New paper resulting from joint collaboration with Base Pair Bio and the Bornhop group at Vanderbilt University. Using aptamers and back scattering interferometry (BSI) to detect small molecules, such as TFV, BPA, and norepinephrine.

1. Characterizing aptamer small molecule
interactions with backscattering interferometry†
Michael N. Kammer,‡a Ian R. Olmsted,‡a Amanda K. Kussrow,a Mark J. Morris,b
George W. Jacksonb and Darryl J. Bornhop*a
Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors,
therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins,
peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because
they can be identified in vitro by SELEX, produced rapidly and relatively economically using
oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and
because of their unique properties they represent an attractive alternative to antibodies. Current assay
methodology for characterizing small molecule–aptamer binding is limited by either mass sensitivity, as
in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a
fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free
and free-solution sensing technique, can be used to effectively characterize SM–aptamer interactions,
providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this
capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A,
tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We
then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All
measurements produced R2 values >0.95 and an excellent signal to noise ratio at target concentrations
that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed,
expediting the assay which is also insensitive to the large relative mass difference between the
interacting molecules.
Analyst
PAPER
Cite this: DOI: 10.1039/c4an01227e
Received 8th July 2014
Accepted 18th August 2014
DOI: 10.1039/c4an01227e
www.rsc.org/analyst
Introduction
Aptamers are small pieces of single-strand DNA or RNA that
have been used in various applications. They can serve as
sensors,1 therapeutics,2 and cellular process regulators,3 as well
as drug targeting.4–7 The diversity of applications and the varied
targets to which aptamers can bind (proteins, peptides, and
small molecules) stem from the ability of aptamers to form
complex three-dimensional shapes including both helices and
single-stranded loops. Aptamers are then able to bind with
targets through a variety of interactions including van der Waals
forces, electrostatic interactions, hydrogen bonding, and base
stacking. Aptamers are selected in vitro and produced rapidly
and inexpensively to bind with high affinity and specicity to a
desired target molecule and as such are a promising alternative
for applications that traditionally use antibodies. To date, there
has been no way to determine the binding affinity between
aptamers and small molecules that is inexpensive, reliable, and
does not require immobilization.
Currently, less than some 25% of existing aptamers have
been generated for SM targets. Some of the earliest aptamers
were selected against small organic dyes,8 which remained the
focus of aptamer development during their infancy. However,
methods were soon developed to easily select aptamers to larger
biomolecules. Later, more complex targets containing more
functional groups and structural motifs yielded a greater
probability of nding sequences that can interact with the
target via hydrogen bonds, electrostatic interactions, and
hydrophobic interactions.9 As a result, the development focus
switched to protein-binding aptamers and development of new
aptamers for small molecules became less prevalent.
Recently, there has been a renewed interest in the design of
aptamers that bind small molecules.10–12 Identifying and char-acterizing
new SM binding aptamers is important because they
play key roles in a diverse range of biological processes, are
involved in the environment, and are oen difficult to quantify.
Small molecules can be harmful toxins, benecial drugs, such
as antibiotics, nutrients, and serve as intercellular signal
mediators or neurotransmitters. The vast majority of our most
important therapeutics are small molecules,13 in part because
aDepartment of Chemistry, Vanderbilt University, 2201 West End Avenue, Nashville,
Tennessee, USA. E-mail: darryl.bornhop@vanderbilt.edu
bBase Pair Biotechnologies, Inc., Houston, Texas, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4an01227e
‡ These co-rst authors contributed equally to the work.
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2. Analyst Paper
they can oen easily diffuse across cellular membranes.14
Aptamers represent a unique class of chemical species because
they can be selected to bind, manipulate, inhibit, control, or
even detect small molecules in a complex matrix.
Currently there are six common methods of measuring
aptamer–target binding affinity. These are surface plasmon
resonance (SPR), biolayer interferometry (BLI), uorescence
anisotropy, isothermal calorimetry (ITC), microscale thermo-phoresis
(MST) and backscattering interferometry (BSI). SPR
measures the localized change in the refractive index (RI) near
the surface of a substrate in order to detect binding15,16 and has
been used in a multiplex format.17 However, SPR is a hetero-geneous
method that requires complicated surface chemistry,
tethering or immobilization of one of the species, typically the
smaller of the two binding partners. Immobilization of one of
the interacting species is known to impact the binding, and the
use of relatively expensive gold-plated slides or gold nano-particles
remains a limitation in SPR. Biolayer interferometry is
another heterogeneous, label-free, optical analytical technique.
