art
as static blockades of the channel current at a fixed level.
Typically, the binding or translocation of an analyte to the
channel, or to the channel/auxiliary-molecule complex, is
associated with a fixed reduction in the observed channel
current.
[0073] The present invention brings a novel modification in the
design and use of the auxiliary molecules. In particular, in the
present invention, the auxiliary molecules produce a "toggling"
blockade between several different levels (with two usually
dominating). The resulting blockade signal for the auxiliary
molecule by itself is no longer at approximately a fixed blockade
level, but now consists of a telegraph-like blockade signal with
stationary statistics. Upon binding of analyte to the auxiliary
molecule the toggling signal is greatly altered, to one with
different transition timing and different blockade residence
levels. Building on this as a biosensing foundation requires
sophisticated computational tools, such as Hidden Markov Models and
Support Vector Machines, but offers at least a hundred-fold
improvement to the sensitivity of the device. This is because the
events of analyte bound versus non-bound (detected or not) can now
be discerned with the much greater information in the
multiple-level residencies and transition timings. This is in sharp
contrast with the sparse information of non-bound having one
blockade level and bound having another, single, blockade level.
Given the noise in the system and the limited dynamic range for
blockades of the open channel current, the device is greatly
restricted if not endowed with the sensitive timing information. It
has even been shown that minor environmental alterations to
temperature, pH, etc., results in the toggle signal produced by
"toggling" type auxiliary molecule being modified significantly--in
essence the channel with toggling-type auxiliary molecules can
provide sensitive biosensing on the solution environment itself
(which, in turn, is why the binding of target analyte to the
toggling-type auxiliary molecule can be so sensitive).
[0074] Most of the prior art has been pursued by biochemists, and
as such has focused on engineered protein pores (genetically, or
via addition of auxiliary "carrier/adapter" molecules). The various
incarnations of the engineered channels, and their binding
(detection) properties, have almost entirely focused on events
defined by a single (static) blockade level. What has not been
appreciated, until now, is what can be accomplished with modern
signal analysis and pattern recognition tools. In essence, by
engineering a channel/auxiliary-molecule system with much greater
complexity in the observed blockade signal, and then resolving that
complexity with the aforementioned methods, a much better detector
results. Now events are no longer defined by static blockade levels
but by multiple blockade levels with internal transitions typically
obeying stationary statistics. Also overlooked are various
electronic modulation techniques for improving the effective
bandwidth of the device.
[0075] Preliminary results clearly demonstrate higher sensitivity
with toggling-type auxiliary molecules, as shown hereafter. This
opens the door to a new, highly precise, means for examining the
binding affinities between any two molecules, all while still in
solution! In brief, the auxiliary molecule can be
rigidly/covalently bound to one molecule of interest, and then
exposed to a solution containing the other molecule of interest.
The transitions between different stationary phases of blockade
then correspond to the bound/unbound configuration between the two
molecules of interest and reveals their binding kinetics (and
binding strength). All the while, the toggling-type auxiliary
molecule is producing its sensitive toggling blockade signal (even
if modified with one species of analyte, or sensing moiety,
rigidly/covalently bound to it).
[0076] Studies, described in what follows, were also done with
toggling-type auxiliary molecules that also had sensing moieties at
their uncaptured end (the end exposed to solution, not entering the
channel). The molecules studied included antibodies and aptamers,
and were chosen to demonstrate the specific utility of this device
in drug candidate screening--antibodies that bind strongly to
target antigen are good antibodies, same for aptamers in many
situations. Sometimes the drug is a toxin, so you want it to bind,
but not for too long, etc. In the larger context of the channel
device itself, the advanced computational tools offer the
possibility of analyzing/characterizing the device channels
themselves (typically pore-forming toxins, such as from staph or
anthrax). The advanced computational tools also offer a platform
for examining the efficacy of certain channel-forming toxins as
cellular syringes for delivery of specific molecules and drugs to
the cellular cytosol (such as antigen delivery to evoke a CTL
response).
EXPERIMENTAL
Nanopore Detector Biophysics Methods
[0077] Nanopore experiments. Each experiment is conducted using one
.alpha.-hemolysin channel inserted into a
diphytanoyl-phosphatidylcholine/hexadecane bilayer across a
25-micron-diameter horizontal Teflon aperture, as described
previously in Akeson M, D. Branton, J. J. Kasianowicz, E. Brandin,
D. W. Deamer, 1999, Microsecond Time-Scale Discrimination Among
Polycytidylic Acid, Polyadenylic Acid, and Polyuridylic Acid as
Homopolymers or as Segments Within Single RNA Molecules, Biophys.
J. 77(6):3227-3233. Seventy microliter chambers on either side of
the bilayer contains a conductive medium in the form of 1.0 M KCl
buffered at pH 8.0 (10 mM HEPES/KOH) except in the case of buffer
experiments where the salt concentration, pH, or identity may be
varied. Voltage is applied across the bilayer between Ag-AgCl
electrodes to provide a source for ionic flow. DNA control probes
are added to the cis chamber at 10 or 20 .mu.M final concentration.
All experiments are maintained at room temperature
(23.+-.0.1.degree. C.), using a Peltier device.
[0078] Controlling Nanopore Noise Sources and Choice of Aperture.
