Downscaling functional bioassays by single-molecule techniques
Transcript of Downscaling functional bioassays by single-molecule techniques
Drug Discovery Today � Volume 11, Numbers 13/14 � July 2006 REVIEWS
Downscaling functional bioassays bysingle-molecule techniquesFeng Hong and Douglas D. Root
University of North Texas, Department of Biological Sciences, Division of Biochemistry and Molecular Biology, PO Box 305220, Denton, TX 76203-5220, USA
In this short review we examine the potential of single-molecule assays in drug development and in basic
research to provide new types of information at the smallest assay scales. A key advantage of many
single-molecule assays is the requirement for conservative amounts of precious sample compared to
conventional assays. In addition, they measure processes that are not observed directly in molecular
ensembles. These advantages are balanced currently by difficulties in assay setup, preparation and
equipment expense. However, future developments will ameliorate these drawbacks with the
production of simpler, less expensive experimental systems for single-molecule assays.
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The advent of single-molecule assays opens new possibilities in
drug development and research. The downscaling of bioassays to
the single-molecule level has the potential to reduce the amounts
of sample required and increase the different types of information
that can be obtained. The difficulties in setting up and performing
single-molecule assays limit their implementation, but this might
improve as the technologies develop. In this review we introduce
some of the diverse single-molecule assays that are available and
discuss the practical aspects of their applications.
What is a single-molecule assay?In the most general sense, investigation of a system of interest at
the level of individual molecules is a single-molecule assay, which
can include detection, spectroscopy, manipulation, imaging and
computational simulation (Figure 1). Detection by optical meth-
ods yields positional information and quantification, often in real
time. Further spectroscopy of the sample can provide information
on structural changes either within a single molecule or relative to
another object, and can even be used to detect catalytic and
binding events. In addition, several experimental-manipulation
devices enable specific mechanical perturbation of the single
molecule. By contrast, high-resolution microscopy, such as elec-
tron microscopy of single molecules, can approach nanometer
resolution and traditionally requires immobilized samples,
although recent developments in atomic force microscopy
Corresponding author: Root, D.D. ([email protected])
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(AFM) might allow real-time imaging in some cases. Although
theoretical in concept, computational simulations provide the
ultimate examination of the atomic resolution of a single mole-
cule. These areas offer new opportunities to test the effects of
drugs and characterize the molecular function of potential drug
targets.
Fluorescence microscopy is one of the most popular techniques
for single-molecule study, should adequate sensitivity be achieved.
Increasing the fluorescent signal from a single molecule by better
optical collection and higher excitation intensities is limited
primarily by photobleaching. Photobleaching can be reduced
substantially in vitro by the use of oxygen-scavenging systems
and reducing reagents. Another limitation is background, the
main sources of which are the fluorescence from residual impu-
rities, optical elements and light scattering. The following are
some basic geometries for increasing the signal:background-noise
ratio to detect single-molecule fluorescence:
(i) T
ee fron
otal internal reflection fluorescence microscopy (TIRFM).
TIRFM, which was developed by Dan Axelrod and colla-
borators [1], affords an important improvement in the
sensitivity of fluorescence microscopy. When high-angle
incident light is totally reflected from an interface between
two media of different refractive indices (e.g. glass and
water), the light (evanescent field) decays exponentially from
the interface and, thus, penetrates only 200 nm into the
optical medium with lower refraction number. This method
can be employed to selectively excite the sample that is only
t matter � 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.drudis.2006.05.003
Drug Discovery Today � Volume 11, Numbers 13/14 � July 2006 REVIEWS
FIGURE 1
Family tree of biological single-molecule assays. Typical single-molecule assays that involve visualization techniques can be described as belonging
to one of four major branches. Electron microscopy was the first technique to clearly visualize single macromolecules and it is an important validation
tool for other single-molecule studies. However, real-time functional applications of this technique are limited. Optical techniques are used mostwidely for functional single-molecule studies. Force microscopy and/or manipulation methods are relatively recent, but many diverse methods are
being developed. Computational single-molecule assays are becoming increasingly common as processing power and efficient algorithms make
them more accurate and practical. Although unlike experimental methods, computational techniques (such as molecular mechanics and dynamics) arepowerful visualization and analysis tools that can either assist other single-molecule assays or be used on their own. In this review, we focus on single-molecule
assays that are derived from visualization methods. Other single-molecule techniques that do not involve visualization, such as patch clamping are
reviewed in [60], which also describes optical trapping and magnetic tweezers.
