DNA-Based Scaffolds for Sensing Applications
Simona Ranallo, Alessandro Porchetta,* and Francesco Ricci*
Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome,
Italy
■
CONTENTS
DNA Nanotechnology for Sensing Applications 44
Optical Structure-Switching DNA Scaffolds 45
Antibody Detection 45
DNA-Based Structure Switching Probes for
Optical Antibody Detection 46
Nucleic Acid-Based Co-Localization Probes for
Optical Antibody Detection 47
DNA-Based Scaffolds for Antibody-Controlled
Reactions, Logic Gates, and Circuits 48
DNA Scaffold Probes as Electrochemical Sensors 50
DNA-Based Switches for Electrochemical Anti-
body Detection 50
DNA Scaffolds for Electrochemical Antibody
Detection Based on Steric Hindrance 50
DNANanostructuresasScaffolds for Sensing
Applications 51
Multienzyme Complexes on Two-Dimensional
(2D) and Three-Dimensional (3D) DNA Origami
Scaffolds 51
DNA Tetrahedral Scaffolds for Sensing Applica-
tions 53
Proximity-Based Assembly of DNA Scaffolds for
Sensing Applications 54
DNA Scaffolds for Creating Synthetic Nanopores
for Single-Molecule Biosensing 56
Conclusions 56
Author Information 56
Corresponding Authors 56
ORCID 56
Notes 56
Biographies 56
Acknowledgments 56
References 56
■
DNA NANOTECHNOLOGY FOR SENSING
APPLICATIONS
DNA nanotechnology employs synthetic nucleic acid strands
to design and engineer nanoscale structural and functional
systems of increasing complexity that may find applications in
sensing,
1−7
computing,
8−10
molecular transport,
11−13
informa-
tion processing,
14
and catalysis.
15,16
Several features make
synthetic DNA a particularly appealing and advantageous
biomaterial for all the applications mentioned above but more
specifically for sensing. First, synthetic DNA sequences,
especially if of limited length (<100 nucleotides), have highly
predictable interactions and thermodynamics. This allows the
development of spatiotemporally controlled nanostructures
with quasi-Armstrong precision and the engineering of
supramolecular devices with well-controlled secondary struc-
tures.
17−22
DNA is a lso quite easy and inexpensi ve to
synthesize: currently the cost of 150 μg of an unmodified
single-stranded DNA strand of 20 nucleotides is ∼8 euros if it
is purchased from one of the many commercial vendors
available in the market. Finally, DNA is relatively stable
compared to other biomolecules like enzymes or antibodies.
The other important feature of synthetic DNA is the wide
range of possibilities that it offers for sensing applications if it is
used as recognition element. Of course, the most obvious use
of a single-stranded synthetic DNA sequence as a recognition
element is for the detection of a specific target complementary
sequence. Countless applications of such a use, especially if
coupled with polymerase chain reaction (PCR), have been
repor ted to date, which resulted in many commercially
available sensing devices.
23,24
Synthetic DNA can also be
used as a recognition element for targets other than DNA. This
is the case, for example, of DNA aptamers, a class of high-
affinity nucleic acid ligands, which are selected through
alternate cycles in vitro to bind a specific target molecule.
25−29
To date, thousands of DNA and RNA aptamers that bind to
specific targets have been selected, including small molecules,
proteins, peptides, bacteria, virus, and live cells.
30−32
Other
aptamers can bind to surface molecules and membrane
proteins of live cells.
33−35
A DNA aptamer is usually a short
DNA sequence (<100 nucleotides) that can bind its specific
target with high affinity (nanomolar to micromolar) and high
specificity. While the affinity of the aptamers is usually not as
high as that of other biomolecular recognition elements (i.e.,
antibodies), there are some advantages connected with their
use, including the lower cost and higher stability. Synthetic
DNA can also be used as a recognition element to detect metal
ions through the use of thymine-thymine (T-T) and cytosine-
cytosine (C-C) mismatches, which specifically bind mercury-
(II)
36−38
and silver(I)
39,40
ions, respectively, or through the
use of copper-dependent DNAzymes.