The accessible detector bandwidth is delimited by noise resulting
from 1/f (flicker) noise, Johnson noise, Shot noise, and membrane
capacitance noise. In FIG. 3a, upper right, the current spectral
density is shown for the typical bilayer, an open .alpha.-hemolysin
channel, and a channel with DNA hairpin blockade. For 1.0 M KCl at
23.degree. C., the .alpha.-hemolysin channel conducts 120 pA under
an applied potential of 120 mV. The thermal noise contribution at
the 1 G.OMEGA. channel resistance has an RMS noise current of 0.4
pA, consistent with FIG. 3a. Shot noise is the result of current
flow based on discrete charge transport. During nanopore operation
with 120pA current (with 10 KHz bandwidth) there is, similarly,
about 0.6 pA noise due to the discreteness of the charge flow. As
with Johnson noise, the Shot noise spectrum is white, consistent
with FIG. 3a. The specific capacitance of lipid bilayers is
approximately 0.8 .mu.F/cm.sup.2 (very large due to molecular
dimensions), and the specific conductance is approximately
10.sup.-6 .OMEGA..sup.-1 cm.sup.-2. In order for bilayer
conductance to produce less RMS noise current than fundamental
noise sources (under the conditions above), the leakage current
must be a fraction of a pA. This problem is solved by reducing to
less than a 500 .mu.m.sup.2 bilayer area, for which less than 0.6
pA leakage current results and for which total bilayer capacitance
is at most 4 pF. This indicates that a decrease in bilayer area by
another magnitude is about as far as this type of noise reduction
can go. Preliminary attempts to do this, however, lead to a very
unpredictable toxin intercalation rate, among other difficulties.
For the experiments considered here, the aperture ranges in size
between 1 microns in diameter and 100 microns in diameter, where
smaller apertures are used in the single channel experiments and
larger apertures in the multi-channel experiments.
[0079] Nanopore-based detection is also limited by the kinetic
nature (time-scale) of the blockade signal itself, since the
molecular blockades typically involve binding and dissociation
(analyte-channel binding, or antibody-antigen binding). It is
hypothesized that it is possible to probe higher frequency realms
than those directly accessible at the operational bandwidth of the
channel current based device, or due to the time-scale of the
particular analyte interaction kinetics, by modulated excitations.
This can be accomplished by chemically linking the analyte or
channel to an excitable object, such as a magnetic bead, under the
influence of laser pulsations. In one configuration, the excitable
object can be chemically linked to the analyte molecule to modulate
its blockade current by modulating the molecule during its
blockade. In another configuration, the excitable object is
chemically linked to the channel, to provide a means to modulate
the passage of ions through that channel. In a third experimental
variant, the membrane is itself modulated (using sound) in order to
effect modulation of the channel environment and the ionic current
flowing though that channel. Studies involving the first, analyte
modulated, configuration (FIG. 6), indicate that this approach can
be successfully employed to keep the end of a long strand of duplex
DNA from permanently residing in a single blockade state. Similar
study of magnetic beads linked to antigen may be used in the
nanopore/antibody experiments if similar single blockade level,
"stuck," states occur with the captured antibody (at physiological
conditions, for example). Likewise, this approach can be considered
for increasing the antibody-antigen dissociation rate if it does
occur on the time-scale of the experiment.
[0080] Control probe design. Since the five DNA hairpins studied in
the prototype experiment have been carefully characterized, they
are used in the antibody experiments as highly sensitive controls.
The nine base-pair hairpin molecules examined in the prototype
experiment share an eight base-pair hairpin core sequence, with
addition of one of the four permutations of Watson-Crick base-pairs
that may exist at the blunt end terminus, i.e.,
5'-G.cndot.C-3',5'-C.cndot.G-3',5'-T.cndot.A-3', and
5'-A.cndot.T-3'. Denoted 9GC, 9CG, 9TA, and 9AT, respectively. (9GC
has SEQ ID NO: 3; 9CG has SEQ ID NO: 4; 9TA has SEQ ID NO: 2; and
9AT has SEQ ID NO: 5.) The full sequence for the 9CG hairpin is 5'
CTTCGAACGTTTTCGTTCGAAG 3' (SEQ ID NO: 4), where the base-pairing
region is underlined. The eight base-pair DNA hairpin is identical
to the core nine base-pair subsequence, except the terminal
base-pair is 5'-G.cndot.C-3'. The prediction that each hairpin
would adopt one base-paired structure was tested and confirmed
using the DNA mfold server
http://bioinfo.math.rpi.edu/.about.mfold/dna/form1.cgi), which is
based in part on data from SantaLucia J. 1998, A unified view of
polymer, dumbbell, and oligonucleotide DNA nearest-neighbor
thermodynamics, Proc. Natl. Acad. Sci. USA 95(4):1460-1465. A
standardized aliquot of antibody is used as the control for
antibody experiments once the kinetics of antibody capture and
antigen-binding events are established and shown to be highly
reproducible.
[0081] Data acquisition. Data is acquired and processed in two ways
depending on the experimental objectives: (i) Using commercial
software from Axon Instruments (Redwood City, Calif.) to acquire
data, where current will typically be filtered at 50 kHz bandwidth
using an analog low pass Bessel filter and recorded at 20 .mu.s
intervals using an Axopatch 200B amplifier (Axon Instruments,
Foster City, Calif.) coupled to an Axon Digidata 1200 digitizer.