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located at the evanescent wave region. Thus, the background
outside of this region is greatly discriminated.
(ii) C
onfocal fluorescence microscopy (CFM). In CFM, theexcitation light is focused to a spot, producing a fluorescence
emission that is refocused to a detector pinhole, so that most
of the signals from other sources are excluded. Thus, the
background is greatly reduced and only the layer of specimen
that is in the confocal plane is imaged [2]. By changing the
focal positions, the whole specimen can be scanned to
reconstruct its 3D image. CFM is especially suitable for
investigating events in living cells and tissues in real time
and with high spatial resolution, but the pinhole limits the
amount of signal collected relative to wide-field microscopy.
(iii) N
ear-field scanning optical microscopy (NSOM). In NSOM, atapered optical fiber with subwavelength aperture size, often
coated with metal, serves as a near-field optical probe and is
scanned in close proximity to the sample [3]. A non-
propagating, oscillating, electric field extends several hun-
dred nanometers beyond the tip. The spatial resolution of
NSOM is limited by the size of the light source rather than by
the diffraction limit, and its optical resolution can be
<70 nm [3]. Only a few other fluorescence techniques, such
as stimulated emission depletion and structured illumina-
tion fluorescence microscopies, report comparable resolu-
tion [4]. A unique advantage of NSOM is that spectroscopic
and topographic information can be measured simulta-
neously. Disadvantages are low-power throughput, poor
reproducibility of the field distribution at the tip and the
possibility of either mechanical or electromagnetic pertur-
bation of the sample by the probe.
Often, fluorescence microscopy and spectroscopy are performed
simultaneously on single molecules, but other spectroscopic
single-molecule assays also exist. Another optical technique that
has been applied to single-molecule assays is surface enhanced
resonance Raman spectroscopy (SERRS). SERRS is a Raman
technique that provides greatly enhanced Raman signal from
Raman active molecules absorbed on certain surfaces. To obtain
single-molecule Raman spectra, individual molecules have to be
adsorbed on metallic nanoparticles, usually silver or gold, at a very
low concentration [5].
A different multifunctional tool for microscopy and spectro-
scopy is AFM. The basis of AFM performance is the repulsive and
attractive force between a nanometer-size tip (probe) and the
sample. A cantilever bends in response to forces between the tip
of the cantilever and the sample. This causes changes in the angle
of reflection of a probe laser beam from the back of the cantilever
and, thus, changes the position where the laser beam strikes the
detector. The sample is mounted on a piezoelectric stage, which
ensures high resolution in 3D positioning [6]. Biological samples
can be imaged in their native state, at a lateral resolution of 0.5–
1 nm and a vertical resolution of 0.1–0.2 nm, in real time and in
physiological conditions [7]. AFM can be more than an imaging
tool because single molecules can be manipulated by providing a
small (pN) force to the sample. A single biomolecule can be either
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FIGURE 2
Practical limits of using small sample sizes. A key advantage of single-
molecule assays is the potential for downscaling to the ultimate level, butseveral practical limitations prevent the complete exploitation of this feature.
The extremely dilute concentrations that are required to achieve single-
molecule discrimination in these assays leads to large amounts of sample
loss, typically by non-specific adsorption to chambers, which increases theamount of sample needed for handling. Future developments might reduce
the amount of sample that can be handled.
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stretched or twisted by the AFM tip to study its mechanical
properties and structures [8]. Computational simulations of such
stretching can reproduce the force–distance curves that are
obtained experimentally, thereby adding atomic-resolution inter-
pretation to the measurements [9].
Even smaller forces than those of the AFM can be applied with an
optical trap. This is a highly focused laser beam that is used to trap
and remotely manipulate refractive particles [10]. A refractive par-
ticle near the focus will experience two types of force: a scattering
force, which is in the direction of light propagation; and a gradient
force, which is in the direction of the spatial light gradient. As the
result of a balance between the scattering force and gradient force,
the equilibrium position of the trapped particle is located slightly
down-beam from the focal point [11]. A small displacement
(<150 nm) from this position, results in a force imbalance that
tends to re-establish equilibrium, so the optical trap acts like a spring
and can be used as a force transducer in the pN range or lower.
Establishing precise position and force calibration is only practical
currently with spherical objects. Thus, microscopic beads are used
either alone or attached to objects of interest as handles to apply
calibrated force [12]. The current resolution is 0.1–0.4 nm and forces
as small as tens of fN have been measured [13,14].