41
Similarly, nonconven-
tional DNA interactions can be used to rationally design pH-
sensitive DNA switches that can be used as nanometer scale
pH meters.
42−44
Such probes typically exploit DNA secondary
structures that display pH dependence due to the presence of
specific pro tonation sites. These structures include I-
motif,
45−50
inter- and intramolecular triplex DNA,
51−55
DNA
tweezers,
56
and catenanes.
57
Recently, we have also reported
on the rational design of programmable DNA-based nano-
switches whose closing and/or opening can be triggered over
specificdifferent pH windows by simply changing the relative
Special Issue: Fundamental and Applied Reviews in Analytical
Chemistry 2019
Published: December 1, 2018
Review
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content of TAT/CGC triplets in the switches.
58
Finally, DNA
can be employed as a convenient recognition element for the
detection of transcription factors, proteins that control the
transcription of genetic information and specifically recognize
double-stranded or single-stranded DNA and RNA sequen-
ces.
59−63
The examples of DNA-based sensors described above, in
which DNA itself is used as recognition element, have been
recently summarized in several extremely complete re-
views.
64−68
Although interesting for sensing applicatio ns,
these examples also demonstrate that the range of targets
that can be detected with DNA as a recognition element is
limited, and this can ultimately hamper further progress in the
field of DNA-based sensors. Recently, however, a novel use of
synthetic DNA has proven to be extremely advantageous for
analytical use. DNA, in fact, contains several functional groups
that make it quite straightforward to modify a synthetic
nucleotide sequence at both ends or internally. A variety of
additional reactive groups can be introduced into DNA
sequences, and most of these modifications are currently
available in the catalogues of synthetic DNA oligonucleotide
commercial vendors. For sensing applications, these functional
groups can be used to conjugate signaling moieties (for
example, fl uorophore/quencher pairs or electrochemical redox
labels) or anchoring tags (for example, thiol groups for
attachment to a gold electrode surface). As we will show
during the course of this review, this chemical versatility can
also be used to attach and conjugate different recognition
elements to a synthetic DNA sequence, thus widely expanding
the range of targets that could be detected with DNA-based
sensors. In these cases, DNA is thus simply used as a versatile
scaffold that allows the attachment and conjugation of a wide
range of small and large molecules with high accuracy and
precision. This review intends to summarize the recent
advancements made in this direction by describing results
achieved in the past 4 years and will serve as an important
demonstration that synthetic DNA can indeed be used as a
versatile scaffold for a wide range of sensing applications that
are not limited to the targets that are usually recognized by
DNA probes. We will not focus on the conjugation strategies
and protocols used; we direct the readers to recent specific
reviews on this subject.
69,70
We will instead focus on the
practical analytical applications of DNA scaffold systems. The
examples we have included in this review can be divided into
three major classes. Initially, we will describe DNA-based
systems belonging to the class of structure-switching probes
that are mostly based on optical detection. We will then
describe DNA-scaffolded electrochemical sensors and finally
show the potential of DNA nanostructures (origami) to
position recognition elements in a highly precise way.
■
OPTICAL STRUCTURE-SWITCHING DNA
SCAFFOLDS
Antibody Detection. The detection of specific antibodies
and other diagnostic proteins plays a crucial role in the
diagnosis of many diseases, infections, and pathologies.