Applied potential is 120 mV (trans side positive) unless otherwise
noted. In some experiments, semi-automated analysis of transition
level blockades, current, and duration are performed using Clampex
(Axon Instruments, Foster City, Calif.). (ii) Using LabView-based
experimental automation. In this case, ionic current is also
acquired using an Axopatch 200B patch clamp amplifier (Axon
Instruments, Foster City, Calif.), but it is then recorded using a
NI-MIO-16E-4 National Instruments data acquisition card (National
Instruments, Austin Tex.). In the LabView format, data is low-pass
filtered by the amplifier unit at 50 kHz, and recorded at 20 .mu.s
intervals. In both fixed duty cycle (i.e., not feedback controlled)
data acquisition approaches, the solution sampling protocol uses
periodic reversal of the applied potential to accomplish the
capture and ejection of single biomolecules. The biomolecules
captured consist of antibodies and antigen, bound together or not,
and in various orientations, and DNA control probes with
stem-capture orientation and are added to the cis chamber typically
in 20 .mu.M concentrations. The time-domain finite state automaton
used in the prototype is used to perform the generic signal
identification/acquisition for the first 100 msec of blockade
signal (Acquisition Stage, FIG. 1b). The effective duty cycle for
acquiring 100 ms blockade measurements, when found to be sufficient
for classification purposes, is adjusted to approximately one
reading every 0.4 seconds by choice of analyte concentration.
Further details on the voltage toggling protocol and the
time-domain FSA are in FIG. 7 and Winters-Hilt et al., 2003.
[0082] Stability of the nanopore. Because the nanopore is in a
lipid bilayer, it is by definition a fluid structure and has a
finite lifetime. Manipulations, especially adding antibody or
antigen to the chamber, have the potential to disrupt the nanopore.
If this occurs, it can greatly delay studies of the
antibody-antigen interaction. But it should still be possible to
perform these analyses. There is the potential for the replacement
of lipid bilayers with more durable films, such as hybrid
bilayer/solid-state systems where the bilayer rests on a thinly
coated solid-state substrate. Alternatively, the protein-channel
basis for the nanoscopic channel can be eliminated entirely, in
favor of a purely solid-state channel (Li, J., D. Stein, C.
McMullan, D. Branton, M. J. Aziz, and J. Golovchenko, Ion Beam
Sculpting at nanometer length scales, Nature 412, 166-169
(2001)).
[0083] Characterization under physiological buffer conditions. The
standard buffer condition for the nanopore detector is 1.0 M KCl
with a pH of 8.0. This buffer was found to be most conducive to
channel formation and to channels that do not gate. At
significantly lower pH the channel is known to gate (Bashford, C.
J., PORE-FORMING TOXINS: Attack and Defense at the cell surface,
Cell. Biol. Mol. Lett. Vol. 6 No. 2A. 2001), if it even forms in
the first place, which complicates use of the nanopore detector at
physiological conditions (pH 7.0, 100 mM NaCl). Since the pH of
blood is usually in the range 7.35 to 7.45, and channel formation
has been observed at 250 mM KCl, nanopore operation at the high pH
and high salt end of the physiological range, relevant for antibody
function in the bloodstream, may be possible with minimal
alteration to the experimental parameters. Evaluation of
antibody/antigen binding efficacy in a physiological buffer
environment is particularly important if the nanopore/antibody
detector is to be used for clinically relevant screening on the
efficacy of antibodies to a given antigen.
[0084] If unable to alter buffer to physiological conditions, this
will alter the on and off rates of antigen binding. But ELISA
studies have shown that the antibodies still bind to the
multivalent forms of the antigen in 1 M KCl, although binding is
diminished, and it appears that one may be able to lower the KCl
concentration to 0.25 M and still get pore formation. Thus while
the validity of rate constants and other parameters of the
interaction may be called into question, one may still be able to
perform channel analyses, and other studies of antibody capture and
alteration of channel blockade by antigen binding.
[0085] Nanopore bio-sensor single-signal saturation. Another
limitation in the utility of the nanopore/antibody detector is that
once antigen is bound by a channel-captured antibody it is very
difficult to effect the release of that antigen. This is a
complicating issue in acquiring a large sample of antibody-antigen
binding observations. Attempts to "shake-off" the antigen by
cycling to higher temperatures only serves to lead to added channel
formation events--even after perfusion-elimination of solution
toxin after the first channel formation. This is understood to
result from higher mobility in the bilayer that allows resident
toxin monomers to coalesce on the timescale of the experiment that
would otherwise not do so. A buffer-based solution to this problem
is already known from purifying antibodies through a column
containing antigen, where the release of antibodies bound in the
column is effected by perfusion with 1.0 M MgCl.sub.2. This
presents the possibility of weakening the antibody-antigen binding
by similar choice of buffer to obtain large sample sets of binding
events. The limitation of this is that the parameters will have
likely deviated substantially from the physiological norm.
Alternatively, a balanced stoichiometric ratio of antibody to
antigen could be rapidly sampled, with lengthy sampling
acquisitions only on antibody captures that occur without bound
antibody and that then wait to observe antigen binding.
[0086] Antibody/Antigen--Design, Synthesis, and Purification. For
most of the experiments, a panel of native and genetically
engineered antibodies to a well defined synthetic polypeptide
antigen are used, (Y,E)-A-K. See Horgan, C., K. Brown, and S. H.
Pincus. 1990. Alteration in heavy chain V region affects complement
activation by chimeric antibodies. J. Immunol. 145:2527; Horgan,
C., K. Brown, and S. H. Pincus. 1992a, Effect of H chain V region
on complement activation by immobilized immune complexes, J.