In BLI the interference pattern of white light is produced from
reection of two surfaces: a layer of immobilized protein on the
biosensor tip, and an internal reference layer. During the
binding event the reference surface region is compared to the
sample region. BLI overcomes some of the limitations of SPR,
yet it still requires surface immobilization. As with SPR, quan-ti
cation of SM–aptamer binding is inherently difficult to
measure with BLI, because it relies on a change in the mass at
the surface upon binding. Typically, the smaller of the inter-acting
partners must be immobilized, so that the binding can
be detected with reasonable nesse.18,19 Attempts to immobilize
small molecules to the surface and detect the larger moiety are
somewhat complicated and have practical limitations, since
minimal changes in the structure can have a signicant impact
on the affinity.20 ITC is a widely accepted method to measure the
rate and affinity of molecular interactions21–23 that has recently
demonstrated the ability to probe aptamer–target interac-tions,
24 however this method suffers from low throughput,
relatively large sample quantities, and poor detection limits.
Fluorescence anisotropy (FA) has been used successfully for the
characterization of aptamer–protein binding,25–27 however the
rotational change upon binding between a large biomolecule
and a SM ligand can be small or not observable. Microscale
thermophoresis (MST) is a relatively new free-solution method
that can be applied to aptamer–SM interaction.28 Like FA, MST
still requires uorescent labeling of the biopolymer and thereby
increases the cost and complexity of the assay.
Backscattering interferometry (BSI) is a label-free, free-solu-tion
technology that can rapidly measure binding with little a
priori knowledge of the system and allows for a broad range of
binding affinities (pM–mM) to be measured.29,30 In the most
common conguration of BSI, a microuidic chip with a
channel etched in glass acts as both the sample holder and the
optics. BSI utilizes a red helium–neon (HeNe) laser (l ¼ 632.8
nm) to illuminate the chip in a simple optical train consisting of
a source, an object, and a transducer. As the beam impinges on
the channel, the beam reects and refracts within the channel
before exiting as a high contrast fringe pattern. When a binding
event occurs it results in a change in the refractive index (RI)
that produces a change in the spatial position of the fringes.
This fringe shi is monitored using a CCD array in combination
with Fourier analysis.31 Because the BSI signal is a product of
inherent properties of the sample (conformation, hydration and
molecular dipole moment), there is no need for surface-immobilization
or labeling to quantify a binding interaction. A
more thorough explanation of BSI and the assay methodology is
presented in our previous work. Specically, these publica-tions30,32
offer a detailed description of the instrument and
would serve as a guide for in-house construction. Furthermore,
the reader is encouraged to contact the Bornhop research group
if questions arise during implementation at their site.
Our group has previously used BSI to measure aptamer–
protein binding affinities in the low nanomolar range,33
showing also that it can quantify allostery in thrombin. Here,
looking at a range of target molecules, we demonstrate the
ability to quantitatively characterize the interaction between SM
and aptamers with BSI. Ampicillin and tetracycline serve as
common antibiotics, epirubicin as an anthracycline drug is
used for chemotherapy, and tenofovir is an antiretroviral drug.
We also studied BPA, which is a chemical toxin commonly used
in plastics in the food and beverage industry. Finally, we studied
the aptamer interaction with the hormone and neurotrans-mitter
norepinephrine. We showed that BSI results compared
well to published affinity values. In other cases, we performed
range-nding experiments to bracket the Kd, before building
saturation isotherm plots with endpoint analysis. Our attempts
to use MST or BLI for these analytes resulted in unreliable Kd
values (data not shown). In all cases, BSI yielded high quality Kd
values for the binding interactions, rapidly and with minimal
sample manipulation.