71,72
Despite their widespread use, however, current methods for
the quantitative detection of specific antibodies, including
enzyme-linked immunosorbent assays (ELISAs) and Western
blots, remain cumbersome, laboratory-bound processes
73,74
because the formation of antibody − antigen complexes is not
linked to any easily measurable output. For this reason, current
Figure 1. DNA-based structure switching probes for optical antibody detection. (A) The optical antibody-switch platform is designed to adopt a
two-tailed stem−loop conformation in the absence of the target antibody. The two single-stranded tails act as an anchoring strand for DNA
complementary sequences that are conjugated at one end with an appropriate recognition element (i.e., digoxigenin antigen) for the target antibody
(anti-digoxigenin antibody). The binding of one copy of a target antibody to the two recognition elements causes a conformational change resulting
in the opening of the stem−loop conformation and in an increase in the fluorescence signal intensity. (B) The antibody-switch sensor detects anti-
digoxigenin antibodies at low nanomolar concentrations. (C−E) The modular nature of the platform allows the detection of different targets by
changing the recognition element [C, dinitrophenol (DNP); D, eight-residue FLAG peptide; E, 13-residue epitope excised from HIV protein p17]
in a nanomolar concentration range, without any significant cross-reactivity with the other nonspecific targets. Reproduced from A Modular, DNA-
Based Beacon for Single-Step Fluorescence Detection of Antibodies and Other Proteins. Ranallo, S.; Rossetti, M.; Plaxco, K. W.; Valle
e-Be
lisle, A.;
Ricci, F. Angew. Chem., Int. Ed. 2015, Vol. 54, Issue 45 (ref 89). Copyright 2015 Wiley.
Analytical Chemistry Review
DOI: 10.1021/acs.analchem.8b05009
Anal. Chem. 2019, 91, 44−59
45
methods for antibody detection typically employ “sandwich
assay” formats in which in a multistep, wash-intensive process a
conjugated secondary antibody or antigen is used to generate
an observable signal.
75−77
While these limitations have only
modestly impacted the use of these approaches in industrial-
ized countries, they significantly limit the applicability of these
techniques in point-of-care applications and in the developing
world.
78,79
To ensure rapid appropriate care for patients,
simple, inexpensive, and quantitative tools for the detection of
specific antibodies are thus urgently needed. Apart from the
obvious application for disease diagnostics, platforms for
antibody detection could also be used for other important
purposes. In recent years, for example, immunotherapy has
attracted a great deal of interest because of its promising
expectations for the treatment of various forms of cancer or
other diseases.
80−82
Indeed , immuno therapy represents a
powerful tool, either as a monotherapy or as a combination
therapy with chemotherapy or radiation. At the end of 2016,
nearly 60 antibody drugs had been approved by the Food and
Drug Administration, and many more are currently in clinical
trials.
83
Recently, bioengineered bispecific antibodies (BsAbs)
containing two di ff erent binding sites within a single molecule
seem to offer the potential to improve therapeutic efficacy and
promise to be the next generation of immunotherapy.
84,85
From this perspective, it would be extremely important to be
able to measure, during immunotherapy treatment, the levels
of therapeutic antibodies at designated time intervals and to
maintain a constant drug concentrationinapatient’ s
bloodstream, thereby optimizing individual dosage regimens
and clini cal outcom es in patients.
86−88
For the reasons
mentioned above, point-of-care (POC) methods for the
detection and quantification of therapeutic antibodies would
improve the characterization and monitoring of immunothera-
pies, improving their efficacy with subsequent great societal
and medical benefits.
Motivated by the considerations mentioned above, re-
searchers have recently been strongly devoted to employing
the advantageous features of synthetic DNA to design and
develop rapid and sensitive analytical platforms for antibody
detection. In all of these examples, DNA is not employed as a
recognition element but merely as a scaffold to attach the
specific recognition element and to signal the presence of the
target antibody through a conformational change that takes
advantage of the spatial geometry common to most antibodies.
All IgG and IgE antibodies, in fact, share the same y-shaped
structure wit h two identical binding sites s eparated by
approximately 10− 12 nm. This simple and yet often
overlooked feature of antibodies has been instrumental in
cleverly designing novel direct systems for antibody detection
based on the use of synthetic DNA scaffolds and different
sensing strategies.