Immunol. 149:127; Horgan, C., K. Brown, and S. H. Pincus, 1992b,
Variable region differences affect antibody binding to immobilized
but not soluble antigen, Hum. Antibodies Hybridomas 3:153; and
Horgan, C., K. Brown, and S. H. Pincus, 1993, Studies on antigen
binding by intact and hinge-deleted chimeric antibodies, J.
Immunol. 150:5400-5407. The antigen-binding characteristics,
ability to form immune complexes, and effector functions of these
antibodies have been carefully studied. Three different antibodies
from this set are utilized in the experiments in this experimental
effort. All have identical variable domains of murine origin, but
one is a murine IgG1, one a human IgG1, and the other a human IgG4.
These are designated T6, 10B, and B11, respectively. In addition to
these antibodies, other antibodies are used to determine whether
the ability of antibodies to be captured is a general phenomenon,
and whether the blockade patterns obtained with antibodies are
dependent on pI, isotype, species origin, or other predictable
characteristics of the antibody or of the method of
preparation.
[0087] All monoclonal antibodies are grown in tissue culture
because ascites preparations are inflammatory exudates subjecting
the antibodies to the potential of proteolytic digestion,
attachment of complement components and so forth. Cells are either
grown in medium containing fetal calf serum adsorbed on protein G
to remove remaining Ig, or in serum free hybridoma medium. To test
the effect of preparation method, murine IgG1 antibody is either
purified by ammonium sulfate precipitation, antigen-affinity
purification or protein G chromatography. This antibody has already
been shown to be susceptible to capture. All other antibodies are
routinely purified on protein G and eluted with 0.5M glycine-HCl pH
2.5, immediately neutralized, and dialyzed into phosphate buffered
saline (PBS). Once antibodies are purified, they are run on
SDS-PAGE to confirm purity and run on IEF prepoured gels (Biorad)
to determine pI. Antigen binding is confirmed by ELISA in PBS and
in 1 M KCl (so long as that buffer is used).
[0088] In addition to T6, 10B, and B11, the following antibodies
are tested: murine and human monoclonal antibodies representing all
of the IgG isotypes, murine IgG1 antibodies having different pIs
(determined experimentally), and polyclonal protein A purified
antibodies from human, rabbit, goat and mouse. The murine and human
antibodies are obtained from an extensive collection of such
antibodies. We have cell lines secreting multiple representatives
of each isotype. Although the pIs of most of the antibodies may not
be known, a collection of anti-DNA antibodies that have been tested
(because of the ability of catioinic antibodies to deposit in
kidneys) and have a range of PIs is used.
[0089] For the antigen-binding studies, different versions of
(Y,E)-A-K are prepared which vary in molecular weight and valency.
Previous studies have demonstrated that the epitope to which the
antibodies bind may be represented by the synthetic peptide
EYYEYEEY (Horgan et al., 1990, supra; Horgan et al., 1992a, supra;
Horgan et al., 1992b, supra; and Horgan et al., 1993, supra). Thus
we have the following antigens: 1. The synthetic polypeptide
(Y,E)-A-K, which is highly multivalent and with different
preparations has a molecular of either 50,000, 125,000, or 300,000
daltons. 2. EYYEYEEY which is monovalent and has a molecular weight
of 1183, and 3. Ovalbumin, molecular weight 43,000 which has been
conjugated to EYYEYEEY at ratios of 1, 3, and 10 peptides per
molecule of OVA. These different antibody preparations allow study
of the effect of antigenic mass and valency of binding upon the
observations.
[0090] Antibody Characterization Experiments. The experiments
characterize the blockade induced by the capture of antibody within
the nanopore. The effects of antibody preparation and structural
characteristics of the antibody are evaluated. Similar,
reproducible, signal traces are believed to be found for antibody
molecule blockades as in the DNA hairpin experiments. The
complication is that the antibody molecule is more complex than the
DNA hairpin molecule, and has several possible capture orientations
instead of one. The objective, initially, is to enumerate the
possible capture orientations. Because the immunoglobulin domain
fold is an elongated barrel, it is likely that capture events will
occur at the narrow ends of the domain, rather than along the side
of the beta-barrel, since we have shown that albumin, a more
globular molecule, cannot be captured. Thus it is possible that we
may capture binding of domains consisting of individual chains,
portions of both chains, within the Fab and Fc, and possibly, the
carbohydrate attached to the heavy chain (although this is usually
oriented internally and not surface accessible). Only some of these
conformations will be able to bind antigen, (as demonstrated in the
preliminary results). Thus, the overall objective in this case is
to fully quantify the various channel captures of antibody alone.
This information, combined with antigen binding data regarding the
relative antigen binding capabilities of antibody in each
conformation, allow one to begin to determine what blockade pattern
is associated with different capture orientations. (To do this will
require acquisition of sufficient amounts of the nanopore/antibody
data to allow the machine learning software to be adequately
trained.)
[0091] The capture of each antibody preparation should be studied
by multiple events. Control software may be designed that
automatically detects the capture event, collects data for a
defined time (100 ms to 1 s depending on experiment), ejects the
antibody from the nanopore by reversing the current, and then sets
up to capture another antibody molecule. Additional software may be
designed to classify the blockade signals obtained. In this way,
one is able to collect data from several hundred capture events for
each antibody preparation, classify them on the basis of channel
blockade produced, and perform statistical analyses defining the
rate for each type, and determine whether consistent results are
obtained on a day-to-day basis, and determining whether the
antibody pI, isotype, preparation, or species origin has an effect
on the frequency of the capture modes.