Materials and methods
Previously unreported aptamers were selected by Base Pair
Biotechnologies, Inc. (Pearland, TX). The sequence of the pub-lished
BPA aptamer “12-5” was obtained from Jo et al.34 All
binding affinity determinations by BSI were performed in an
end-point format as previously described,29,33,35,36 with the
aptamer diluted in PBS buffer containing 150 mM NaCl and 1
mM MgCl2. In short, the end-point (mix-and-read) assay
consists of mixing a series of samples containing increasing
concentrations of the SM target, from 0 nM to saturation, with a
constant amount of aptamer, followed by 2 hour incubation,
and the subsequent BSI was read. Samples are prepared in a
manner such that the background solution composition is
constrained throughout.
BSI provides a relative output using a series of blanks con-sisting
of the same concentrations of the target, in the absence
of an aptamer, as a reference allowing for correction for the
analyte-induced bulk RI shi. A non-binding SM is also incu-bated
with each aptamer, serving as a control and providing a
measure for nonspecic-binding. Typically, 1 mL of sample is
injected into the instrument and measured for 30 seconds, the
channel is then evacuated, rinsed if needed and the next sample
is introduced. The entire assay is run in triplicate, over a large
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3. Paper Analyst
ligand concentration range, allowing a saturation isotherm to
be constructed and providing the most conservative outcome
with respect to system dri.We calculate the difference between
the binding sample and the blank and plot these values versus
concentration. The data are t to a single site saturation
isotherm model using GraphPad Prism 4 (GraphPad Soware,
Inc., La Jolla, CA). Using the coefficients of this nonlinear
regression t, Kd and Bmax are calculated.
Results and discussion
Six aptamers were provided in a blinded fashion by Base Pair
Biotechnologies to the Bornhop laboratory for binding affinity
measurements. The binding affinity of various aptamers to their
respective target was measured by BSI with no a priori knowl-edge
of the binding system and only an estimate of the possible
Kd. Three of the aptamers were studied previously by other
methods (Table 1), tenofovir, epirubicin and bisphenol A (BPA);
the other three have remained uncharacterized until this report.
With a motivation to provide true Kd values37,38 we used
concentrations of the aptamer at or signicantly below the Kd
value.
Fig. 1A illustrates the saturation isotherm plot for the
aptamer TFV_G2:125:256 binding to the antiretroviral drug
(M.W. ¼ 287 g mol1) tenofovir. The experiment yielded an R2
value of 0.9885 for triplicate binding assay determinations. Our
SM control experiment consisted of quantifying the
TFV_G2:125:256 aptamer binding to 40 nM of ampicillin. At this
concentration, the highest used for tenofovir, the aptamer
showed a relatively modest binding signal suggesting decent
specicity versus a commonly used SM drug. Table 1 illustrates
the binding affinity of 9.0 1.4 nM for tenofovir determined by
BSI (a Kd of approximately 1 nMwas determined by MST). While
the affinity determinations were not designed to measure the
limit of detection, we estimate these values to be 2.5/6.2 nM for
tenofovir. The limit of detection (LOD) is dened as 3 times the
standard deviation of each individual phase reading, aer
signal settling, over 20 seconds, divided by the slope of the
linear region of the saturation isotherm. In our case we calcu-late
the limit of quantication (LOQ), as 3 times the pooled
(average) standard deviation for replicate measurements
divided by the slope. In our serum biomarker work we have
found that the LOQ for an assay is typically 10 to 20-fold better
than the Kd, thus a quantitative assay with an LOQ of 300 pM is
well within reach for BSI using this aptamer–SM interaction.
The BSI determination of the Kd for Epirubicin286, the
aptamer for the anthracycline drug used for chemotherapy
epirubicin, is shown in Fig. 1B. Our rapid, label-free and free-solution
evaluation gave a binding affinity of 626 121 nM.
Again our results compare well with the MST-measured value of
711 30 nM for the binding of this 285 g mol1 chemotherapy
drug to the aptamer. In this case the control ampicillin had no
quantiable binding to the aptamer giving a BSI signal below
the S/N threshold of 5 milli-radians (mrad) fringe shi. While
the specicity of this aptamer, at least with respect to another
common heterocyclic SM is quite high, the affinity would likely
not be sufficient for a number of analytical applications.