DNA-Based Structure Switching Probes for Optical
Antibody Detection. We have recently rationally designed a
DNA-based platform for the optical single-step and quantita-
tive detection of antibodies based on a target-induced
conformational change mechanism.
89
Our analytical platform,
which we named antibody-switch (Figure 1A), is comprised of
a 66-nucleotide scaffold DNA sequence (black) containing two
five-nucleotide internal, complementary regions. This scaffold
forms, in the absence of the target antibody, a two-tailed
stem−loop structure ( Figure 1A). A fluorophore/quencher
pair was internally conjugated at two thymines at the end of
the stem portion so that the scaffold provides a weak
fluorescent signal in the stem−loop configuration. The two
single-stranded tails of the scaffold act as anchoring strands for
the hybridization of two DNA complementary sequences that
are conjugated at one end with an appropriate recognition
element (antigen) for the target antibody. To avoid the use of
two different antigen-conjugated strands, the scaffold strands
were designed to have a 5′−3′ frame inversion at one end of
the stem. This ensures that the two tails meet “head to head”
(3′-end to 3′-end), thus allowing a single recognition element-
modified strand sequence to populate both recognition sites.
The binding of one copy of a target antibody to the two
recognition elements on the scaffold causes the opening of the
stem−loop conformation, thus resulting in an increase in the
intensity of the fluorescence signal due to the fact that the
fluorophore is forced away from the quencher. In this example,
the modularity of DNA is thus used to design a sensor that is
composed of multiple units each with a different purpose. The
scaffold unit p rovides signaling and the conformational
switching mechanism. The antigen-conjugated strands, instead,
provide the recognition ability for the target antibody. While
the sensing idea of such a platform is simple, its rational design
and optimization is not straightforward and requires the careful
observation of the following general rules. The first rule applies
to all structure-switching sensors and is related to sensitivity.
The thermodynamic switching equilibrium of the stem−loop
scaffold between the closed and open conformation is in fact
crucial to achieving a high sensitivity and a good signal-to-
noise ratio. In this specific case, a weak background and a large
signal change can be achieved when the switching equilibrium
is shifted toward the closed conformation (i.e., when the
equilibrium switching constant, K
S
= [open conformation]/
[closed conformation], is <0.1).
90
However, an overstabiliza-
tion of the closed conformation also increases the energetic
barrier that antibody binding must overcome to cause the
conformational switch, thus affecting sensitivity. A compromise
in the optimization of the switch is thus required, and usually,
the lowest limits of detection can be achieved with a K
S
of ∼1,
which gives a maximum signal gain of 50% (because 50% of
the switch is already in the “open” conformation in the absence
of input) while decreasing the observed affinity only 2-fold
compared to the intrinsic affinity of the target antibody.
61,91
The second rule that needs to be considered in the design of
antibody switches is the distance that the two recognition
elements should span in the open conformation to allow
optimal binding of the antibody. In this regard, we note that
the hinge region that links the Fc and Fab portions of an
antibody is a flexible tether that allows a quite independent
movement of the two Fab arms, thus making the distance
between the two binding sites (present at the end of the Fab
arms) quite variable.
92,93
Despite this, the common y-shaped
structural view of an IgG or IgE antibody shows the two
binding sites separated by approximately 10−12 nm. This
distance should thus be taken as a reference for the rational
design of the antibody-switch.
Both of the design rules described above can be easily met
by taking advantage of the versatility of synthetic DNA, which
allows one to fine tune the stability of the stem−loop
conformation by simply changing the length of the stem and
its TA versus GC content and to span a quite precise distance
between the recognition elements by changing the number of
nucleotides in the loop. To optimize the switch according to
the rules mentioned above, digoxigenin was initially used as
antigen and anti-digoxigenin antibodies were employed as
Analytical Chemistry Review
DOI: 10.1021/acs.analchem.8b05009
Anal. Chem. 2019, 91, 44−59
46
targets. While we note that this is not a clinically relevant
target, the use of the digoxigenin/anti-digoxigenin couple is
perfect for optimization purposes for the following reasons.