Channel Current Cheminformatics (Signal Analysis & Pattern
Recognition) Methods.
[0092] Extraction of kinetic information begins with identification
of the main blockade levels for the various blockade classes
(off-line). This information is then used to scan through already
labeled (classified) blockade data, with projection of the blockade
levels onto the levels previously identified (by the off-line
stationarity analysis) for that class of molecule. A time-domain
FSA performs the above scan (FIG. 8), and uses the information
obtained to tabulate the lifetimes of the various blockade levels.
Once the lifetimes of the various levels are obtained, information
about a variety of kinetic properties is accessible. If the
experiment is repeated over a range of temperatures, a full set of
kinetic data is obtained (including "spike" feature density
analysis, as shown in FIG. 9). This data may be used to calculate
k.sub.on and k.sub.off rates for binding events, as well as
indirectly calculate forces by means of the van't Hoff Arrhenius
equation.
[0093] Signal Preprocessing Details. Each 100 ms signal acquired by
the time-domain FSA consists of a sequence of 5000 sub-blockade
levels (with the 20 .mu.s analog-to-digital sampling). Signal
preprocessing is then used for adaptive low-pass filtering. For the
data sets examined, the preprocessing is expected to permit
compression on the sample sequence from 5000 to 625 samples (later
HMM processing then only required construction of a dynamic
programming table with 625 columns). The signal preprocessing makes
use of an off-line wavelet stationarity analysis (Off-line Wavelet
Stationarity Analysis, FIG. 1b).
[0094] HMMs and Supervised Feature Extraction Details. With
completion of preprocessing, an HMM is used to remove noise from
the acquired signals, and to extract features from them (Feature
Extraction Stage, FIG. 1b). The HMM is, initially, implemented with
fifty states, corresponding to current blockades in 1% increments
ranging from 20% residual current to 69% residual current. The HMM
states, numbered 0 to 49, corresponded to the 50 different current
blockade levels in the sequences that are processed. The state
emission parameters of the HMM are initially set so that the state
j, 0<=j<=49 corresponding to level L=j+20, can emit all
possible levels, with the probability distribution over emitted
levels set to a discretized Gaussian with mean L and unit variance.
All transitions between states are possible, and initially are
equally likely. Each blockade signature is de-noised by 5 rounds of
Expectation-Maximization (EM) training on the parameters of the
HMM. After the EM iterations, 150 parameters are extracted from the
HMM. The 150 feature vector components are extracted from
parameterized emission probabilities, a compressed representation
of transition probabilities, and use of a posteriori information
deriving from the Viterbi path solution (further details in
Winters-Hilt et al., 2003). This information elucidates the
blockade levels (states) characteristic of a given molecule, and
the occupation probabilities for those levels (FIG. 3a, lower
right), but doesn't directly provide kinetic information. The
resulting parameter vector, normalized such that vector components
sum to unity, is used to represent the acquired signal during
discrimination at the Support Vector Machine stages.
[0095] A combination HMM/EM-projection processing followed by
time-domain FSA processing allows for efficient extraction of
kinetic feature information (e.g., the level duration
distribution). FIG. 10 shows how HMM/EM-projection might be used to
expedite this process. The objective of the HMM/EM processing is to
reduce level fluctuations, while maintaining the position of the
level transitions. The implementation uses HMM/EM parameterized
with emission probabilities as gaussians, which, for
HMM/EM-projection, is biased with variance increased by
approximately one standard deviations (see results shown). This
method is referred to as HMM/EM projection because, to first order,
it does a good job of reducing sub-structure noise while still
maintaining the sub-structure transition timing. The benefit of
this over purely time-domain FSA approaches is that the tuning
parameters to extract the kinetic information are now much fewer
and less sensitive (self-tuning possible in some cases).
[0096] The classification approach adopted in (Winters-Hilt et al.,
2003) is designed to scale well to multi-species classification (or
a few species in a very noisy environment). The scaling is possible
due to use of a decision tree architecture and an SVM approach that
permits rejection on weak data. SVMs are usually implemented as
binary classifiers, are in many ways superior to neural nets, and
may be grouped in a decision tree to arrive at a multi-class
discriminator. SVMs are much less susceptible to over-training than
neural nets, allowing for a much more hands-off training process
that is easily deployable and scalable. A multiclass implementation
for an SVM is also possible--where multiple hyperplanes are
optimized simultaneously. A (single) multiclass SVM has a much more
complicated implementation, however, is more susceptible to noise,
and is much more difficult to train since larger "chunks" are
needed to carry all the support vectors. Although the "monolithic"
multiclass SVM approach is clearly not scalable, it may offer
better performance when working with small numbers of classes. The
monolithic multiclass SVM approach also avoids a combinatorial
explosion in training/tuning options that are encountered when
attempting to find an optimal decision tree architecture. It was
revealed in (Winters-Hilt et al., 2003), however, that the SVM's
rejection capability often leads to the optimal decision tree
architecture reducing to a linear tree architecture, with strong
signals skimmed off class by class. This would prevent the
aforementioned combinatorial explosion if imposed on the search
space, and that should be the first architecture attempted for the
signal classifications.