Nevertheless, the interaction produces a robust signal indi-cating
that binding produces a signicant change in confor-mation
and hydration, suggesting that further renement of the
basic structure could produce an aptamer with higher affinity
and robust S/N. These results are encouraging because label-free
SM quantication has been challenging, yet ongoing
studies are needed to determine if this level of performance will
enable the construction of an assay for drug dosing and efficacy
monitoring in cancer patients.
An inexpensive and robust assay for Bisphenol-A (BPA, M.W.
¼ 228 g mol1), a once-common food-grade plastic resin now
considered harmful to human health, is quite desirable.
Current assays for BPA levels in human blood utilizing LC-tandem
mass spectrometry have detection limits ranging from
0.43 nM (ref. 39) to 64 nM.40 In an attempt to simplify the
determination for BPA, aptamers with Kd values in the nano-molar
range, directed against BPA have been recently described
by Jo et al.34 These aptamers were also found to be highly
specic for BPA, and did not bind to structurally related
compounds bisphenol B or 4,40-bisphenol.34 Here we measured
a binding affinity for the same clone, the “12-5” described by Jo
et al., allowing further benchmarking of our methodology.
Fig. 1C presents the BPA–aptamer saturation isotherm
produced in our BSI experiments, illustrating that we measured
a binding affinity of 20.2 2.1 nM. This value compares well to
the 10 nM Kd reported (no technical replicates) by Jo et al. using
equilibrium lter binding. In this case, 10 mM of the control
molecule ampicillin showed no appreciable binding to the
aptamer. As noted previously, affinity determinations are not
designed to measure LOD/LOQ. Given our limited data, we
estimate the LOD and LOQ to be 2.2/7.3 nM BPA. In our serum
biomarker work we have found that the LOQ for an assay is
Table 1 Binding affinity of six aptamers to their respective SM targets
Aptamer ID Target Aptamer BSI Kd Literature Kd R2
TFV_G2:125:256 Tenofovir 1 nM 9.0 1.4 nM 10 nM 0.989
Epirubicin286 Epirubicin 50 nM 626 121 nM 711 nM 0.983
BPA published Bisphenol A 1 nM 20.2 2.1 nM 10 nM 0.979
Ampicillin284 Ampicillin 50 nM 402 99 nM 569.8a nM 0.978
Tetracyc_11C02 Tetracycline 50 nM 2.94 0.28 mM N/A 0.993
Norepi_3932c_Prim Norepinephrine 50 nM 188 64 nM N/A 0.955
a The value for the Ampicillin literature Kd comes from an MST measurement included in the ESI (Fig. S1).
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4. Analyst Paper
typically 10 to 20-fold better than the Kd. Given our recent
experience with serum and tissue,41 the magnitude of the signal
for this assay at low concentrations and that the LOQ for an
assay is typically 10 to 20-fold better than the Kd, we are con-
dent that a label-free BSI assay would be quantitative for BPA at
the 100's of the pM level.
As Fig. 1 and Table 1 illustrate, collectively there is an
excellent agreement between the two solution phase measure-ments,
BSI and MST over a relatively wide range of Kd values.
The three other SM targets studied here represent species
that are relatively difficult to measure, most typically by ELISA
requiring multiple binding and wash steps.42–44 In this case, the
mix-and-read operation by BSI is particularly advantageous
because it allowed rapid Kd range nding, on constrained
sample quantities (microliter volumes), with no a priori infor-mation
about the species.
Ampicillin is a widely used beta-lactam antibiotic (M.W. ¼
349 g mol1) and represents a potential contributor to over-use
antibiotic resistance. Here our saturation isotherm (Fig. 2A)
yielded a Kd of 402 99 nM for the aptamer “ampicillin284”.
For many applications, ampicillin is detected in the environ-ment
and biological samples by ELISA, capitalizing on enzy-matic
signal amplication, but needing a cold-train to ensure
Fig. 2 BSI Kd determinations for three aptamer targets with no
previously reported values. (A) Ampicillin binding to Ampicillin284, (B)
tetracycline binds to tetracyc_11C02, and (C) norepinephrine binds to
norepi_3932c_Prim.
protein functionality. lWhile the typical detection limit using
commercial ELISA kits is approximately 300 pM, no simple
assay–analyzer combination exists that is capable of identifying
a wide range of drugs at both low (e.g. 0.1 mg ml1) and high
concentrations (100 mg m1) as desired by the research
community. Since these assays were designed to measure
affinity, the aptamer concentration was considerably lower than
the Kd. For a detection limit/quantitation assay,41 we would
employ an aptamer concentration in excess to allow the
dynamic range and the lower limit of detection to be extended.