First, digoxigenin is a small molecule that does not affect the
overall stability of the stem−loop scaffold. Second, digoxigenin
is a widely used hapten in biotechnology
94
and contains more
than one functional group that allows a simple and inexpensive
conjugation to synthetic DNA (Figure 1B).
Once the antibody-switch is optimized, its modular nature
offers the possibility of generalizing the platform for the
detection of other antibodies. Indeed, by simply changing the
recognition element on the scaffold, one can create a platform
for the measurement of potentially any antibody. This was
demonstrated by using as recognition elements not only small
molecules but also peptides. Specifically, the small molecule
dinitrophenol (DNP),
95
which is recognized by the anti-DNP
antibody and the Flag
96
and p17 peptides,
97
recognized by the
anti-Flag antibody and the anti-HIV antibody, respectively,
were employed (Figure 1C−E). All the switch variants respond
rapidly (<10 min) to their specific targets with low nanomolar
affinity for their targets, with a comparable efficiency in 90% of
blood serum, and very important for sensing purposes, they did
not exhibit any significant cross-reactivity with the other
targets. Such an antibody-switch platform presents several
advantages that make it well-positioned among other direct
assays and suggest that it may be of utility in a range of
different applications such as point-of-care diagnostics and in
vivo imaging. It is versatile and can be easily adapted to the
detection of a wide range of antibodies. It is rapid and
reagentless and does not require washing steps. The use of
synthetic DNA makes it also quite stable and cost-effective.
Obviously, together with the positive features mentioned
above, there are limitations that should be considered. First,
the lack of any amplification step makes the detection limit
achievable with this platform not comparable to that observed
with other homogeneous assays for the detection of antibodies
based on enzyme amplification steps (e.g., ELISA). In this
regard, we note that the detection limit achieved with the
antibody-switch is in the lo w nanomolar range, which
represents the limit of detection of commonly used
fluorescence detectors. Second, the platform is obviously
sensitive to the size of the antigen employed, the largest
antigens employed so far being peptides of <13 residues (p17).
The use of larger recognition elements (for example, entire
proteins) would surely result in the need for reoptimization of
the entire scaffold unit.
Nucleic Acid-Based Co-Localization P robes for
Optical Antibody Detection. To overcome the practical
limitations affecting the platform described above and to
improve the advantages of using DNA-based switching probes
for diagnostic applications, a new approach that couples the
positive features of DNA-based conformational switching
Figure 2. Nucleic acid-based co-localization probes for optical antibody detection. (A) Here the DNA scaffold is employed in conjunction with an
antibody-induced co-localization mechanism. The modular nature of the recognition platform allows for the detection of any antibody for which an
antigen can be conjugated to a nucleic acid strand. (B) Simultaneous orthogonal multiplexed detection of anti-DIG, anti-DNP, and anti-HIV-1 p17
antibodies using DNA scaffolds modified with three different fluorophore/quencher pairs. (C) Signal gain of the three nanoswitches obtained by
adding each antibody (50 nM) in different combinations. Filled and empty circles are used to identify the antibody added in solution. Reproduced
from Porchetta, A.; Ippodrino, R.; Marini, B.; Caruso, A.; Caccuri, F.; Ricci, F. Programmable Nucleic Acid Nanoswitches for the Rapid, Single-Step
Detection of Antibodies in Bodily Fluids. J. Am. Chem. Soc. 2018, 140, 947−953 (ref 98). Copyright 2015 American Chemical Society.
Analytical Chemistry Review
DOI: 10.1021/acs.analchem.8b05009
Anal. Chem. 2019, 91, 44−59
47
probes (nanoswitches) with those of co-localization ap-
proaches was recently proposed by our group.