[0097] SVMs use variational methods in their construction and
encapsulate a significant amount of discriminatory information in
their choice of kernel. In reference (Winters-Hilt et al., 2003)
one of the present inventors used novel, information-theoretic,
kernels for notably better performance than standard kernels. The
kernels studied were not limited to those satisfying Mercer's
conditions, although they might, since they can be described as
metrics "regularized" by incorporation as positive arguments in a
decaying exponential. The commonly used Gaussian kernel, which does
satisfy Mercer's conditions, has such an exponential form, and was
outperformed in all cases studied by the entropic and variation
kernels used in the prototype (FIG. 11, Winters-Hilt et al., 2003).
The original motivation for working with the entropic kernel was to
obtain a faster, more noise-resistant, kernel for information
obtained via an HMM feature extractor, instead of the theoretically
attractive, but less noise resistant, choice of directly
integrating the two via a Fisher Kernel. This led to a general
formulation where feature extraction was designed to arrive at
probability vectors (i.e., discrete probability distributions) on a
predefined, and complete, space of possibilities. (The different
blockade levels, and their frequencies, for example.) This turns
out to be a very general formulation, wherein feature extraction
makes use of signal decomposition into a complete set of separable
states. A probability vector formulation also provides a
straightforward hand-off to the SVM classifiers since all feature
vectors have the same length with such an approach. What this means
for the SVM is that geometric notions of distance are no longer the
best measure for comparing feature vectors. For probability vectors
(i.e., discrete distributions), the best measures of similarity are
the various information-theoretic divergences: Kullback-Leibler,
Renyi, etc. By symmetrizing over the arguments of those divergences
a rich source of kernels is obtained that works well with the types
of probabilistic data obtained.
[0098] The SVM discriminators are trained by solving their KKT
relations using the Sequential Minimal Optimization (SMO) procedure
(Platt, 1998). A chunking variant of SMO also is employed to manage
the large training task at each SVM node. The multi-class SVM
training generally involves thousands of blockade signatures for
each signal class. The data cleaning needed on the training data is
accomplished by an extra SVM training round.
[0099] Re-establishing the .alpha.-hemolysin channel on a
day-to-day basis presents a major complication to the pattern
recognition task. SVM classification in such circumstances faces
weaker training convergence and poorer signal calling. For the
blockade signals to be considered, two stabilization approaches are
employed. (i) a passive stabilization approach that optimizes the
kernels for high rejection. And, as needed, (ii) active,
computationally-based, stabilization methods that track control
signature samples that are intermixed with the target analyte
signal (see description of DNA hairpin controls in the Nanopore
Experimental Design).
[0100] Development of a Class Independent HMM to extract kinetic
information from channel current data. Two, critical, engineering
tasks must be addressed in a practical implementation of this
objective: (i) the software should require minimal tuning; and (ii)
feature extraction must be accomplished in approximately the same
100 ms time span as the blockade acquisition. (The latter,
approximate, restriction was successfully implemented for the 300
ms voltage-toggle duty cycle used in the prototype.) The feature
extraction tools used to extract kinetic information from the
blockade signals will include finite-state automata (FSAs),
wavelets, as well as Hidden Markov Models (HMMs). Extraction of
kinetic information from the blockade signals at the millisecond
timescale for objectives (i) and (ii) are addressed by use of HMMs
for level identification, HMM-EMs for level projection, and
time-domain FSAs for processing of the level-projected
waveform.
[0101] Development of Class Dependent HMM/EM and NN algorithms to
extract transient-kinetic information. If separate HMMs are used to
model each species, the multi-HMM/EM processing can extract a much
richer set of features, as well as directly provide information for
blockade classifications. The multiple HMM/EM evaluations, however,
on each unknown signal as it is observed, represent a critical
non-scaling engineering trade-off. The single-HMM/EM approach
adopted in Winters-Hilt et al., 2003, on the other hand, scales
well to multiple species classification (or a few species in a very
noisy environment) because a single HMM/EM was used, and the entire
discriminatory task was passed off to a decision tree of Support
Vector Machines (SVMs). Another benefit of incorporating SVMs for
discrimination was that they provided a robust method for rejecting
weak data. The software design in some embodiments of this
invention retains the clear benefits of the SVM-based
classification approach, but now incorporates a richer set of
information from multi-HMM/EM processing. The challenge in doing so
is to perform the extensive feature extraction in the allotted
sampling timeframe. Where real-time processing can't be
accomplished with species-specific multi-HMM/EM processing, an
alternative approach is to augment the feature extraction with
neural net (NN) feature extraction methods (not discrimination)
that can learn and extract features on-line. In this approach,
information about the level states, and their time-constants, is
obtained in a much cruder form.
[0102] Development of multi-class SVMs and develop SVM Kernels
specialized for discrete probability feature vectors. This signal
processing classifies molecular blockades based on kinetic
profiles, and recognizes transitions between kinetic profiles.
Initially this is accomplished by extending the Support Vector
Machine (SVM) Decision Tree approach. There are two, critical,
engineering hurdles for a practical implementation of this aim: (i)
automation in training of the SVM architecture; and (ii)
optimization over a class of relevant SVM Kernels. For problem (i),
a great deal of automation in training has already been
accomplished in the construction of the prototype SVM Decision
Tree. In that effort, the decision tree training and testing
problem was reduced to specifying the topology of the tree and the
location of the files used in training each of its nodes. Scripts
were written to process a specified list of different decision
trees and distribute the processing for those trees over a network
of computers. If the SVM Decision Tree approach fails at the
training stage, an alternate approach involving a monolithic
multiclass SVM (with no decision tree) is possible. For problem
(ii), the search for a suitable kernel, the proposed SVM classifier
implementation significantly benefits from the extended class of
divergence kernels pioneered in developing the prototype SVM. In
that work the divergence kernels were designed to match with the
probability vector data obtained with the feature extraction
approach, and appear to be very well suited to signal analysis on
channel current blockades.