Future investigations will be directed toward target quantica-tion,
but in this case we predict that an optimized BSI assay
based on this target–probe interaction would have sensitivity in
the 100s of pM range in a complex milieu.
Tetracycline is a broad-spectrum polyketide antibiotic (M.W.
¼ 444 g mol1) that continues to be a challenge to quantify42
and given the rise in antibiotic resistance,45 the potential to
quantify this important drug with a simple method, in food or
in the near-patient setting could be of great value. In an initial
set of experiments we evaluated the affinity of our aptamer
Fig. 1 BSI Kd determinations for the three aptamer targets with a
previously reported Kd. (A) Tenofovir binding to TFV_G2:125:256, (B)
Epirubicin binding to the Epirubicin286 aptamer and (C) BPA binding to
the BPA aptamer.
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5. Paper Analyst
“tetracyc_11C02” with BSI. In free-solution, the saturation
isotherm for tetracycline binding to this early generation
aptamer yielded a Kd of 2.94 0.28 mM. Admittedly, improve-ment
on a Kd in the micromolar range to approximately 1.4 mM
would be required to facilitate a widely deployable assay for
food and serum. Yet, even at this level, our BSI would be
competitive with the microbiological assay in milk and could be
used to detect the drug in waste water.
Detection of norepinephrine is important because high
plasma levels may be associated with reduced survival rates of
otherwise healthy older individuals46 as well as patients with
congestive heart failure.47 Because norepinephrine is also
secreted in urine, it may serve as a convenient biomarker as well
for monitoring general neurotransmitter disruption and/or for
monitoring treatments.48 Fig. 2C presents the binding assay for
our norepinephrine aptamer “Norepi_3932c_Prim”. Our
aptamer for this SM hormone and neurotransmitter target
(M.W. ¼ 169 g mol1), gave a binding affinity of 188 64 nM.
Given our recent biomarker results,41 at this affinity a BSI assay
could be used for the detection of norepinephrine with high
condence and minimal optimization in the range of 0.5–10 nM
(0.85–1.7 ng ml1) in serum. By comparison, a commercially
available ELISA for norepinephrine (Abnova, Taipei, Taiwan)
reports a lower calibration range of 20 ng ml1 in urine and 318
pg ml1 in plasma (LOD ¼ 118 nM in urine; 1.8 nM in plasma).
Alternatively, a LC-mass-spec based approach achieved a LOD of
1.23 ng ml1 in urine.49 Thus, using BSI in conjunction with
aptamers and small molecules has the potential to rapidly assay
these analytes without the multiple wash and enzymatic
amplication steps of ELISA nor the complicated instrumen-tation
required for LC-MS approaches. Mix-and-read, label-free
operation also facilitates accelerated assay validation, impor-tant
when attempting to translate a procedure to the clinical
setting.
Conclusion
It has been shown here that BSI can be used to effectively
measure the interaction between an array of small molecules
and aptamers selected to bind to these targets. The results were
quantitative, giving Kd values for the molecular interactions in a
label-free, free-solution format. With the mix-and-read BSI
assay, no immobilization of aptamers or small molecules is
required, nor is any chemical modication such as labeling
needed for either of the interaction species. Binding determi-nations
took hours to complete, with no prior knowledge of the
binding system. Where Kd was previously known, Kd values
determined by BSI agreed well with the literature. BSI also
worked well for binding systems with unknown Kd values, and
where we were unable to successfully obtain reliable values with
other methods. These results were easily produced and gave
reproducible Kd values within the expected range of affinities.
Given our results, we predict that going forward BSI will enable
the rapid characterization of high affinity aptamers, particularly
those with pM to nM affinity values, helping to expedite their
use in the main-stream.
Acknowledgements
This research was supported in part by a grant from the
National Science Foundation (Grant CHE-0848788) and the
Vanderbilt University Institute of Chemical Biology. D.J.B. and
A.K. have a nancial interest in a company that is commer-cializing
BSI. The other authors declare that they have no
competing nancial interests.
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