98
In this
specific case, the spatial geometry of the target antibody is in
fact used to induce an increase in the effective concentration of
two DNA-based modules each conjugated with a recognition
element (i.e., an antigen). Considering also in this case a
distance of approximately 10−12 nm between the two binding
sites of a single antibody, one can predict that the two moieties
bound to a single antibody will be confined in a zeptoliter
volume thus leading to an effective concentration in the high
micromolar range.
99
This increase in effective concentration
can be employed to trigger a signaling reaction that, otherwise,
will be silent. The system is composed of two synthetic nucleic
acid modules ( Figure 2A). A first module (reporter module)
that comprises a fluorophore/quencher-modified DNA hairpin
(#1) is designed to hybridize with a synthetic nucleic acid
strand (#2) conjugated to an appropriate recognition element
(an antigen). The second module (input module) is
conjugated with another copy of the antigen and contains a
domain complementary to the loop sequence of strand #1.
Upon binding to the target antibody, the reporter and input
modules are co-locali zed in a confined volume, thereby
increasing their local concentrations and allowing their efficient
hybridization. Such antibody-induced hybridization forces the
opening of the hairpin, enhancing the switch’s fluorescence
(300-fold increase) and allowing for the rapid (within 2 min)
and sensitive detection of the antibody in the low nanomolar
range. The modular nature of the platform permits one to
easily change the recognition element in the two modules,
allowing the detection of different antibodies (Figure 2B). By
using different fluorophore/quencher pairs, the multiplexed
detection of different target antibodies in an orthogonal way
was also demonstrated (Figure 2C). The platform has been
employed to monitor the immune response elicited from HIV-
positive patients enrolled in a medical trial and treated with a
peptide-based (AT20 peptide) phase I therapeutic vaccine.
Recently, the use of this platform has also been demonstrated
for the detection of small molecules through a competitive
assay. Sp ecifically, a competitive fluorescence single-step
detection of environmentally relevant small target analytes
was developed.
100
The modular system consists of the same
two modules described above that are designed to be co-
localized in a confined volume in the presence of a target
antibody. The presence of free recognition element molecules
(i.e., antigen) competing for the same antibody binding
prevents the reporter and input modules from being in the
proximity, which leads to a decreased fluorescence emission.
Similar proximity-based strategies have been demonstrated
in the past 20 years for analytical purposes. In this regard, one
of the main representative examples is the protein-fragment
complementation assay that allows one to monitor bimolecular
interactions through the use of a reporter protein initially split
into two halves, each one tethered to a specific recognition
element. The interaction between the two re cognition
elements, or with a third interactive species, induces the co-
localization and the assembly of the two protein halves to
constitute the active signaling protein. Inspired by this, an
antibody-templated assembly of a functional RNA structure
was recently proposed by our group.
101
To do so, a Spinach
aptamer (a GFP-like RNA mimic),
102
which specifically binds
to a synthetic copy [3,5-difluoro-4-hydroxybenzylidene imida-
zoline (DFHBI)] of the natural GFP fluorophore, leading to
the display of GFP-like fluorescence properties, has been split
and conjugated with a pair of antigens. The binding of the
target antibody to the two RNA-conjugated antigen strands
allows the constitution of the active conformation of the
Spinach aptamer and leads to efficient binding of DFHBI with
a consequent increase in the intensity of the fluorescence
signal. The templated assay was tested for the detection of two
different antibodies (anti-digoxigenin and anti-dinitrophenol
antibodies), producing an affinity in the low nanomolar
range.
101
The performance of the assembly process was also
tested in RPMI cell culture medium and HeLa cell whole
lysates, and this test revealed similar performances in terms of
the sensitivity and observed signal, suggesting the potential use
of this assay for bioimaging and bioanalytical purposes.