Antibody Experimental Results.
[0103] The first introduction of antibody into the nanopore
detector analyte chamber was meant to examine the time-scale before
disruption of the channel current. This was expected to occur
because the antibody had been designed to target the epitopes of
the alpha-hemolysin monomer. The unexpected result was that the
antibody appeared to be drawn into the channel, just like dsDNA,
with result a similar binding-kinetics "telegraph" signal in the
channel current. This experiment has since been repeated hundreds
of times, with a variety of different antibodies. Most of the
antibodies examined have identical variable domains of murine
origin.
[0104] Preliminary results indicate that single antibody molecules
can be drawn into the nanopore detector in a manner analogous to
the DNA hairpin studies. In those DNA hairpin blockade studies it
was found that DNA hairpins, with stem lengths ranging between
eight base-pairs and twelve base-pairs, produced blockade traces
with transitions between a limited number of blockade states that
were highly sensitive to changes in voltage and temperature.
Analysis of those, reproducible, DNA hairpin signal traces led to a
consistent picture for the binding and conformational kinetics for
the captured ends of those molecules (Winters-Hilt et al., 2003;
Vercoutere, W., S. Winters-Hilt, V. S. DeGuzman, D. Deamer, S.
Ridino, J. T. Rogers, H. E. Olsen, A. Marziali, and M. Akeson,
2003, Discrimination Among Individual Watson-Crick Base-Pairs at
the Termini of Single DNA Hairpin Molecules, Nucl. Acids Res. 31,
pg 1311-1318; Winters-Hilt, S. 2003, Highly Accurate Real-Time
Classification of Channel-Captured DNA Termini, Third International
Conference on Unsolved Problems of Noise and Fluctuations in
Physics, Biology, and High Technology, pg 355-368; Winters-Hilt, S,
and M. Akeson, "Nanopore cheminformatics," DNA and Cell Biology,
October 2004; Winters-Hilt, S., "Nanopore detection using channel
current cheminformatics," SPIE Second International Symposium on
Fluctuations and Noise, 25-28 May, 2004). As with those DNA
studies, where only a single channel was conducting current through
a bilayer, an antibody blockade can be seen as a clearly
discernible event (see FIG. 4). Instead of the open channel current
being the baseline reference in the nanopore detector, however,
there is now the possibility that the nanopore/antibody blockade
may form a reference channel current signal in its own right. An
antigen-binding event then perturbs the channel current signal,
resulting from the antibody blocking the channel alone, and is the
basis of the antibody-antigen binding-event observation (FIG. 5).
The lifetimes of the different channel blockades associated with
the antibody-antigen binding (and dissociation) can then be used to
understand the kinetics of that binding, including its strength,
with implications as to the efficacy of the antibody for that
antigen.
[0105] The blockade signals for murine IgG1 (denoted T6) has been
examined most extensively thus far. The antibody exhibits more than
one blockade signal, suggesting that the different parts of the
molecule can be drawn in. Since the immunoglobulin domain fold is
an elongated barrel, it is possible that capture events could occur
at the narrow ends of the domain, as individual chains, or portions
of both chains, within the Fab and Fc, and possibly, the
carbohydrate attached to the heavy chain. FIG. 4 shows other
blockade signals that have been observed, along with images of
possible capture orientations that might accompany them (all images
are overlaid to scale).
[0106] It was hypothesized that the antibody blockade signal should
alter shortly after introduction of antigen if antigen binding were
to occur and alter the channel blockade. This hypothesis was partly
based on previous results with DNA hairpin blockades, that also
exhibited the telegraph blockade signal, and that were found to be
very sensitive to environmental conditions. This hypothesis proves
to be correct, as FIG. 5 shows upon addition of moderately high
concentration (100 .mu.g/ml) of 200 kDa multivalent synthetic
polypeptide (Y,E)-A-K. The timescale before the blockade signals
are altered is also interesting, it can range from seconds to
minutes, which may help to resolve which blockade signal goes with
what type of blockade in FIG. 4.
[0107] Once antigen is bound by a channel-captured antibody it is
very difficult to effect the release of that antigen. This is a
complicating issue when seeking to obtain a large sample of
antibody-antigen binding observations. Another complicating issue
is that an antibody might form a blockade that does not exhibit the
multi-level kinetic signal, residing in a "stuck" state instead.
This is already a problem that is observed to occur for longer
dsDNA molecules. The solution may be to periodically knock the
system to keep it out of its "stuck" state in order to have it
continue to exhibit its terminus binding kinetics. Recent results
with a laser excitation of a magnetic bead that is attached to a
DNA hairpin with 20 base-pair stem length (attachment at the
hairpin loop) is shown in FIG. 6. This preliminary result holds
great promise for keeping analyte blockades in their most sensitive
regime, as well as offering prospects for injecting modulatory side
information.