Thediversityof single-molecule assays enables severalparameters
to be determined in a broad range of systems. By using these
techniques, the following properties can be measured in a single-
molecule assay: (i) localization of an individual molecule (even in a
living cell) [15,16], a site on an individual molecule [17], and
colocalization of more than one molecule [18]; (ii) fluorescence
intensity of dye molecules in living cells [15]; (iii) fluorescence
lifetime [19]; (iv) image and nano-structure of molecules [20,21];
(v) dynamics (reaction and conformation) [22–25]; (vi) intermole-
cular and intramolecular reactions [26,27]; (vii) distance between a
donor and an acceptor [28–30] or a donor and two acceptors [31];
(viii) orientation and rotation of molecules [32,33]; (ix) mechanical
properties of molecules [34–36]; and (x) folding and unfolding
intermediate states of macrobiomolecules [37]. Examples of experi-
mental systems that are currently studied at the single-molecule
level include, but are not limited to: (i) protein folding and unfold-
ing; (ii) enzyme catalysis; (iii) ion channels; (iv) signaling; (v) DNA,
RNA and their binding proteins; (vi) membrane structure;
(vii) molecular motors; and (viii) complex cellular structures. Thus,
there are many opportunities to exploit single-molecule assays.
How much sample is required for a single-moleculeassay?One attractive feature of using single-molecule assays for drug
development is the small size of the sample and valuable reagents,
which can enable a significant downscaling of conventional
assays. Several ml of a nanomolar concentration (or less) of sample
is sufficient for some single-molecule assays (Figure 2). However,
because single-molecule assays often have a low level of signal and
might have heterogeneous sample molecules, it usually takes more
repetitions of experiments (dozens to hundreds of repeats) than
ensemble experiments for satisfactory statistics and distribution
data for analysis. Also, the handling of small volumes of dilute
concentrations of sample can lead to large fractional losses of the
sample, especially from non-specific adsorption to vessel walls
during handling and storage. For small proteins and nucleic acids,
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the feasibility of chemically synthesizing single molecules in situ,
such as by modifying photolithographic techniques used on some
gene chips but with only a single molecule at each illuminated
spot, might reduce handling and storage problems. New oppor-
tunities exist for the development of technologies to deal with
such handling and storage problems (Figure 2).
In addition to analytes, other specialized materials, for exam-
ple, sensitive fluorescent dyes and modified surfaces, are needed
for a single-molecule assay. Fluorescent beads [38], dye-doped
nanoparticles [39] and quantum dots offer enhanced fluorescent
signals when larger probe sizes are tolerable [40,41]. Centroid
determinations on larger particles, such as microspheres, can
provide strong sensitivity even with brightfield microscopy
[42]. The chemical modification of molecules, particularly with
large probes, can sometimes require substantial quantities of
sample. Furthermore, dilute samples are often not suitable for
such processing, which provides a practical limit on the amount
of sample that is needed.
In experiments in which single molecules are immobilized on
surfaces, conjugating materials (e.g. BSA, biotin, streptavidin and
metal particles), and suitable surfaces (e.g. quartz cover slip) might
be required. Dilute samples typically adsorb slowly and ineffi-
ciently to surfaces, so that only a small fraction of the material
might be coated. In principal, if the sample quantity is highly
limited and not too fragile, the remaining sample can be recovered
and reapplied to a new area. Reducing the area over which the
sample is immobilized might be feasible for some single-molecule
assays. Microfluidics and spotting-array techniques reduce the area
over which a sample is applied to further improve sample utiliza-
tion [43]. The production of kits that facilitate single-molecule
assays has the potential to improve throughput, if sufficiently
reliable sources are available. AFM requires extremely flat, mod-
ified surfaces. Although these can be procured, they are usually
prepared fresh by the experimenter for optimum performance. The
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size and quality of the surface preparation will determine, in part,
the amount of sample that is required for a particular assay.
How fast are single-molecule assays?When planning a single-molecule assay, one must consider setup
and preparationtime,andthe timeneededtorecord and analyze the
data (Table 1). When planning high-throughput, single-molecule
assays, it is essential to identify steps that can be automated. Steps in
single-molecule assays differ widely in speed, depending on the type
of assayandthe breadth of information required.When implement-
inga newassay, setup is typically the rate-limiting step.However, for
computational single-molecule assays on large systems, data record-
ing is usually the slowest phase. Alternatively, if the data generated
are too complex to be analyzed easily and automatically, the ana-
lysis phase frequently becomes the longest part.