DNA-Based Scaffolds for Antibody-Controlled Reac-
tions, Logic Gates, and Circuits. Apart from the analytical
applications described above, DNA-based scaffolds that
respond to antibodies or other macromolecular targets can
also be used to control reactions, molecular circuits, logic gates,
and load/release of molecular cargos. Gothelf and co-workers
proposed a general method that employs DNA-based strand
displacement competition reactions (SDCs) for the detection
of small molecules and proteins.
103
The assay principle is based
on the conjugation of a recognition element to a DNA strand
involved in a classic strand displacement reaction. The binding
of a macromolecular target (i.e., a protein or an antibody) to
such a recognition element shifts the equilibrium of the DNA-
based strand displacement competition reaction, and this
results in a measurable optical output. The authors
demonstrated nanomolar detection of antibodies or protein
targets and, through a competitive approach, the nanomolar
detection of small molecules such as biotin, digoxigenin,
vitamin D, and folate, in buffer and in plasma. The method is
flexible and provides an interesting way to use DNA as a
scaffold for sensing applications.
In a follow-up work, Gothelf and co-workers demonstrated
the development of hybridization chain reaction (HCR) for
the detection of different targets.
104
The sensing principle
relies on the use of DNA reaction initiator strands conjugated
with small molecule ligands. The binding of the target protein
to this recognition element provides a steric hindrance to the
initiator strand resulting in a retarded HCR. This assay allows a
response within nanomolar c oncentrations of the small
molecules in <5 min even in 50% human plasma.
The group of M. Merkx, which pioneered the development
of protein-switches for antibody detection,
105−108
has recently
demonstrated the design of bivalent peptide−DNA conjugates
that can act as molecular locks to control the activity of an
antibody. They originally designed dsDNA bivalently con-
jugated with two antigen peptides spanning a distance of 10−
12 nm. These bivalent peptide−dsDNA conjugates could bind
to the target antibody 500-fold more strongly than a
monovalent peptide, allowing effective blocking of the antigen
binding sites in a noncovalent manner.
109
The cleavage of the
linker between the peptide epitope and the DNA could restore
the activity of the antibody. The same group extended this
approach by designing novel DNA−peptide conjugates that
could block the antibody activity through hybridization. This
allowed the easy activation and/or inhibition of the antibody
by toehold strand displacement reactions (Figure 3A).
110
Employing yeast as a cellular model system, reversible control
of antibody targeting was demonstrated with low nanomolar
concentrations of peptide−DNA locks and oligonucleotide
displacer strands (Figure 3B). Introduction of two different
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![Figure 1. DNA-based structure switching probes for optical antibody detection. (A) The optical antibody-switch platform is designed to adopt a two-tailed stem−loop conformation in the absence of the target antibody. The two single-stranded tails act as an anchoring strand for DNA complementary sequences that are conjugated at one end with an appropriate recognition element (i.e., digoxigenin antigen) for the target antibody (anti-digoxigenin antibody). The binding of one copy of a target antibody to the two recognition elements causes a conformational change resulting in the opening of the stem−loop conformation and in an increase in the fluorescence signal intensity. (B) The antibody-switch sensor detects antidigoxigenin antibodies at low nanomolar concentrations. (C−E) The modular nature of the platform allows the detection of different targets by changing the recognition element [C, dinitrophenol (DNP); D, eight-residue FLAG peptide; E, 13-residue epitope excised from HIV protein p17] in a nanomolar concentration range, without any significant cross-reactivity with the other nonspecific targets. Reproduced from A Modular, DNABased Beacon for Single-Step Fluorescence Detection of Antibodies and Other Proteins. Ranallo, S.; Rossetti, M.; Plaxco, K. W.; Valleé-Beĺisle, A.; Ricci, F. Angew. Chem., Int. Ed. 2015, Vol. 54, Issue 45 (ref 89). Copyright 2015 Wiley.](/figures/figure-1-dna-based-structure-switching-probes-for-optical-12br9byv.webp)