[0108] Examples of excitable objects include microscopic beads,
magnetic and non-magnetic, and fluorescent dyes and the like. Bead
attachments can couple in excitations passively from background
thermal (Brownian) motions, or actively, in the case of magnetic
beads, by laser pulsing and laser-tweezer manipulation. Dye
attachments can couple excitations via laser or light (UV)
excitations to the targeted dye molecule. Large, classical,
objects, such as microscopic beads, provide a method to couple
periodic modulations into the single-molecule system. The direct
coupling of such modulations, at the channel itself, avoids the low
Reynolds number limitations of the nanometer-scale flow
environment. For rigid coupling on short biopolymers, the overall
rigidity of the system also circumvents limitations due to the low
Reynolds number flow environment. Similar consideration also come
into play for the dye attachments, except now the excitable object
is typically small, in the sense that it is usually the size of a
single (dye) molecule attachment. Excitable objects such as dyes
must contend with quantum statistical effects, so their application
may require time averaging or ensemble averaging, where the
ensemble case involves multiple channels that are observed
simultaneously--which relates to the platform of the multi-channel
configuration of the experiment. Modulation in the third,
membrane-modulated, experiment also avoids quantum and low Reynolds
number limitations. In all the experimental configurations, a
multi-channel platform may be used to obtain rapid ensemble
information. In all cases the modulatory injection of excitations
may be in the form of a stochastic source (such as thermal
background noise), a directed periodic source (laser pulsing,
piezoelectric vibrational modulation, etc.), or a chirp (single
laser pulse or sound impulse, etc.). If the modulatory injection
coincides with a high frequency resonant state of the system, low
frequency excitations may result, i.e., excitations that can be
monitored in the usable bandwidth of the channel detector.
Increasing the effective bandwidth of the nanopore device greatly
enhances its utility in almost every application.
[0109] Biotechnology Application: Increasing the effective
bandwidth of the nanopore device greatly enhances its utility in
almost every application, particularly those, such as DNA
sequencing, where the speed with which blockade classifications can
be made (sequencing) is directly limited by bandwidth
restrictions.
Aptamer Experimental Results.
[0110] An aptamer is a nucleic acid (DNA or RNA) that has strong
binding affinity for a target protein, DNA/RNA, or other
biomolecule. The binding affinity is selected for by PCR
amplification of the most fit molecules, with fitness given by
binding strength. Since the DNA hairpins with the desired toggle
blockade properties can be viewed as "dumb aptamers," i.e.,
aptamers with no binding target, selection of DNA-hairpin variants
with the desired binding properties is a logical starting point for
the search process (constrained in both "toggler" captured-end
parameters and sensing moiety parameters). FIG. 12 shows an aptamer
with a "toggler"-type nine-base-pair stem captured end and a
ssDNA-overhang for complement ssDNA detection (a "pseudo-aptamer").
FIG. 13 shows a collection of blockade signal traces. The short
blockades in the upper left slide of FIG. 13 result from ssDNA
translocation by the un-annealed ssDNA components. The long
blockade in the upper left, and all right, panels is for capture
with the overhang end entering first. The panel third down on the
left shows the desired toggler-type signal. FIG. 14 shows the
modification to the toggler-type signal upon addition of 5-base
ssDNA. The observed change is hypothesized to represent annealing
by the complimentary 5-base ssDNA component, and thus detection of
the 5-base ssDNA molecule.
[0111] Each and every patent, patent application and printed
publication referred to above is incorporated herein by reference
in toto to the fullest extent permitted as a matter of law.
[0112] This invention is susceptible to considerable variation in
its practice. Therefore, the foregoing description is not intended
to limit, and should not be construed as limiting, the invention to
the particular exemplifications presented hereinabove. Rather, what
is intended to be covered is as set forth in the ensuing claims and
the equivalents thereof permitted as a matter of law.
Sequence Listing Free Text
[0113] SEQ ID NO: 1: DNA hairpin used in antibody experiments as a
control.
[0114] SEQ ID NO: 1: DNA hairpin used in antibody experiments as a
control.
[0115] SEQ ID NO: 2: DNA hairpin used in antibody experiments as a
control.
[0116] SEQ ID NO: 3: DNA hairpin used in antibody experiments as a
control.
[0117] SEQ ID NO: 4: DNA hairpin used in antibody experiments as a
control.
[0118] SEQ ID NO: 5: DNA hairpin used in antibody experiments as a
control.
[0119] SEQ ID NO: 6: Synthetic peptide to which the antibodies may
be represented.
[0120] SEQ ID NO: 7: Peptide used for antigen binding studies.
Sequence CWU 1
1
6 1 20 DNA Artificial DNA hairpin used in antibody experiments as a
control. 1 gtcgaacgtt ttcgttcgac 20 2 22 DNA Artificial DNA hairpin
used in antibody experiments as a control. 2 tttcgaacgt tttcgttcga
aa 22 3 22 DNA Artificial DNA hairpin used in antibody experiments
as a control. 3 gttcgaacgt tttcgttcga ac 22 4 22 DNA Artificial DNA
hairpin used in antibody experiments as a control. 4 cttcgaacgt
tttcgttcga ag 22 5 22 DNA Artificial DNA hairpin used in antibody
experiments as a control. 5 attcgaacgt tttcgttcga at 22 6 8 PRT
Artificial Synthetic peptide to which the antibodies may be
represented. 6 Glu Tyr Tyr Glu Tyr Glu Glu Tyr 1 5
* * * * *
References