The setup of a single-molecule assay generally requires consid-
erable attention to both assay standardization and material pre-
paration. Assay standardization is most difficult during the initial
setup phase. Standardization is complicated by the observation
that different individual molecules seem to exhibit wide variations
in their responses. By contrast, ensemble measurements, by nat-
ure, contain a great deal of averaging that can reduce detection
variability. Consequently, more single-molecule observations rela-
tive to ensemble measurements are required to achieve a similar
level of confidence.
In many cases, the most problematic aspect of standardization is
determining the conditions under which a single molecule, rather
than an ensemble, is observed. Various arguments have been used
to support the singularity of the observed molecule including
sample dilution, frequency of response, photobleaching to zero
emission, anticipated magnitude of a signal, lowest unitary
response, lack of complexity in the data and microscopic visuali-
zation at high resolution [44]. Commonly, much time is spent
searching for a suitable single molecule to measure. The more
demanding the criterion for identifying single molecules, the
slower the assay. Often, not all observations need to be on a single
molecule; in such cases, occasional measurements of small groups,
in addition to single molecules, provide the desired information.
In some types of assays, calibration is a time-consuming com-
ponent of standardization. Optical-trapping devices, magnetic
traps and microneedle systems usually require extensive calibra-
tion, often for each measurement [45]. Calibration is somewhat
less demanding with atomic-force microscopes and other canti-
TABLE 1
Developments to increase throughput
Bottlenecks Possible developments
Setup Kit assays
Sample preparation Chemical synthesis
Observations Robotics
Analysis Automation
Computational speed Fast algorithms
Integration Inline coupling
Sensitivity Robust labels
Equipment availability Lower cost equipment
Validation Molecular imaging
lever-based systems, but is still quite demanding if the cantilever is
either changed or modified frequently. For higher-throughput
systems, designs and instruments should require minimal recali-
bration. Fluorescence measurements are usually the fastest to
standardize unless highly precise intensity comparisons between
widely differing microscopic fields are required.
Preparation of material is typically faster for single-molecule
assays than for bulk measurements because of the smaller sample
requirements. Chromatographic procedures of an analytical scale
are sufficient for many single-molecule assays, so intensive upscal-
ing of preparations can be avoided. Such analytical-scale prepara-
tions are also automated more easily, which can further reduce
time expenditures. However, if the sample must be analyzed by
slow procedures such as peptide mapping, as in some fluorescence
applications, then the time advantage is reduced or lost because of
difficulties in handling small sample amounts.
The time to record data can sometimes be faster for single-
molecule assays than for bulk measurements, such as in some force
measurements. Once a suitable molecule has been identified, the
recording of a mechanical measurement is not rate limiting and
occasionally is quicker than conventional force–transducer mea-
surements on larger samples. In AFM on a well-prepared sample,
hundreds of recordings can often be made in minutes. However,
many other single-molecule assays require longer acquisition times
that are either comparable to or exceed those of ensemble measure-
ments. The increased need for sensitivity might prolong exposure
times. If multiple types of measurements are to be performed on the
same single molecule, the manipulations require more care, and
repetitive measurements can be more crucial for single-molecule
assays.
Recording the data is not the rate-limiting step in most single-
molecule assays, but analysis of the data can be. The increased
simplicity of the single-molecule system can make the analysis per
measurement faster than that of comparable bulk samples. Con-
sequently, automation can greatly increase the speed the analysis
of single-molecule data. By contrast, more measurements and
increased noise in the data offset these speed advantages. In
particular, the sorting of the data to remove false positives, arti-
facts and contaminants becomes time-consuming if they either
occur frequently or are difficult to resolve from the intended data.
Why are single-molecule assays useful?A primary advantage of single-molecule assays is that they provide
unique information that cannot be obtained from ensemble mea-
surements (Figure 3). Consequently, comparing an ensemble mea-
surement with extrapolations from a single-molecule assay can
reveal complex interactions that result in differences between the
two. For instance, comparing recent studies of blebbistatin (which
inhibits myosin II) during in vitro motility assays of single actin
filaments sliding over myosin with its effects in live cells reveals
that blebbistatin produces high levels of free radicals when
exposed to blue light [46]. Also, the antibiotic microcin J25
increases pauses in transcription by bacterial RNA polymerase,
which is not resolved using ensemble assays [47].
The observation of single-molecule dynamics provides insight
into enzymatic mechanisms that are not obvious from ensemble
measurements [34]. Step sizes of motor proteins have been eval-
uated in greater detail by single-molecule assays [12,16,48]. The
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FIGURE 3
New information from single-molecule assays. One of the major
advantages of downscaling assays to the single-molecule level is the ability to
extract types of information that is lost in measurements from ensembles.This new information might contribute to drug testing and target-system
characterization by, for example, enabling a more complete assessment of
the variability in responses and identifying transient structures that might
provide targets for drugs. Correlations between results from different types ofsingle-molecule assays are crucial to verify these essential new data.
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rotation of ATP synthase has been demonstrated in textbook
fashion [49]. Spontaneous unraveling of the myosin coiled coil
has been visualized in real time [50] and its flexibility measured in
multiple orientations [51]. Several nucleic acid binding proteins
including hexameric helicases, lambda exonuclease, topoisome-
rases and RNA polymerases have been shown to move along
nucleic acids with complex directional mechanisms [52–55].
The forces that are involved in unfolding proteins and nucleic
acids provide unique insights into the structure of these macro-
molecules and can be used to fingerprint various domains. Pro-
teins such as titin, myosin and b-amyloid each exhibit unique
force–distance curves that vary depending on the domain struc-
ture that is stretched [6,45,56]. Such quantification of the unfold-
ing and unbinding forces correlates with known biological
functions. Potentially, these molecular fingerprints can be used
to identify domain folds in otherwise uncharacterized structures.
Diversity in a molecular species is most evident when examined
at the single-molecule level. Variations in protein folding are often
detected less easily using ensemble averages. The variations can give
rise to a broad range in measurements of binding, catalysis, velocity,
metrology and flexibility, even among proteins with identical
sequences [57]. It is possible that drugs might be developed that
interact only with divergent states of the protein. Such states would
not be identified easily by determining average structures.
In some cases, simply observing and counting single molecules
with high precision might be an important task. Microscopic
fluorescence-autocorrelation methods facilitate counting, enzy-
matic analysis and diffusion characteristics of single molecules
in very small sample volumes that are not immobilized, such as
living cells [58]. Although the dispersion of single molecules across
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a surface is not determined readily by bulk measurements, it is
observed directly by single-molecule visualization. Substrate
release from single molecules can also be observed directly by
fluorescence, giving a measurement of kinetics. Unique photo-
physical properties, such as the blinking of green fluorescent
protein, have also been determined by single-molecule observa-
tion and this property might enable super-resolution imaging in
living cells by permitting saturated excitation at lower light inten-
sities [4].
In addition to providing new insights into the structure and
function of a molecule and its interaction with a drug, single-
molecule assays provide a great cost saving in some situations.
Most notably, if only small quantities of protein are available,
some types of single-molecule assays are the only feasible option.
For example, measuring the enzyme activity of some proteins
requires that they are expressed in mammalian cells in culture,
but the yield is far less than can be achieved in bacterial expression
systems. Many native proteins are only expressed in small quan-
tities, so their proteomic analysis would be enhanced greatly by
single-molecule analysis.
Molecular dynamics and mechanics simulations are performed
typically at the single-molecule level and offer great cost savings in
preliminary drug screening and the detailed atomic analysis of
structure. Large libraries of drugs can be screened for interaction
with a target atomic model at reasonable speeds, thereby eliminat-
ing the most unlikely candidates and reducing the costs of synth-
esis and animal testing. Although still less accurate than
experimental results, increasingly promising correlations between
macromolecular simulations and experimental results have been
identified for some types of simulations [59].
Single-molecule assays are not always cost-effective. In many
cases the equipment used is more expensive than for comparable
bulk measurements because more sensitive, delicate instruments
are needed. In addition, a higher level of operator expertise is often
required, which can lead to greater personnel expenditures, espe-
cially if there is an increase in the setup and processing time for the
given experimental design. Frequently, substantial changes in
setup and analysis are incurred when switching between assays
of different types of single molecules.
Concluding remarksDespite some drawbacks, single-molecule assays have several vir-
tues that make them either competitive with or superior to tradi-
tional assays for some applications. When sample quantities are
either limited or extremely expensive, single-molecule assays
usually have an advantage. If ensemble averaging prevents detec-
tion of the desired property, the single-molecule assay is the only
resort. Future increases in the diversity of single-molecule assays
will expand the types of properties that can be observed and the
speed and precision with which they can be analyzed, as well as
decrease the expense. The future will see rapid expansion of single-
molecule assays.
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